PET and MRI of Prostate Cancer - DiVA portal953219/... · 2016-09-06 · Lymph node metastasis in...

ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2016

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1248

PET and MRI of Prostate Cancer

CATRIN VON BELOW

ISSN 1651-6206ISBN 978-91-554-9662-3urn:nbn:se:uu:diva-300940

Dissertation presented at Uppsala University to be publicly examined in Rosénsalen, Ing.95/96 NBV, Akademiska sjukhuset, Uppsala, Friday, 30 September 2016 at 09:15 for thedegree of Doctor of Philosophy (Faculty of Medicine). The examination will be conductedin Swedish. Faculty examiner: Professor Katrine Åhlström Riklund (Umeå universitet).

Abstractvon Below, C. 2016. PET and MRI of Prostate Cancer. Digital Comprehensive Summaries ofUppsala Dissertations from the Faculty of Medicine 1248. 75 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-554-9662-3.

Prostate cancer (PCa) is the most common non-skin malignancy of men in developed countries.In spite of treatment with curative intent up to 30-40% of patients have disease recurrence aftertreatment, resulting from any combination of lymphatic, hematogenous, or contiguous localspread.

The concept of early detection of PCa offer benefits in terms of reduced mortality, but at thecost of over-diagnosis and overtreatment of indolent disease. This is largely due to the randomnature of conventional biopsies, with a risk of missing significant cancer and randomly hittingindolent disease.

In the present thesis, diagnostic performance of MRI DWI and 11C Acetate PET/CT lymphnode staging of intermediate and high risk PCa, was investigated, and additionally, predictivefactors of regional lymph node metastases were evaluated. Further, additional value of targetedbiopsies to conventional biopsies, for detection of clinically significant PCa, was investigated.

In paper one and two, 53 and 40 patients with predominantly high risk PCa underwent 11CAcetate PET/CT and 3T MRI DWI, respectively, for lymph node staging, before extended pelviclymph node dissection (ePLND). The sensitivity and specificity for PET/CT was 38% and 96%respectively. The sensitivity and specificity for MRI DWI was 55% and 90% respectively.

In paper three, 53 patients with newly diagnosed PCa were included. All patients underwentmulti-parametric MRI, followed by two cognitive targeted biopsies. Five more clinicallysignificant cancers were detected by adding targeted biopsies to conventional biopsies.

In paper four the value of quantitative and qualitative MRI DWI and 11C Acetate PET/CT parameters, alone and in combination, in predicting regional lymph node metastases wereexamined. ADCmean in lymph nodes and T-stage on MRI were independent predictors of lymphnode metastases in multiple logistic regression analysis.

In conclusion the specificity of diffusion weighted MRI and 11C Acetate PET/CT for lymphnode staging was high, although the sensitivity was low. Predictive factors of regional lymphnode metastases could be retrieved from diffusion weighted MRI and 11C Acetate PET/CT.By combining targeted biopsies with conventional biopsies the detection rate of clinicallysignificant PCa could be increased.

Keywords: Prostate cancer, lymph nodes, lymph node staging, PET/CT, MRI DWI, extendedpelvic lymph node dissection, targeted biopsies, multi-parametric MRI, TRUS guided biopsy,multiple regression analysis

Catrin von Below, Department of Surgical Sciences, Radiology, Akademiska sjukhuset,Uppsala University, SE-75185 Uppsala, Sweden.

© Catrin von Below 2016

ISSN 1651-6206ISBN 978-91-554-9662-3urn:nbn:se:uu:diva-300940 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-300940)

To Lisa, Clara, Erik and David

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Daouacher G, von Below C, Gestblom C, Ahlström H, Grzegorek R, Wassberg C, Sörensen J, Waldén M. Laparoscopic extended pelvic lymph node dissection as validation of the performance of [11C]- acetate-PET/CT in detection of lymph node me-tastasis in medium and high-risk prostate cancer. BJU Int. 2016 Jul;118(1):77-83

II von Below C, Daouacher G, Wassberg C, Grzegorek R, Gestblom C, Sörensen J, Ahlström H, Waldén M. Validation of 3T MI including diffusion weighted imaging for nodal staging of newly diagnosed intermediate- and high-risk prostate cancer. Clin Radiol. 2016 Apr; 71(4):328-334

III von Below C, Wassberg C, Norberg M, Tolf A, Kullberg J, Ladjevardi S, Häggman M, Bill Axelson A, Ahlström H. Additional value of magnetic resonance targeted biopsies to standard trans-rectal ultrasound guided biopsies for detection of clinical signifi-cant prostate cancer. Submitted

IV von Below C, Wassberg C, Grzegorek R, Kullberg J, Gestblom C, Sörensen J, Waldén M, Ahlström H. Quantitative and qualitative analysis of diffusion weighted MRI and 11C Acetate PET/CT: Predictive factors for regional lymph node metastasis in newly diagnosed prostate cancer of intermediate and high risk. Submitted

Reprints were made with permission from the respective publishers.

Contents

Introduction ................................................................................................... 11Risk assessment of prostate cancer ........................................................... 11Lymph node metastasis in prostate cancer ............................................... 12Mechanism of lymph node metastasis ...................................................... 13Sentinel node technique ............................................................................ 14Prostate anatomy ....................................................................................... 14Prostate biopsies ....................................................................................... 15Positron emission tomography/computed tomography ............................ 17Diffusion weighted imaging (DWI) ......................................................... 19Specific background to the studies ........................................................... 20

Paper I .................................................................................................. 20Paper II ................................................................................................. 21Paper III ............................................................................................... 21Paper IV ............................................................................................... 22

Aims .............................................................................................................. 24

Materials and methods .................................................................................. 25Prospective studies on lymph node staging of intermediate and high risk PCa (Study I,II and IV) ............................................................. 25

Patients ................................................................................................. 25Imaging ................................................................................................ 26Surgical technique ................................................................................ 27Histopathological evaluation ............................................................... 28Image analysis ...................................................................................... 29Statistical analysis ................................................................................ 31

Prospective study on the added value of MRI targeted biopsies (study III) .................................................................................................. 32

Patients ................................................................................................. 32Multiparametric MRI data acquisition and processing ........................ 35Multiparametric MRI evaluation and MRSI data interpretation .......... 36MRI-targeted biopsies .......................................................................... 36Pathology ............................................................................................. 38Statistical analysis ................................................................................ 38

Results ........................................................................................................... 39Lymph node staging with ePLND as reference standard ......................... 39Quantitative and qualitative lymph node analysis .................................... 43Targeted biopsies ...................................................................................... 47

Discussion ..................................................................................................... 49Summary of findings ................................................................................ 49Prostate lymph node metastases evaluated with 11C Acetate PET/CT ..... 50The importance of ePLND ....................................................................... 51Biological border ...................................................................................... 51Prostate lymph node metastases evaluated with MRI DWI ..................... 52USPIO and PSMA .................................................................................... 53Apparent diffusion coefficient .................................................................. 53Predictive factors of regional lymph node metastases .............................. 54MRI-targeted biopsies in PCa ................................................................... 55Methods for targeting MRI lesions ........................................................... 56Likert and PIRADS .................................................................................. 57Definition of clinically significant cancer ................................................ 58Limitations ................................................................................................ 58

Conclusions ................................................................................................... 60

Acknowledgements ....................................................................................... 61

References ..................................................................................................... 62

Abbreviations

PCa Prostate cancer PSA Prostate specific antigen DRE Digital rectal examination TRUS Trans-rectal ultrasound RP Radical prostatectomy EAU European Association of Urology ePLND Extended pelvic lymph node dissection PLND Pelvic lymph node dissection BPH Benign prostatic hyperplasia MRI Magnetic resonance imaging CEUS Contrast-enhanced ultrasound mpMRI Multi-parametric magnetic resonance imaging AS Active surveillance PET/CT Positron emission tomography/computed tomography SUV Standardized uptake value ROI Region of interest DWI Diffusion weighted imaging ADC Apparent diffusion coefficient ESUR European Society of Urogenital Radiology DCE Dynamic contrast enhancement MRSI Magnetic resonance spectroscopic imaging SB Standard trans-rectal ultrasound guided biopsies MRI-TB Magnetic resonance imaging-targeted biopsies FAS Fatty acid synthase pathway T Tesla T1W T1 weighted T2W T2 weighted TSE Turbo spin echo TR/TE Repetition time/Echo time FOV Field of view NSA Number of signal averages SE-EPI Spin echo single shot echo planar imaging GMP Good manufacturing practice LN Lymph node PPV Positive predictive value NPV Negative predictive value

ROC Receiver operating curve AUC Area under the curve RF Radiofrequency BW Bandwidth SPAIR Spectral selective attenuated inversion recovery SENSE Sensitivity encoding PRESS Point resolved spectroscopy ISUP International Society of Urological Pathology USPIO Ultra small iron oxide particles PSMA Prostate specific membrane antigen SNR Signal to noise ratio BI-RADS Breast Imaging Reporting and Data System PREDICT Prostate Diagnostic Imaging Consensus Meeting PI-RADS Prostate Imaging Reporting and Data System

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Introduction

Risk assessment of prostate cancer Prostate cancer (PCa) is the most common tumor among European men and the third most common cause of death (1) PCa is the most common malig-nancy in Swedish men, accounting for over 30 percent of all male cancer. In 2014, 10985 new cases of PCa were diagnosed and there were 2398 deaths from PCa (Cancer incidence in Sweden 2014, The national board of health and welfare).

The first suspicion of prostate cancer (PCa) is almost always due to an el-evated Prostate specific antigen (PSA) test and or a pathological digital rec-tal examination (DRE). The actual diagnosis is made with trans-rectal ultra-sound (TRUS) guided core biopsies. The Gleason score is the recommended methodology for grading PCa. According to current international conven-tion, the (modified) Gleason score (2) of cancers detected in a prostate biop-sy consists of the Gleason grade of the dominant (most extensive) carcinoma component plus the highest grade, regardless of its extent, there is no 5% rule (3). The lowest score is Gleason 6 (3+3) and the highest score Gleason 10 (5+5). Gleason pattern 6 has an exceedingly low risk of leading to death from PCa, i.e. cannot metastasize to lymph nodes or further (4). On the other hand Gleason score 4 has metastatic potential (5). In most cases, the molecu-lar abnormalities associated with cancer are absent in Gleason pattern 3 and present in Gleason pattern 4 (6-12).

Along with Gleason score, Pretreatment PSA and clinical T category have been shown to be independently predictive of various combinations of pros-tate cancer related endpoints in the non-metastatic setting (13-15).

In 1998, D’Amico et al introduced a risk stratification system to predict post-treatment biochemical failure after radical prostatectomy (RP) and ex-ternal-beam radiotherapy (16). This system divided non-metastatic patients into low-, intermediate-, and high-risk based on initial PSA, clinical T stage and biopsy Gleason score. Low-risk prostate cancer was defined as T1/T2a, and PSA ≤10 ng/ ml, and Gleason score ≤6. Intermediate-risk prostate can-cer was defined as T2b, or PSA 10–20 ng/mL or Gleason 7 disease. High-risk disease was defined as ≥T2c or PSA >20 ng/mL or Gleason 8–10 dis-ease. The same risk stratification system is recommended in the European Association of Urology (EAU) guidelines 2015 with the addition of locally advanced category in the High risk group: T3-T4 or N+.

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Another risk assessment system in prostate cancer is the utility of differ-ent nomograms, that incorporates multiple risk variables in mathematical models that calculate the probability of disease recurrence or progression. Each nomogram is developed using a different set of patients in a different setting. Only a few of the nomograms are based on extended pelvic lymph node dissection (ePLND), One of those, the Briganti Karakiewicz nomogram (17, 18) has been externally validated in both open and robot-assisted RP series and showed the highest predictive accuracy when compared with other prognostic tools (19, 20).

There are many other predictive tools used in prostate cancer, but a re-view of these is beyond the scope of this thesis.

Lymph node metastasis in prostate cancer Most cancer related deaths are a result of metastasis (21). Lymph nodes close to the primary tumor are commonly the first metastatic site (22, 23). Lymph node metastases are themselves seldom life threatening, but of major prognostic significance for prostate cancer and other cancers (22, 24-27). This is demonstrated by a large study from the Mayo Clinic of 3463 prostate cancer patients, which revealed that 10-year cancer-specific survival was significantly worse in those with positive lymph nodes after radical prosta-tectomy and regional lymph node staging (28). On the other hand it has been shown that patients with a low volume of nodal burden (two or fewer posi-tive nodes) have favorable long-term outcomes, which is significantly better than patients with a higher volume of lymph node invasion. This suggests that patients with a high nodal burden had systemic and more advanced dis-ease (29-32). There is also evidence that the degree of lymph node involve-ment is a strong predictor of cancer specific survival (33, 34).

The gold standard for N-staging is open or laparoscopic lymphadenecto-my. Many studies have shown that the rate of lymph node invasion in PCa patients almost linearly increases with the extent of pelvic lymph node dis-section (PLND) (35-38).

A limited PLND should be abandoned due to the high rates of false-negative findings, a node dissection limited to the obturator fossa will miss ~50% of metastases (35, 37). For this reason, general agreement has been reached on the indication for performing ePLND whenever PLND is indicat-ed (39, 40). Extended lymph node dissection includes removal of the nodes in the external-iliac, obturator- and internal iliac anatomic region. Some studies have advised to extend the dissection to include the common iliac lymph nodes up to the crossing of the ureter, with this approach 75% of all potential lymph node metastases are removed (41).

Sentinel node mapping studies have shown that aside from above men-tioned lymph node regions, the prostate also drains to the pre-sacral nodes

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(41, 42). Adding pre-sacral area to the template decreased the risk of incom-plete clearance to only 3% (42).

Mechanism of lymph node metastasis The lymphatic system is part of the circulatory system and an important part of the immune system. Lymph nodes are immune organs that react to pe-ripheral infections through antigen-presenting dendritic cells that prime lym-phocytes to produce antibodies in response to antigen (43). When lymphatic fluid has passed through the lymph node sinuses it subsequently drains into further lymph nodes proximally in the lymph node chain, before entering the venous blood mainly via the thoracic duct. In the cancer setting, the sentinel nodes are the first few lymph nodes into which a tumor drains. The sentinel node concept was first described by Gould et al. 1960 in a patient with a parotid carcinoma (44).

The sentinel lymph node is influenced by tumor-derived factors such as cytokines (45, 46), and studies have discovered that the immune function of the lymph node is severely impaired in cancer even before tumor cells are colonizing the lymph node. This immunosuppression might promote the formation, growth and transformation of tumor cells to develop metastatic potential (47-50).

A number of studies have examined the relationship between lymphangi-ogenesis and prostate cancer metastasis, and there is evidence of lymphangi-ogenesis but not angiogenesis in prostate cancer lymph node metastasis and systemic metastasis (51, 52). Interestingly, lymphangiogenesis in the sentinel node develop before the appearance of metastasis (53).

Angiogenic growth factors secreted from the primary tumor alter the lymph node structure by inducing lymphatic vessel formation (54, 55), and thereby facilitating tumor cell transport to the lymph nodes and establishing a favorable microenvironment for disseminated tumor cells (53).

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Sentinel node technique The sentinel node technique has been used to detect regional lymph node metastases with varying degrees of success in a number of cancers. For ex-ample the technique is established in early breast cancer (56). The concept of the sentinel node technique is to find the first lymphatic draining site of a primary tumor, which would be the first lymph node to be affected by metas-tasis, before involving other lymph nodes. Consequently, if the sentinel node is negative, the higher drainage stations should not harbor metastasis and a regional lymph node dissection could be omitted. However in PCa there are multiple primary lymphatic draining sites (sentinel nodes) (57) and that along with the fact that metastatic tissue in lymphatic vessels and lymph nodes may hinder the uptake of 99mtechnetium and dye, can lead to false negative results. The EAU guidelines for PCa 2016 state that due to the lack of data from large multicenter cohorts, the sentinel node technique remains experimental (58).

Prostate anatomy The prostate gland is an organ that is part of the urinary and reproductive systems in males. The prostate has an oval shape with a rounded base and consists of 70% glandular tissue and 30% fibro muscular or stromal tissue. The size of the prostate varies between individuals, ranging between the size of a walnut to the size of a small apple.

The prostate gland is divided into three zones: peripheral, transition, and central zones. The relationship of these zones is illustrated in figure 1. The peripheral zone is the largest zone situated posteriorly, laterally and in the apical part of the gland. Approximately 75% of prostate cancers originate in the peripheral zone (59-61). The central zone is located posterior to the prox-imal part of the urethra and prostate cancer is uncommon in this zone, only 5% occurs in this zone. The transition zone is situated anterior and lateral to the proximal urethra, 25% of prostate cancer originates from this zone. The transition zone constitutes 20% of the prostate gland until the age of 40, dur-ing aging the transition zone enlarges, and grows into the largest zone of the prostate gland. A condition known as benign prostatic hyperplasia (BPH). The central zone and the transition zone cannot be separated on magnetic resonance imaging (MRI), therefore these zones are referred to as the central gland. The anterior fibro-muscular stroma is a region of non-glandular tis-sue, located in the most anterior part of the gland. This region thins as it extends laterally and posteriorly and forms the fibro-muscular pseudo cap-sule that surrounds the prostate gland (62).

In the cranio-caudal direction the gland is divided in base, midgland and apex.

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Figure 1. Illustration of anatomy of the prostate in the axial, sagittal and coronal plane. CZ central zone, PZ peripheral zone, TZ transition zone, U urethra, 1: anterior fibromuscular stroma, 2: ejaculatory ducts, 3: neurovascular bundle, 4: verumonta-num, 5: seminal vesicle 6: periurethral tissue. Courtesy of Yacoub et al. Radi-ographics 2012; 32: 819-837. Reprint with permission from RSNA.

Prostate biopsies The standard approach for prostate biopsy is TRUS guided biopsy. The orig-inal systematic sextant biopsy was described by Hodge et al 1989 (63). Six biopsies were obtained bilaterally in the gland, three on each side, in equally spaced regions along the parasagittal plane halfway between the midline and the most lateral part of the prostate (Figure 2).

Later in the mid 1990´s Stamey et al (64) suggested a modified sampling of the midgland by directing the core needle more laterally (Figure 2). This modification increased the diagnostic yield. Extended biopsy schemes have subsequently been developed, in which eight, 10, or 12 cores were obtained (Figure 2), which have resulted in improved detection of prostate cancer (65-67).

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Figure 2. (a) Coronal illustration show the regions sampled in the prostate with standard sextant biopsy. (b) Coronal (left) and axial (right) illustrations of the modi-fied sextant biopsy, more lateral positions of the needle at the midgland (M). (c) Coronal illustration show the added prostate regions (open circles) that may be sam-pled in an extended biopsy scheme, for eight, 10 or 12 cores. Courtesy of Yacoub et al. Radiographics 2012; 32: 819-837. Reprint with permission from RSNA.

In contrast to image-guided biopsies in other cancers e.g. breast cancer and kidney cancer, TRUS guided biopsy is not targeted, this is due to the fact that the majority of prostate cancers are not visible on TRUS (68). Various ultrasound modalities have been developed to overcome the low sensitivity of grey-scale ultrasound for PCa, including Doppler techniques, contrast-enhanced ultrasound with gas-filled micro bubbles (CEUS) and sonoelas-tography (69). However there are no recommendations for the routine use of these novel imaging techniques.

Due to the high false negative rate of TRUS guided biopsy, repeat biopsy is often indicated, if clinical suspicion of PCa persists. Saturation biopsy technique, with 20-50 samples, has been proposed to increase the diagnostic yield with 18-34 % (70, 71), however saturation biopsy does not increase detection rate of high Gleason grades compared to 12-core standard TRUS biopsy (72). A limitation of the standard TRUS guided biopsy approach is that it may lead to underestimation (25-42%) and overestimation (14%) of the Gleason score and in addition provide uncertain information about the volume of PCa (73, 74). Further the anterior part as well the apex and the base of the prostate gland are under-sampled at TRUS guided biopsy.

These limitations emphasize the need of an imaging modality that is ca-pable of detecting significant PCa, while overlooking low risk tumors.

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Multi-parametric MRI (mpMRI) including T2-weighted imaging with dif-fusion-weighted imaging, dynamic contrast-enhanced imaging, or spectros-copy, of the prostate has evolved over the past decade and there is increasing evidence that targeted biopsies toward areas of suspicion on mpMRI has a high accuracy for detection of clinically significant PCa (75-79). Further correlation with radical prostatectomy shows that mpMRI, has excellent detection rate for Gleason 7 cancers (80-83).

In current practice guidelines mpMRI targeted biopsy is attributed a lim-ited diagnostic role. According to EAU guidelines 2016, mpMRI and target-ed biopsies may be used after previous negative TRUS biopsies when clini-cal suspicion of cancer persists (76, 84). Further EAU concluded that there was insufficient evidence to recommend mpMRI in biopsy naïve men (85).

Concerning repeat biopsies in active surveillance (AS) the EAU guide-lines stated that mpMRI imaging is of interest due to the high negative pre-dictive value for anterior cancers (86) but this strategy is not yet recom-mended to replace repeat TRUS guided biopsies (87).

In the National guidelines on PCa published by the Swedish National board of Health and Welfare 2014, recommendations on using mpMRI tar-geted biopsies in the setting of previous negative TRUS biopsies has a low degree of priority: level eight on a scale from one to ten. Targeted mpMRI biopsies in AS and biopsy naïve men was not addressed in the Swedish Na-tional guidelines.

The limited role for mpMRI targeted biopsies in EAU and Swedish Na-tional guidelines reflects that additional evidence is needed before a mpMRI targeted approach will be implemented in the diagnosis and management of PCa.

Positron emission tomography/computed tomography Positron emission tomography (PET) is a molecular imaging modality in which biologically active molecules labeled with positron emitting radioiso-topes are used to measure biological processes. The positrons released by the radioisotope annihilates with an electron. Each annihilation produces two 511 keV photons, travelling in opposite directions, the photons are detected by a ring of detectors inside the PET camera (Figure 3). The detectors are scintillation crystals connected to photomultiplier tubes, which detect and amplify the photons emitted and convert those into electrical signals. These electrical signals are manipulated and converted into a PET image.

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Figure 3. Positron emission and annihilation coincidence detection in PET. Reprint with permission from Springer Verlag.

The CT in the combined PET/CT scanner provides detailed anatomic infor-mation and attenuation correction. The standardized uptake value (SUV) is a semi-quantitative assessment of the radiotracer uptake in tissue. SUV is normalized to the injected radiotrac-er dose and the weight of the patient. The SUV is usually reported as the, maximum pixel value or the mean value of a region of interest (ROI). PET/CT is mainly used in oncology imaging but other indications exist, for example in neurology, cardiology and inflammation/infection.

e-β+

Detector

DetectorPositronrange

180°± 0.25°

Annihilationphoton

(511 k eV)

Annihilationphoton

(511 k eV)

β+

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Diffusion weighted imaging (DWI) DWI is a functional MRI sequence that produce images with the use of dif-fusion of water in biologic tissues. Functional and quantitative information about tissue cellularity is acquired with this technique. DWI images are pro-duced on the basis of random microscopic (Brownian) motion of free water molecules. In contrast to free diffusion, diffusion of water molecules in bio-logic tissues is impeded by their interaction with cell membranes and intra-cellular organelles. The signal is derived from the motion of water molecules in intracellular, extracellular, and vascular spaces. The degree of diffusion restriction is inversely related to tissue cellularity and cell membrane integri-ty (88).

The biologic mechanisms are not clearly understood; however, an in-crease in the number of cells for example in cancer or inflammation, increas-es the total amount of intracellular space and decreases the extracellular space, leading to restricted diffusion (Figure 4) (89, 90).

Figure 4. Illustration of the diffusion of water molecules. (a) Restricted diffusion in tissue with increased cellularity: water molecules (arrows) in intracellular space, extracellular space and intravascular space, all of which contributes to overall diffu-sion signal. (b) Free diffusion in tissue with low cellularity and defective cell mem-branes. Reprint with permission from AJR/ARRS.

The standard method for diffusion weighted imaging is a single shot spin echo T2-weighted sequence with two symmetric diffusion-sensitizing gradi-ents on each side of the 180° refocusing pulse (Stejskal-Tanner sequence).

The level of diffusion weighting can be adapted by modifying the ampli-tude and duration of the diffusion gradient and the time between the first and second gradients. The b-value is an index of the degree of diffusion weighting, determined by the gradient characteristics described above. The diffusion restriction can be quantified by scanning with different b-values to calculate the apparent diffusion coefficient (ADC) map. The ADC map is

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calculated during post-processing with use of at least two different b-values, although three or more b-values are commonly used to improve noise. By plotting the logarithm of the signal intensity on the y-axis against the b val-ues on the x-axis, the ADC value is the slope of the line fitted to the expo-nential function. By placing a ROI within a lesion, the ADC value for the lesion can be determined.

DWI is integrated with standard magnetic resonance imaging, and does not require any extra equipment. DWI is indispensable in oncology imaging and in diagnosis of early brain infarction.

Specific background to the studies Paper I Detection of PCa lymph node metastases may be of importance in the cura-tive strategy if involved lymph nodes are surgically resected or incorporated into the radiation therapy dose plan (91-93). Further early and prolonged hormonal therapy in the curative setting can improve survival and reduce risk of relapse in patients with positive regional lymph nodes (92, 94, 95). In a study of long term follow up (ten years) of curative strategies where no or limited pelvic lymph node dissection (PLND) was performed, high rate of disease relapse (>50%) was reported for T3 cases (96).

Radical prostatectomy with ePLND in intermediate- and high-risk pa-tients can increase the PSA progression-free survival (93), indicating the importance of diagnosing the patients with lymph node metastases.

Extended PLND is recommended in EAU guidelines for patients with in-creased risk of lymph node metastasis. A limited dissection in the obturator area misses 50-70% of lymph node metastases (35, 37, 41).

Conventional CT and MRI have low accuracy as N staging tools (97, 98). Choline is the most documented radioisotope for PET/CT imaging of pros-tate cancer, but few studies compare imaging results to histological findings (99-101).

11C Acetate is another radioisotope under investigation in prostate cancer. Compared with 11C and 18F Choline PET/CT, the detection rate of metastases and localization of biochemical relapse after curative treatment appear to be equivalent (102, 103). 11C Acetate PET/CT has been studied predominantly in the context of biochemical relapse after curative treatment and it is used clinically at several imaging centers. 11C Acetate PET/CT has not previously been validated to a histological reference from ePLND, at initial diagnosis of PCa.

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Paper II In developed countries PCa is the most common non-skin malignancy in men (104). Knowledge about the N stage is important for treatment planning and presence of lymph node metastases is a critical prognostic factor (105). In PCa patients treated with curative intent, recurrence occur with biochemi-cal relapse in 30–40% (106). There is a clearly a need for diagnostic meth-ods to identify patients with lymph node positive disease.

Nomograms based on clinical variables such as prostate biopsy Gleason score, proportion of positive biopsies, PSA level and clinical tumor stage, can predict a patients risk of having N1disease (107). The reference standard method for lymph node staging in patients at in-creased risk of N1 disease, is ePLND with histopathological diagnosis (108). However, the procedure is associated with morbidity and relatively high cost (109).

Previously imaging criteria has relied on lymph node size and morpholog-ical criteria, however there is a significant overlap between the size of nor-mal and malignant lymph nodes. Resulting in poor sensitivity of convention-al CT and MRI (110-113).

Functional imaging might have the capability to increase the accuracy of lymph node staging. MRI including DWI (MRI DWI) has shown promise in detecting malignant lymph nodes (114-117), but only a few articles have been published on lymph node staging of early PCa (118-121).

According to European Society of Urogenital Radiology (ESUR) guide-lines (122) a standard MRI of the prostate and lesser pelvis, should include T2 weighted imaging (axial, coronal, sagittal) and T1 weighted imaging with a larger field of view together with DWI and ADC maps. Dynamic contrast-enhanced MRI or spectroscopy may be added to this clinical protocol.

Due to different ADC thresholds between tumor types and between dif-ferent MRI systems, quantification of ADC is difficult to implement in the clinical setting.

Paper III Early detection of PCa has advantages in terms of reduced mortality, but at the cost of over diagnosis and overtreatment of indolent disease (123-126). There is clearly a need for an improvement in diagnostic strategies. Multi-parametric MRI (mpMRI) of the prostate has received increased attention in the scientific literature and there is growing evidence that mpMRI can relia-bly identify clinically significant prostate cancer (75, 84, 127-130) and in addition have a low detection rate of low risk PCa (131). Targeted biopsies toward mpMRI suspicious lesions is therefore an interesting diagnostic ap-proach.

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Prostate MRI has developed over the past decade. The detection of cancer lesions has improved considerably with the introduction of functional imag-ing with DWI and ADC. A few studies have shown that ADC correlates with Gleason score (77, 132, 133). Standard anatomical T2 and T1 weighted im-aging and DWI with ADC map is recommended as obligatory in the Europe-an guidelines (108, 122), second functional imaging technique i.e. dynamic contrast enhancement (DCE) or magnetic resonance spectroscopic imaging (MRSI) is optional. For larger prostate lesions the sensitivity for significant cancer is high but for smaller lesions the detection rate for significant cancer is less, particularly in the central gland. Further the specificity may be re-duced due to false positive findings such as prostatitis, benign hyperplasia, calcifications and scars. TRUS guided biopsies (standard biopsies - SB) with a total of 10-12 cores is the conventional pathway of prostate cancer diagno-sis. The cores are typically taken systematically from each sextant region at mid, base, and apex on both right and left sides.

The targeted approach with MR guided biopsies (MRI-TB) differs sub-stantially from the SB approach.

SB has reformed the diagnosis and management of PCa, but due to its random nature conventional SB may miss cancer, requiring re-biopsy, possi-bly lead to underestimation of significant cancer and may randomly hit in-significant disease (134, 135). The drawback of SB to correctly diagnose clinical significant cancer is confirmed by a 40% underestimation of Gleason grade on biopsies, in comparison with subsequent prostatectomy (136). Re-cent studies have demonstrated that MRI-TB can be very useful, particularly in patients with negative SB but with clinical suspicion of PCa (137).

Paper IV Presence of regional lymph node metastases in PCa is a prognosticator of significantly decreased disease-specific survival rates and biochemical recur-rence-free rates (24). Consequently detection of N1 disease is of considera-ble importance. The reference standard for regional lymph node staging in patients at increased risk of lymph node metastases is ePLND (108). Limited dissection of the obturator fossa misses 50% of metastases (35) hence an extended approach is recommended. Nonetheless ePLND is associated with high cost, hospitalization and possibly postoperative complications. Imaging may have a role to select patients suitable for ePLND. Conventional CT and MRI depend on morphological criteria, mainly size and shape of lymph nodes, in the evaluation of lymph node metastases, and thus have limited N-staging value (98, 111, 138). Functional imaging techniques have received increased attention in the scientific literature. MRI DWI has been investigat-ed by several researchers in PCa (118-121, 139, 140) as well as 11C and 18F

Choline PET/CT (99, 141-143). The PET radiotracer 11C Acetate has been studied by a few researchers in the lymph node staging setting (144, 145).

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Acetate can be metabolized in different ways, the most important in PCa is the fatty acid synthase pathway (FAS), as this pathway is overexpressed in PCa (146-148). The standardized uptake value (SUV) is a semiquantitative measure of the uptake of this tracer.

A few publications have indicated that ADC measurements in MRI DWI can discriminate prostate cancer from benign prostatic lesions, but with a significant overlap (149-151).

To the best of our knowledge no study has combined quantitative and qualitative analysis of 11C Acetat PET/CT and MRI DWI for lymph node staging, with histopathology from ePLND as reference standard.

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Aims

The aims of the studies described in this thesis were:

I To validate 11C Acetate PET/CT for lymph node staging of inter-mediate- and high-risk PCa with ePLND as reference.

II To validate 3 Tesla (T) MRI DWI for lymph node staging of in-termediate- and high-risk PCa with ePLND as reference.

III To evaluate the additional value of MRI targeted biopsy to stand-ard TRUS guided biopsy for detection of clinically significant PCa (Gleason ≥7). An additional aim was to compare the biopsy results to MRI evaluation using the Likert scale.

IV To examine the value of quantitative and qualitative 3T MRI DWI and 11C Acetate PET/CT parameters in predicting regional lymph node metastasis of newly diagnosed PCa of intermediate and high risk.

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Materials and methods

Prospective studies on lymph node staging of intermediate and high risk PCa (Study I,II and IV)

Patients In study I and IV, 53 consecutive patients with intermediate (n=6) and high risk (n=47) prostate cancer according to D´Amico risk categories (152) were prospectively included, patient characteristics are listed in table 1. In study IV, 40 underwent 3T MRI DWI and 11C Acetate PET/CT, the remaining 13 had 11C Acetate PET/CT only. In study I and II, 11C Acetate PET/CT and 3T MRI DWI were studied separately. All patients underwent imaging within two weeks before ePLND. Inclusion criteria were a negative bone scintigra-phy and a risk of lymph node spread of >20% according to the Briganti nomogram (153). Exclusion criteria were contraindication to laparoscopy, contraindication to MRI examination (eg, pacemaker, magnetic implants) and hip replacement or (previous hip or lower pelvis fractures.) The studies were approved by the regional ethics committee and radiation ethics com-mittee (study I and IV). Informed consent was obtained in all patients before participation.

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Table 1. Patient characteristics.

Value Number of patients 53 Mean (range) age, years 67 (54-76) Mean (median; range) PSA level, ng/mL 24 (19; 3-112) Clinical T-stage, n (%)

T1c 1 (1.9) T2 11 (20.8) T3 41 (77.4)

Biopsy Gleason sum, n (%) 6 5 (9.4) 7 39 (73.6) 8 5 (9.4) 9 4 (7.5)

D’Amico risk stratification, n (%) Intermediate 6 (11.3) High 47 (88.7)

Percentage risk of LN invasion*, n 19–59 27 ≥60 26

*Calculated according to the Briganti et al. nomogram.

Imaging In study II and IV patients were examined with a 3T scanner (Achieva, Philips Medical Systems, Best, The Netherlands) using a two-channel whole body coil for excitation and a six-element phase-array coil for receiving. MRI from apex of the prostate to the aortic bifurcation was performed using T1- (T1W) and T2-weighted (T2W) turbo spin echo (TSE) sequences in axial plane. The main T1W acquisition parameters were as follows: repetition time/echo time (TR/TE), 670/10 ms; field of view (FOV) 260 x 260 mm2; acquisition matrix 150 x 186; number of signal averages (NSA), 2. T2W TSE scans were acquired with TR/TE 7000/120 ms; FOV 260 x 260 mm2; acquisition matrix 166 x 173; number of signal averages (NSA), 2. Axial fat suppressed DWI was performed using the spin-echo single-shot echo-planar imaging (SE-EPI) technique (TR/TE, 2450/55 ms; FOV 220 x 280 mm2; acquisition matrix 73 x 94; diffusion encoding gradients b = 0, 100, 200, 400, 500 s/mm2; NSA, 3). The ADC maps were obtained with mono-exponential fitting. Separate DWI imaging with single b value (1000 s/mm2) was performed for qualitative diagnostics Axial T1W (TR/TE, 500/8 ms) and T2W TSE (TR/TE, 3000/100 ms) images of the prostate gland and vesi-cles were obtained with FOV 160 × 160 and acquisition matrix 182 x 200 and 160 x 200, respectively. NSA = 3 for both acquisitions. Axial DWI/ADC SE-EPI scans used following parameters: TR/TE, 1800/55 ms; FOV, 220×220 mm2; acquisition matrix 98 x 126; NSA, 4; diffusion encoding gradients b = 0, 100, 200, 400, 500 s/mm2. The ADC maps were obtained

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with mono-exponential fitting Separate DWI imaging with single b value (1000 s/mm2) was performed for qualitative diagnostics.

In study I and IV, 11C Acetate was synthesized according to in-house good manufacturing practice (GMP) procedures. Patients were fasted for at least 4 hours prior to PET. Five MBq/kg body weight of 11C Acetate was injected intravenously in an antecubital vein 10 min prior to PET acquisition. PET/CT was performed on a GE Discovery ST16 (GE Healthcare, Waukesha, ML) hybrid scanner. A venous phase contrast-enhanced CT used both for morphologic analysis and for attenuation correction (140 kV, auto-mA 10-80 mA), and PET with 3 min per bed position, covering regions from the upper thighs to the neck, typically obtained in 6 bed positions. Total PET acquisition time was 18 min. Total effective dose of both PET and CT with this protocol was approx. 9mSv. PET images were corrected for attenuation, dead-time, scatter and decay, and reconstructed to a 50 cm field of view in a 128 x128 matrix using an iterative reconstruction algorithm with 2 iterations and 21 subsets as supplied by the manufacturer.

Surgical technique A trans-peritoneal laparoscopic ePLND was performed, first from the exter-nal and common iliac artery and vein from the ureter and to the deep circum-flex vein, respecting the genitofemoral nerve, secondly from the obturator fossa (the space between the external iliac vein down to the obtura-tor nerve), and lastly the internal iliac area from the obturator nerve down to the internal iliac artery and to the deep pelvic floor (Figure 5). The three specimens from each side were sent to the pathologist in separate containers. The surgery was performed by the same team with 4 years of prior experi-ence of 130 standardized laparoscopic ePLNDs.

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Figure 5. ePLND sites: 1, external and common iliaca vessels; 2, Obturator fossa; 3, internal iliaca vessels. The diagram used with permission from Department of Urol-ogy, Bern, Switzerland.

Histopathological evaluation Samples from separate anatomical regions were handled in separate contain-ers. The specimens were received in formalin (4% buffered formaldehyde) of adequate volume, approximately 5-10 times the weight/volume of the tissue, to ensure proper fixation. The number of embedded lymph nodes was recorded for each site. Lymph nodes <8-10 mm were embedded whole. Lymph nodes >10 mm around the equator were dissected longitudinally through the hilum or sliced serially at approximately 3mm intervals, depend-ing on size. All lymph nodes were embedded in total and dehydrated in al-cohol for 21 hours to facilitate sectioning, and thereafter embedded in paraf-fin and sectioned (4 µm) in two levels. Sections were stained with haema-toxylin and eosin. For each anatomical region, the number of positive lymph nodes and the proportion of positive lymph nodes were reported. Extra glan-dular extension was noted if identified. The largest dimension was recorded for every metastasis. Immunohistochemistry with pan-cytokeratin (AE1/AE3) was used infrequently, when needed to confirm a metastasis.

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Image analysis In study I a radiologist (C.v.B.) specialized in nuclear medicine and oncolog-ical radiology, with > 10 years of experience in Acetate PET/CT reading, analyzed the images qualitatively for any focally increased, non-physiological 11C Acetate uptake and size. If the uptake in a lymph node was intense compared with background activity, it was reported as positive re-gardless of the size and shape. If the uptake was moderate the lymph node was reported positive if enlarged (> 6 mm, short axis), round or oval (Figure 6). For each of the six anatomical sites applied for lymph node dissection the presence of lymph node metastases at PET/CT was rated yes or no, as a part of a clinical report to the surgeon before ePLND. One additional radiologist (C.W.) specialized in nuclear medicine and oncology imaging repeated the PET/CT analysis, blinded to all other information in order to calculate in-terobserver variability. No second opinion was allowed.

Figure 6. Example of bilateral true-positive lymph nodes (arrows) with focal uptake of 11C Acetate at PET/CT.

In study II, MRI evaluation was done by radiologist C.W. and the readings were performed according to the clinical routine with no blinding to the clin-ical information in the referral form.

The readings were performed analyzing both functional and morphologi-cal parameters together. First the functional parameters indicative of malig-nancy, very high signal and very low signal (compared to signal intensity in apparently normal lymph nodes) on DWI and ADC maps, respectively, were analyzed visually for detection of suspected lymph node metastases. There-

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after morphological criteria for metastases of the corresponding lymph nodes on T2W images were assessed. The following morphological criteria of ma-lignancy were analyzed; size and shape (round, irregular and ill defined bor-ders), T2 signal intensity (heterogeneous or similar to primary tumor) or cluster of grouped lymph nodes (≥ 3). Size criteria of ≥ 6 mm (154), short axis, was used for oval shaped lymph nodes with no clear fatty hilum. Lymph nodes measuring more than 6 mm but with a clear elongated shape and fatty hilum was not reported as suspicion of metastases; typically located along the external iliac vessels.

The lymph nodes were assessed as normal or malignant. To be reported as malignant the following criteria were requested: positive functional criteria together with at least one morphological criterion as described above.

For each of the six anatomic regions applied for lymph node resection the number of malignant lymph nodes was noted, as part of a clinical report. Anatomical region on MRI were matched with the corresponding region on surgery/histology, for each of the six anatomic regions.

Surgeons were not masked to the clinical reports. The imaging part of this study was based solely on the preoperative clinical MRI reports.

In study IV radiologist C.v.B. analyzed the images, blinded to histo-pathology results and clinical information. At least 6 months passed between the non-quantitative analyses in study I and II and the quantitative analysis in the present manuscript.

MRI DWI and 11C Acetate PET/CT were reviewed side by side, the lymph node (LN) with the visually most suspicious findings with regard to diffusion restriction, PET activity, shape and size, were chosen from any of the anatomical regions included in an ePLND, for each patient. Diffusion restriction and PET activity weighed heavily in the selection of the visually most suspicious LN, secondly LN shape and thirdly LN size. In case of nor-mal LN findings, the largest LN was assessed. The chosen lymph nodes were assessed for the following features; SUVmax measured by placing a region of interest (ROI) encompassing the uptake, the maximum pixel value representing SUVmax, ADCmean measured by placing a ROI within the LN contour in the LNs largest axial section, size was measured in the LNs short axis and LN shape was registered as oval or round. MRI DWI was also ana-lyzed for primary tumor T stage; obvious extra capsular extension was regis-tered as MRI-T3a, obvious spread to seminal vesicles was registered as MRI-T3b, if non of this features were present the T stage was registered as ≤ MRI-T2. The index lesion, i.e. the largest lesion, in the primary tumor was also assessed; tumor-SUVmax and tumor-ADCmean were measured by the same method described for lymph nodes above.

In all studies (I, II and IV) image analysis was done using Carestream Vue PACS with built in PET/CT as software (Carestream Health, Inc, Roch-ester, NY, USA).

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Statistical analysis In study I and II, power calculations were performed. With a level of signifi-cance of 5%, a power of 80% and a clinical relevant difference of ±10%, 17 lymph node positive patients were needed to test for sensitivity other than 70%. Based on local experience approximately 43% of patients were lymph node positive after ePLND, requiring a minimum of 40 patients for the stud-ies. Sensitivity, specificity, positive predictive value (PPV), negative predic-tive value (NPV) and accuracy for the detection rate with MRI DWI and 11C Acetate PET/CT were calculated with standard mathematical models on both patient and anatomical region level. The lymph node positive patients de-tected with MRI DWI and 11C Acetate PET/CT were compared with those not detected with imaging, concerning PSA, Gleason score, lymph metasta-sis burden and size, calculated risk of lymph node involvement and curative treatment decisions.

Continuous variables were compared by independent T-test, if data had a normal distribution, for non-normally distributed data the Mann-Whitney U test was applied. Categorical variables were compared by Fischer Exact P test (one tailed). A P value of <0.05 was considered statistically significant. Statistical analysis was performed with Statistica 12® (Statsoft Inc Tulsa, OK).

In study IV receiver operating curve (ROC) analysis of LN-ADCmean, LN-SUVmax, LN-size, tumor-ADCmean and tumor-SUVmax was per-formed to determine optimal cut-off from which the variables were dichoto-mized. Variables were then analyzed in simple logistic regression analysis to determine their significance. Different combinations of the significant varia-bles in the simple analysis were then included in multiple logistic regression models but the number of observations in each variable did not allow us to use more than two variables at the same time. For each model the predicted values were compared with the observed values, area under the curve (AUC), sensitivity, specificity, positive and negative predictive value (PPV, NPV), Accuracy, pseudo R2 (Nagelkerke) and Hosmer-Lemeshow statistic were calculated to determine their classification performance. Multicolline-arity between variables was measured with Cramer´s V.

A p-value less than 0.05 was considered statistically significant. Statisti-cal analysis was performed with Dell Inc. (2015). Dell Statistica (data analy-sis software system), version 13.

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Prospective study on the added value of MRI targeted biopsies (study III) Patients Patients newly diagnosed with localized PCa by clinical routine SB, and the majority scheduled for curative therapy, were included and recruited from the Urology Department at Uppsala University Hospital between Nov 2011 and Nov 2014. A total of 75 patients were included, and out of these, 53 patients underwent both SB and MRI-TB. Figure 7 graphically illustrates a flow chart of included patients that went to either: 1). Treatment (radical prostatectomy n=34, curative radiation therapy n=11) or 2). Active surveil-lance (n=8). Patient characteristics are shown in table 2. After written in-formed consent, the patients underwent standard mpMRI (see below) fol-lowed by MRI-TB (2 biopsies). Exclusion criteria were previous history of PCa, contraindication to MRI examination (eg, pacemaker, magnetic im-plants), contraindication to endorectal coil (eg anorectal inflammation or recent surgery), hip replacement or previous hip or lower pelvis fractures. The study was approved by the local Research Ethical Committee.

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Figure 7. Flowchart of included patients.

Eligable patients with newly diagnosed prostate cancer

(n = 75)

Abstained MRI (n = 5)

3T mpMRI with endorectal coil (n = 70)

Abstained targeted biopsies (n = 14)

Scheduled for cognitive targeted biopsies

(n = 56)

Anterior index lesions unreachable by biopsy needle

(n = 2)Large postbiopsy bleeding

obscuring index lesion on mpMRI (n = 1)

Cognitive targeted biopsies (n = 53)

Curative radiotherapy (n 11)Active surveillance (n = 8)

Radical Prostatectomy with whole mount pathology

(n = 34 )

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Table 2. Patient characteristics and findings (n =53).

Patient characteristics in agreement with START criteria, mpMRI- and biopsy core findings All patients RP patients Men included in analysis, n 53 34 Age, median (range) 64 (46-74) 64 (48-74) Prebiopsy PSA level, ng/ml, median (range) 6.4 (1.3-21) 6.0 (1.3-21) PSA density, median (range) 0.18 (0.06-0.63) 0.16 (0.06-0.63) Prostate volume, ml, median (range) 33 (13-123) 32 (13-123) Positive DRE findings (≥T2), n (%) 24 (45) 16 (47) TRUSGB per patient, median (range) 10 (5-12) 10 (5-12) TRUSGB Gleason 3+3, n (%) 21 (39.6) 7 (20.6) TRUSGB Gleason 3+4, n (%) 16 (30.1) 12 (35.3) TRUSGB Gleason 4+3, n (%) 8 (15.1) 8 (23.5) TRUSGB Gleason 3+5, n (%) 4 (7.5) 3 (8.8) TRUSGB Gleason 4+4, n (%) 2 (3.8) 2 (5.9) TRUSGB Gleason 4+5, n (%) 2 (3.8) 2 (5.9) Targeted biopsies per patient, median (range) 2 (2-2) 2 (2-2) Targeted biopsy negative, n (%) 15 (28.3) 7 (20.6) Targeted biopsy Gleason 3+3, n (%) 18 (34.0) 13 (38.2) Targeted biopsy Gleason 3+4, n (%) 8 (15.1) 6 (17.6) Targeted biopsy Gleason 4+3, n (%) 5 (9.4) 3 (8.8) Targeted biopsy Gleason 3+5, n (%) 2 (3.8) 2 (5.9) Targeted biopsy Gleason 4+4, n (%) 1 (1.9) 1 (2.9) Targeted biopsy Gleason 4+5, n (%) 4 (7.5) 2 (5.9) Proportion of positive cores TRUSGB, median (range) 3 (1-8) 3 (1-8)

Proportion of positive cores targeted biopsies, median (range) 1 (0-2) 2 (0-2)

TCCL TRUSGB, mm, median (range) 10 (0.5-58) 12 (0.5-58) TCCL targeted biopsier, mm, median (range) 7 (0.5-30) 7 (1.5-30) MCCL TRUSGB, mm, median (range) 5 (0.5-16) 5 (0.5-16) MCCL targeted biopsies, mm, median (range) 5 (0.5-12) 5 (1-12) Likert score 1 on mpMRI, n 2 1 Likert score 2 on mpMRI, n 9 4 Likert score 3 on mpMRI, n 16 12 Likert score 4 on mpMRI, n 10 7 Likert score 5 on mpMRI, n 16 10 DRE digital rectal examination, TRUSGB trans-rectal ultrasound-guided biopsy, TCCL total cancer core length, MCCL maximum cancer core length, RP radical prostatectomy, mpMRI multiparametric MRI

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Multiparametric MRI data acquisition and processing Magnetic resonance (MR) examinations were performed using a 3 T scanner (Achieva, Philips Medical Systems, Best, The Netherlands) equipped with a dual-source parallel radiofrequency (RF) transmission system. The whole body coil was used for excitation. A six-element receiver phase array (cardi-ac) coil together with a disposable endorectal coil (MEDRAD Inc., Indiano-la, PA, USA) was used for signal reception. T2W TSE images were obtained in the axial, sagittal and coronal orientations, TR/TE 3000/100 ms, FOV 160 x 160 mm2, acquisition matrix 200x154, spatial resolution 0.8x1x3 mm3, interslice gap 0 mm, bandwidth per pixel (BW) 217.8 Hz, NSA 1, scan dura-tion 2 min 19 sec). Axial T1W TSE images were measured with the follow-ing parameters: TR/TE 519/8 ms, FOV 160 x 160 mm2, acquisition matrix 200x181, spatial resolution 0.8x0.9x3 mm3, interslice gap 0.3 mm, BW 290.2 Hz, NSA 3, total scan time 3 min 4 sec. Axial images were taken per-pendicular to the rectal wall. Coronal slices were orthogonal to the axial.

Axial DWI were acquired using identical slice position as T2W TSE im-ages. Fat suppressed DWI was performed using the single-shot spin echo echo-planar sequence. Spectral selective attenuated inversion recovery (SPAIR) RF prepulse was used for fat suppression. The following parame-ters were used for measurement of ADC maps: TR/TE 1700/60 ms, FOV 200x200 mm2, acquisition matrix 112x110, spatial resolution 1.8x1.8x3 mm3, interslice gap 0 mm, BW 12.3 Hz, NSA 6, scan duration 5 min 28 sec. Diffusion encoding gradients (b = 0, 100, 200, 400, and 500 s/mm2) were sequentially applied along the three orthogonal directions. The maps of (iso-tropic) ADC were automatically calculated by the scanner, with mono-exponential fitting. In addition, two extra DWI acquisitions with single b value 1000 s/mm2 and 1500 s/mm2 were used for imaging (TR/TE 1800/60 ms, FOV 200x263 mm2, acquisition matrix 88x115, spatial resolution 2.3x2.3x3 mm3, interslice gap 0 mm, BW 28.6 Hz, NSA 12, sensitivity-encoding (SENSE) factor 2, scan duration 3 min 28 sec).

Three-dimensional magnetic resonance spectroscopic imaging (3D MRSI) of the prostate was performed using point-resolved spectroscopy (PRESS) localization technique (TR/TE 1300/140 ms, spectral bandwidth 2000 Hz, 1024 points, spectral matrix 10x10x8, nominal voxel size 7x7x7 mm3, NSA 1, scan duration 15 min 20 sec). Fat suppression was performed by a frequency selective inversion recovery prepulse. A dual BASING-pulse was used for water and residual fat suppression. Outer volume water and fat saturation slabs were not needed. The measured time domain data were fil-tered in all phase-encoding directions with a Hanning filter. Intensity of spectral lines was estimated by spectral fitting using LCModel and a simu-lated basis set.

There were at least four weeks between SB and mpMRI.

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Multiparametric MRI evaluation and MRSI data interpretation Two experienced radiologist (C.W. and C.v.B.) with >five respectively >four years of experience in oncology imaging and reading prostate evaluat-ed the mpMRI for detection of index lesions to be biopsied based on mor-phological T2 WI criterias, DWI, ADC and MRSI data evaluation. If equiv-ocal findings, a consensus was taken for which was the most likely index lesion to be targeted for biopsy and one lesion was selected for MRI-TB. Both radiologists were blinded to pre-imaging serum PSA, clinical T stage and biopsy status of SB. The mpMRI examinations were also reviewed and graded accordingly to the 5-point Likert scale, ranging from very low suspi-cion to very high suspicion of clinically significant prostate cancer (155, 156). A physicist (J.W.) with >15 y of experience evaluated the prostate MRSI data sets and provided the location and total number of suspicious voxels, (choline+polyamines+creatine)/citrate spectral intensity ratio (CC/C), CC/C max, and CC/C average for the index lesions. There were at least four weeks between SB and MRI.

MRI-targeted biopsies Targeted biopsies were performed by a radiologist with > 10 years of experi-ence in ultrasound and prostate biopsy procedure (M.N.). The biopsies were based on mpMRI index lesion findings taken with ultrasound guidance, so called cognitive fusion, i.e. biopsy cores were aimed using cognitive regis-tration of the index lesion on the basis of zonal anatomy and imaging land-marks (e.g. nodules, cysts). Example of mpMRI index lesion and corre-sponding whole mount pathology cross serial section are shown in figure 8. Two biopsies were taken per patient, even in those patients who had low to very low suspicion of significant PCa on mpMRI One of the radiologists who had evaluated the mpMRI was present during the biopsy procedure, demonstrating the location of the index lesion selected.

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Figure 8. A 60 year old male with T1c prostate cancer, PSA 14 ng/mL. Standard trans-rectal ultrasound guided biopsy: Gleason 3+4 in two out of eight cores. MRI targeted biopsy: Gleason 3+4 in two out of two cores. Whole mount pathology: Gleason 3+4. (a) Diffusion weighted image (b 1500) show a focal, strongly in-creased signal intensity in the left central gland (arrow). (b) Apparent diffusion coef-ficient map shows a focal decreased signal intensity in the left central gland (arrow). (c) T2 weighted image show a focal area of diffuse low signal intensity in the left central gland (arrow). (d) Whole mount pathology section with the 21 x 16 mm index lesion in the left central gland (encircled) corresponding to the MRI findings described above. A few non-index lesions (Gleason 3+3) in right central gland and one non-index lesion in the peripheral zone.

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Pathology An experienced uro-pathologist (A.T.) reviewed both the SB, MRI-TB and if radical prostatectomy, whole mount pathology cross serial section for prima-ry and secondary Gleason grades according to International Society of Uro-logical Pathology (ISUP) 2005 consensus criteria (2). Significant PCa in this study was defined as Gleason score ≥7. When combining SB and MRI-TB, significant cancer detected on either test was regarded as significant cancer.

Statistical analysis Descriptive statistics were used to describe patient characteristics including biopsy results. Cohen’s kappa statistic were used to calculated inter-reader agreement of the 5-point Likert score. A p-value of ≤0.05 was statistically significant. Statistical analysis was performed with Statistica 12®, Statsoft Inc 2300 East 14th St. Tulsa, OK 74104, USA

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Results

Lymph node staging with ePLND as reference standard In all, 969 lymph nodes were harvested in study I, with a mean (range) of 18.2 (9– 40) lymph nodes harvested per patient. According to the anatomical location, 373 (38%), 385 (40%) and 211 (22%) were found along the com-mon and external iliac artery, obturator fossa, and internal iliac artery, re-spectively (Table 3). In all, 26 (49%) of 53 patients were found histological-ly to be lymph node positive one of which was in the intermediate-risk group (17%) and the remaining 25 in the high-risk group (55%). The results pre-sented in detail below pertain to reader one only. For reader one, 10 patients (19%) were true positive, 16 (30%) were false negative and one (2%) was false positive with PET/CT. On an anatomical site level 76 of 318 sites were lymph node positive, 25 of which were identified by PET/CT. In six PET/CT positive sites the histology was negative. All PET/CT scans were considered interpretable.

Table 3. Investigational results.

Value Patients, n (%) 53 (100) LN+ patients, n (%) 26 (49) Acetate-PET/CT true LN+ patients, n (%) 10 (19) Acetate-PET/CT false LN+ patients, n (%) 1 (2) Mean (range) number of LNs taken/patient 18.2 (9–40) Harvested LNs, n (%)

Total 969 (100) External iliac artery 373 (38) Obturator fossa 385 (40) Internal iliac artery 211 (22)

Histologically confirmed LN+, n (%) 116 (12) Acetate-PET/CT true LN+, n (%) 31 (3.1) Acetate-PET/CT false LN+, n (%) 6 (0.6)

There was a statistically significant difference in the number of metastatic lymph nodes (7.9 vs 2.4) and the maximum cancer size (14.1 vs 4.9 mm) between the PET/CT positive and negative cases, respectively. On the other hand, no difference was found between the groups for PSA level, Gleason sum, and the nomogram-calculated risk of lymph node invasion (Table 4) that could affect the performance characteristics of the testing. The largest

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lymph node metastasis that PET/CT could not detect was 12 mm. The study revealed 96% specificity, 38% sensitivity, and 68% accuracy at the individu-al level and 98% specificity, 27% sensitivity, and 81% accuracy at the site level (Table 5).

Table 4. PET/CT detected or missed N+ patients’ characteristics.

Characteristic PET/CT positive PET/CT negative P Number of patients 10 16 Mean (IQR)

LN+ LNs/patient 7.9 (6) 2.4 (3) <0.001 Maximum cancer diameter, mm 14.1 (13.5) 4.9 (3.5) Percentage risk for LN+ estimated by nomogram*

63 (15) 57 (29) 0.474

PSA level, ng/mL 28 (24) 32 (26) 0.786 Curative treatment, n/N (%) 4/10 15/16 (94) 0.005 Mean (SD) Gleason sum 7.2 (0.4) 7.3 (0.7) 0.615 Gleason sum, %

7 80 81 8 20 6 9 0 12

*Briganti et al. nomogram.

Table 5. Sensitivity, specificity, PPV, NPV and accuracy of acetate-PET/CT LN staging on an individual and site level when compared with histological staging.

Positive Negative Total Individual LN-status*

PET/CT+ 10 1 11 PET/CT– 16 26 42

Total 26 27 53 Site LN-status†

PET/CT+ 20 7 27 PET/CT– 55 236 292

Total 75 243 318 *Sensitivity = 38%, specificity = 96%, PPV = 91%, NPV = 62%, and accuracy = 68%. †Sensitivity = 27%, specificity = 98%, PPV = 74%, NPV = 81%, and accuracy = 81%.

A sub-analysis was performed on eight patients with some detectable lymph node tracer uptake on PET/CT but considered unspecific and judged PET/CT-negative. In this group, unspecific tracer uptake in normal-sized and normal-shaped lymph nodes were found at 21 sites. In three of these sites the histology was positive but also in one site with no uptake. The counts fell to: specificity 59%, sensitivity 25%, PPV 14%, and accuracy 33% on site level analysis.

Interobserver analysis was performed at the individual level and for read-er two, 10 of the histologically lymph node positive cases were found posi-tive and the remaining 16 lymph node positive cases were PET/CT negative, identical to reader one. Reader two reported two false positive cases, includ-

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ing the one identified by reader one. For reader two specificity was 93%, sensitivity was 38%, and accuracy was 66% with no statistical difference compared with reader one.

In study II a total 728 lymph nodes were harvested, with a mean number of 18 lymph nodes per patient (range 11-40), 100 (13.7%) of the harvested lymph nodes contained cancer. Twenty (50%) out of 40 patients included in the study had lymph node positive disease. Patient characteristics are listed in table 6.

Table 6. Clinical and pathological characteristics.

Patient characteristics All patients (n=40) Age (years)

Mean 68 ±SD 5.3

Clinical T-stage T1c 1 (2) T2 9 (23) T3 30 (75)

Biopsy Gleason score 6 4 (10) 7 30 (75) 8 4 (10) 9 2 (5)

Risk for lymph node invasiona 21 – 59% 20 ≥ 60% 20

Numbers in parentheses are percentage of total patients (n=4). aCalculated according to Brig-anti-Karakiewicz nomogram.

MRI DWI was true positive in 11 (27.5%) patients, false negative in 9 (22.5%) patients, false positive in 2 (5%) patients and true negative in 18 patients (55%). Sixty-three anatomical regions of 240 (26%) contained can-cer positive LNs, 26 regions (11% of total) were identified by MRI DWI. In 11 (5% of total) MRI DWI positive regions the histology was negative (Ta-ble 7). The study revealed 90% specificity, 55% sensitivity and 72,5% accu-racy for patients and 94% specificity, 41% sensitivity and 80 % accuracy for anatomical regions. Example of true positive and true negative lymph nodes is shown in figure 9. Example showing the minimum lymph node DWI ab-normality regarded as MRI positive is shown in figure 10.

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Table 7. Diagnostic performance of patient based analysis and lymph node anatom-ical region analysis.

Patient based analysis Anatomical region based analysis No. of true positive cases 11 26 No. of true negative cases 18 166 No. of false positive cases 2 11 No. of false negative cases 9 37 Sensitivity (%) 55 41 Specificity (%) 90 94 PPV (%) 85 70 NPV (%) 67 82 PPV, positive predictive value; NPV, negative predictive value.

Figure 9. Images in a patient with high-risk PCa (stage T3) Gleason 3+4=7, PSA 48 ng/ml. (a) T2W image shows two true-positive lymph nodes (arrows) of 6 mm (oval shape) and 3.5 mm (round shape) longest short axis, respectively, in the left obtura-tor region. (b) DWI (b-value 1000 s/mm2) shows the corresponding very hyperin-tense structures (arrows). (c) The ADC map (b-values 0, 100, 200, 400, 500 s/mm2) shows the corresponding very hypointense structures (arrows). True negative lymph nodes (arrowheads).

Figure 10. Images in a patient with high-risk PCa (stage T3) Gleason 4+3=7, PSA 14 ng/ml. Example showing the minimum DWI abnormality, regarded as MRI posi-tive. (a) T2W image shows one true-positive lymph node (arrow) with a 4.5 mm (round shape) longest short axis in the left internal iliac region. (b) DWI (b-value 1000 s/mm2) shows the corresponding hyper intense structure (arrow). (c) The ADC map (b-values 0, 100, 200, 400, 500 s/mm2) shows the corresponding hypo intense structure (arrow).

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The true positive patients had, compared with the false negative group, sig-nificantly more involved lymph nodes, with larger diameter and fewer were treated with curative intent (Table 8). On the other hand no difference was found between the groups regarding PSA, Gleason sum and the preoperative calculated risk for lymph node invasion (Table 4). The largest lymph node metastasis not detected by MRI DWI was 10 mm.

The pelvic bones were assessed in all patients and no bone metastases were detected.

Table 8. Subgroup analysis of lymph node positive (LN+) patient characteristics, MRI positive cases versus MRI negative cases.

Patient characteristics MRI positive MRI negative p-Value Patients, n 11 9 Na LN+ nodes/patient, mean (±SD) 6.9 (4.64) 2.7 (1.41) 0.017b Diameter of the largest involved lymph node, mm, mean (±SD )

12 (8) 5 (3) 0.048c

Risk for LN+ estimated by nomograma %, mean (±SD)

63.6 (16.3) 48.6 (19.6) 0.087c

Curative treatment N (%) 6 (30) 9 (45) 0.030d PSA total ng/ml, mean (±SD) 28.5 (18.4) 38.7 (33.2) 0.676c Gleason score mean (±SD) 7.2 (0.4) 7 (0) 0.290d

7 82% 100% 8 18% 0% 9 0% 0%

aKarakiewicz nomogram. bIndependent t-test. cMann-Whiney U-test. dOne-tailed Fischer’s exact p-test.

Quantitative and qualitative lymph node analysis Of the 53 patients included in study IV, 26 (49%) had lymph node metastases at ePLND (Table 1) Among the 40 patients that had 3T MRI DWI plus 11C Acetate PET/CT 50% had lymph node metastasis (Table 6). The variables MRI-T-stage, LN-ADCmean and tumor-ADCmean had 40 observations, the remaining variables tumor-SUVmax, LN-SUVmax, LN-size and LN-shape had 53 observations. The smallest lymph node in the material was 3.8 mm, the median ROI size for ADC measurements in lymph nodes was 42 mm2, with range 16-334 mm2, the ROI size for ADC measurements in primary tumor was ≥ 80 mm2 (Table 9).

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Table 9. Investigational findings at DWI MRI and 11C Acetate PET/CT

DWI MRI and 11C Acetate PET/CT findings LN-ADCmean 10⁻6 mm² /s, mean (SD) range 917 (191) 582-1398 LN-SUVmax, mean (SD) range 1.8 (1.2) 0.7-5.9 LN size mm, mean (SD) range 6.6 (3.7) 3.8-28.3 Proportion of LNs with round shape, n 19 Proportion of LNs with oval shape, n 34 MRI T-stage, n < T3 25 T3a 14 T3b 14 LN ADC Roi size mm2, median (range) 42 (16-334) Tumor ADC Roi size mm2 ≥ 80 LN: lymph node, MRI T-stage: determined with MRI, only clear cut cases were reported as T3a and T3b, ADC: Apparent diffusion coefficient b0-b500, SUV: Standardized uptake value, ROI: Region of interest.

The ROC analysis of Tumor-ADCmean and Tumor-SUVmax showed insuf-ficient classification with AUC of 0.53 and 0.49 respectively. For LN-SUVmax, LN-ADCmean and LN-size the corresponding AUC were 0.69, 0.72 and 0.62 respectively, these variables were dichotomized using optimal thresholds calculated from the ROC curve and included in simple logistic regression along with MRI-T-stage and LN-shape (Table 10). All variables included in simple logistic regression analysis were significant predictors of LN metastasis and therefore included in multiple logistic regressions models in combination of two (Table 11). Ten combinations were calculated and in model one to three both variables appeared as independent predictors of LN metastasis: LN-ADCmean in combination with LN-SUVmax, LN-shape and MRI-T-stage respectively where the best combinations for prediction of LN metastasis (Table 11). Model three (LN-ADCmean and MRI-T-stage) was the model with highest AUC and pseudo R2, 0.81 and 0.39 respectively, which was higher than the AUC of 0.65 and pseudo R2 of 0.12 for LN-ADCmean alone and the AUC of 0.69 and pseudo R2 of 0.17 for MRI-T-stage alone.

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46

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In model one to three both variables were significant, multicollinearity between the predictors were weak, Cramers´V of 0.09, 0.15 and 0.16 respec-tively. This, in combination with adequate goodness of fit show the validity of the models.

Targeted biopsies In study III, a total of 32 significant PCa cases were detected by SB alone and SB + MRI-TB detected additionally 5 significant cancers (Table 12). MRI-TB alone detected 20 significant cancers but missed 17 significant can-cers. Ten and seven of the significant cancers undetected with MRI-TB, had equivocal findings (Likert of ≤3) and suspicious findings (Likert ≥4) on mpMRI, respectively. Comparison of different Likert scores with biopsy results for MRI-TB and SB is shown in table 13. Table 14 shows Gleason score results of SB and MRI-TB. Inter-reader agreement for Likert score was good with kappa value of 0.77 (95% CI, 0.63-0.92), p<0.0001.

Table 12. Comparison of the detection rate of insignificant and significant prostate cancer, mpMRI targeted biopsies + standard TRUS guided biopsies vs standard TRUS guided biopsies.

MRI-TB + SB SB

Totals Insignificant PCa Significant PCa Insignificant PCa 16 0 16 Significant PCa 5 32 37

Totals 21 32 53 SB trans-rectal ultrasound guided biopsies, MRI-TB: MRI targeted biopsies, PCa prostate cancer.

Table 13. Comparison of different Likert scores with biopsy results for mpMRI targeted biopsies and standard TRUS guided biopsies.

Likert 1/2 Likert 3 Likert 4/5 Totals Significant PCa SB 5 10 17 32 Insignificant PCa SB 6 6 9 21 Significant PCa MRI-TB 1 6 13 20 Insignificant PCa MRI-TB 10 10 13 33 SB trans-rectal ultrasound guided biopsies, MRI-TB: MRI targeted biopsies, PCa prostate cancer.

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Table 14. Comparison of pathology results of standard TRUS guided biopsies and mpMRI targeted biopsies.

MRI-TB Gleason score

SB Gleason 3+3

SB Gleason 3+4

SB Gleason 4+3

SB ≥ Gleason 8 Totals

No Cancer 7 4 1 3 15 3+3 9 7 1 1 18 3+4 3 2 2 1 8 4+3 2 1 2 - 5 ≥ Gleason 8 - 2 2 3 7

Totals 21 16 8 8 53 SB trans rectal ultrasound guided biopsies. MRI-TB: MRI targeted biopsies.

The pathology results from the combination of SB and MRI-TB were com-pared against whole mount pathology in the subgroup of patients that un-derwent radical prostatectomy (n 34). In this subgroup one additional true positive significant cancer was detected by MRI-TB compared to SB alone (Table 15).

Table 15. Comparison of the detection rate of no cancer, insignificant and significant cancer; standard TRUS guided biopsies and the combination of standard TRUS guided biopsies and mpMRI targeted biopsies against whole mount pathology.

Whole Mount Pathology

SB / SB+ MRI-TB No Cancer Insignificant Cancer Significant Cancer Totals

No Cancer 0/0 0/0 0/0 0 Insignificant Cancer 0/0 1/1 4/4 5 Significant Cancer 0/0 6/5 23/24 29

Totals 0 7/6 27/28 34 SB trans rectal ultrasound guided biopsies, MRI-TB: MRI targeted biopsies.

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Discussion

Summary of findings 11C Acetate PET/CT lymph node staging, of patients with predominantly high risk PCa demonstrate a relatively low sensitivity but a high specificity. The diagnostic criteria were rigid, requiring distinct activity in lymph nodes to indicate metastasis, resulting in a high specificity and a separation of ad-vanced node positive cases from less severe cases with a high degree of in-terobserver concordance. The sub analysis of eight cases with unspecific lymph node activity and normal sized and shaped lymph nodes on CT, showed a marked fall of sensitivity, specificity, PPV and accuracy.

MRI DWI lymph node staging of patients with predominantly high risk

prostate cancer, demonstrates a quite low sensitivity while the specificity was high on a per patient level and anatomic region level. When a lymph node was assessed as positive, the anatomical region given was correct in a high degree. Hence, 3 Tesla MRI DWI is specific in terms of localizing and ruling in lymph node metastasis. Moreover, the lymph node positive patients detected with MRI DWI had a high burden of lymph node metastases asso-ciated with poorer prognosis and less benefit from treatment with curative intention (30).

Five more significant PCa cases were detected by adding MRI-TB to

standard TRUS guided biopsies. More than half (58.8%) of the 17 significant PCa cases missed with MRI-TB had mpMRI with Likert score ≤3. The study design was to include all patients with SB biopsy proven PCa followed by MRI-TB independent of the Likert score. This makes our study not really comparable with MRI-TB studies on active surveillance, biopsy naïve males and men with previous negative SB and/or studies of MRI targeted biopsies towards lesions with PIRADS or Likert score of 3 or more.

Quantitative and qualitative analysis of lymph node and primary tumor

findings at MRI DWI and 11C Acetate PET/CT can provide a range of single and combined parameters to help radiologists evaluating the probability of regional lymph node metastases. LN-ADCmean, LN-SUVmax and LN-size were significant predictors of lymph node metastases as were lymph nodes with round shape and stage T3b at MRI, while Tumor-ADCmean and Tu-

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mor-SUVmax had insufficient classification properties. In multiple logistic regression analysis the best combination was LN-ADCmean and MRI-T-stage (model three), this combination increased the predictive value com-pared to that of each parameter alone.

Prostate lymph node metastases evaluated with 11C Acetate PET/CT Study I prospectively evaluated the potential of PET/CT with 11C Acetate for N staging with ePLND as reference standard in a sufficient cohort of patients.

Acetate is the pivotal carbon donor in the intermediary metabolism of the

cell. The retention seen in Acetate PET images of cancers is primarily relat-ed to incorporation of the tracer in anabolic processes (157), and signal am-plitude hence depends on active growth. Studies investigating the potential staging utility of Acetate PET/CT in prostate cancer were recently summa-rized in a systematic review and meta-analysis (158). Most studies concern primary tumors of the prostate or lymph node restaging at PSA-relapse after failure of curative treatments. The diagnostic yield appears to be equivalent to Choline PET/CT (102, 103) but the true accuracy of Acetate PET/CT compared with ePLND remains largely unknown. Recently, Haseebuddin et al. (144) reported a sensitivity of 68% and specificity of 78% on the patient level in a large cohort of 107 patients with intermediate- or high-risk prostate cancer, who underwent PLND, but only a fraction had ePLND. The patients were followed after curative treatment, and treatment failure-free survival was worse in patients with false-positive Acetate PET/CT than in true-negative patients, suggesting that nodal disease was not consistently detected by surgery or pathology. Schumacher et al. (159) compared centralized ePLND and histology to Acetate PET/CT in 19 patients, but only nine of these were primary cancers. In this smaller group, Acetate PET/CT was true positive in three and false negative in one case. There were no false posi-tives, resulting in a sensitivity of 75% and specificity of 100% on the patient level based on only four cases. In a slightly underpowered study, Heck et al. (141) N staged 33 intermediate- and high-risk patients with primary prostate cancer using diffusion weighted MRI and 11C Choline PET/CT. Subsequent ePLND detected 14 lymph node positive patients, eight of which were de-tected with any imaging method tested. They reported sensitivity of 57% and specificity of 79% for DWI, and sensitivity of 57% and specificity of 90% for 11C Choline PET/CT. This may reflect that there is no superior imaging method yet. Other comparable published papers on PET/CT (143, 160) also reveal lower detection rates. Those papers either lack the foundation of a

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solid ePLND with a high number of examined lymph nodes (98) or patients, lack PLND, or provide no information about the number of harvested lymph nodes (160).

The importance of ePLND When evaluating novel imaging techniques attempting nodal staging it is important to use ePLND as gold standard. Most of the nomograms used to estimate the risk of metastasis are based on historical material with PLND limited to the obturator fossa with a total of five to seven lymph nodes from both sides (161, 162). Studies that evaluate therapeutic strategies with or without the presence of lymph node metastases, share the same weakness. Even studies on the sensitivity of imaging techniques may have the same weakness if the confirming surgery is based on a limited number of lymph nodes (143). With ePLND a significantly higher incidence of lymph node spread is found, having a decisive role in the selection of curative strategies. Briganti et al. (153, 163) created a nomogram using open ePLND in order to evaluate the risk of lymph node metastases. This is a validated nomogram (www.nomogram.org) where the risk of having lymph node invasion is cal-culated by specifying the clinical T stage, PSA level, and Gleason sum score obtained by TRUS biopsy. In study I, II and IV, surgery was performed in a single center, were laparoscopic ePLND had been performed since 2007. Prior local experience in approximately 130 cases revealed 43% lymph node positive cases in intermediate- and high-risk patients, which exceeded the Briganti et al. (153) nomogram. The ePLND dissection template covers 75% of the primary lymph node spread sites (41).

Biological border As curative therapies have side effects, the ideal situation would be to have a reliable imaging technique to individualize therapy, on one hand to identify patients with a need to eradicate lymph node spread, and on the other hand to map any spread that has gone too far for curative intent. The number of in-volved lymph nodes is also relevant to a biological border beyond which a curative strategy gives no survival benefit. This cutoff appears to be between two and three involved lymph nodes (30, 33, 164).

In study I and II, the false negative cases had about two to three involved nodes, while the true positive cases had about seven to eight involved nodes (Table 3, Table 4), raising the question if in clinical praxis the true positive cases had such a high burden of lymph node metastases associated with a

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poor prognosis, potentially indicating too large a nodal involvement for a curative strategy (30). Acetate PET/CT and MRI DWI seems to miss most of the cases of lymph node spread where the detection would be of most value in a curative perspective. For these questions, larger and more longitudinal studies are needed.

Prostate lymph node metastases evaluated with MRI DWI There are only a few previous published studies in prostate lymph node me-tastases evaluated with MRI and DWI. A recent study on 3 Tesla published by Thoeny et al investigated normal sized pelvic lymph nodes in prostate (63%) and bladder cancer (37%) patients, with histopathology from ePLND as reference standard (165). Of the 120 included patients, 33 were lymph node positive, sensitivity was 72.7% and specificity was 86.2%. Since 37% of the included patients had bladder cancer it is not possible to do a direct comparison with the results in study II.

Another study by Heck et al (2013) investigated N staging in 33 interme-diate- and high-risk PCa patients scheduled for RP and ePLND, with 1.5 Tesla MRI DWI and 11C Choline PET/CT (141). The majority of patients were at high risk, DWI/ADC evaluation was made using a visual approach and histopathology was the reference standard. The total number of metastat-ic lymph nodes was 92 with reported sensitivity of 57% and specificity of 79% for MRI DWI. The specificity is slightly lower compared to our results in study II, but this may be explained by the fact that Heck et al only used the ADC maps to distinguish between malignant and benign nodes. In study II, we analyzed size, shape, and grouping of nodes along with DWI and ADC maps interpretation. Budiharto et al (2011) undertook N staging in 36 PCa patients with 1.5 Tesla DWI and 11C Choline PET/CT with histopathol-ogy from ePLND as the reference standard (119). Included patients had an increased risk of lymph node metastasis ≥10 but ≤ 35 according to the Partin nomogram and similar patient characteristics, apart from preoperative medi-an PSA: 10.4 ng/ml as compared with 22 in study II. Despite the fact that only patients with no pelvic lymph node involvement on contrast enhanced CT, where included, diagnostic performance of DWI are comparable to our results in study II, with a sensitivity of 42.9 and specificity of 81.8 The slightly better performance in study II is probably due to the inclusion of size and shape of lymph nodes in the analysis.

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USPIO and PSMA Studies of MRI contrast media in the form of ultra-small iron oxide particles, USPIO, on lymph node involvement in PCa have produced promising re-sults. Sensitivity and specificity of lymph node involvement in these studies range between 82–100% and 93–98% (166-168). It is an accurate method for detecting metastases in normal-sized pelvic lymph nodes of patients with localized PCa (169). Unfortunately USPIO in Sweden is only available in research settings.

Recently 68Ga PSMA PET/CT emerged as a promising new tracer binding to the prostate specific membrane antigen (PSMA). PSMA is highly ex-pressed in almost all prostate cancers (170). Promising results with this trac-er has been reported in PCa patients with biochemical recurrence (171). We have found only one publication on N staging at initial diagnosis of PCa: Budäus et al (2015) retrospectively compared imaging findings of 30 pa-tients, examined with 68Ga PSMA PET/CT at 5 different imaging centers, with histology from ePLND as the reference standard (172). All 30 patients had a risk of lymph node invasion >20% according to nomogram (153). Re-sulting sensitivity and specificity was 33.3% and 100%, revealing that 68Ga PSMA PET/CT is limited in detecting lymph node metastases at initial diag-nosis. Further prospective studies of this tracer are warranted.

Apparent diffusion coefficient It is rather a rule than an exception that MRI DWI and ADC maps are in-cluded in clinical oncology imaging protocols (122) and in general a non-quantitative approach is used. The use of ADC measurements in a clinical setting is challenging due to variation of ADC threshold between tumor types and lack of existing consensus on which thresholds to be used. Further the ADC value is largely dependent on the diffusion weighting factors (b values) used in the protocol, variability of the ADC value of up to 40% has been described with the use of different b values (173). To a lesser degree, ADC values can differ between MRI systems (174). This explains why cut-offs for ADC values cited in the literature vary greatly. For example in study IV the lymph node ADCmean cutoff obtained with ROC curve analysis was 800 x 10⁻6 mm2/s, based on the b values 0,100, 200, 400, 500. In another study the cutoff of the ADCmean value was 910 x 10-6 mm2/s based on b values 500, 800, 1000 and 1500 (140). In three studies with the following b values 50, 300, 600, the reported pelvic lymph node ADC mean cutoff were 1430 x 10-6 mm2/s, 1010 10-6 mm2/s and 1300 10-6 mm2/s respectively (118) (120, 121). There is clearly a need for standardization of MRI DWI acquisi-tions to enable comparisons of ADC values between reports (175).

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Up to 80% of regional lymph node metastases in PCa are located in nor-mal sized lymph nodes (168) it is therefore unavoidable to measure ADC in normal sized lymph nodes when evaluating MRI DWI in nodal staging of PCa. This is reflected by the ADC ROI size of pelvic lymph nodes in study IV ranging from 16-334 mm2 with median size of 42 mm2. In a study by Thoeny et al (165) investigating normal sized pelvic lymph nodes in bladder cancer and PCa, the ADC ROI size ranged from 2.8-40.7 mm2 Obviously there is a risk of partial volume effect when measuring ADC in small lymph nodes. However Eiber et al (120) showed that partial volume effects, even in lymph nodes down to 6 mm, do not substantially distort measurements of ADC.

Study II, III and IV were conducted on a 3 Tesla scanner. With the ad-vantages of almost doubled signal to noise ratio (SNR), compared with im-aging at 1.5T increased spatial resolution and decreased acquisition time can be achieved. Imaging at 3T is especially beneficial for diffusion weighted imaging, because DWI is a technique with inherently low SNR. Image dis-tortion from increased artifacts related to increased field strength can con-tribute to a loss of image quality at 3T (176). These artifacts can be reduced by using parallel imaging and parallel transmission technology, both used in study II, III and IV (177).

Predictive factors of regional lymph node metastases To the best or of our knowledge study IV is the first study investigating quantitative and qualitative predictors of regional lymph node metastases from MRI DWI and 11C Acetate PET/CT, with histopathology as reference standard.

A recent retrospective study by Park et al (178) investigated 101 PCa pa-tients with normal sized lymph nodes undergoing ePLND, with MRI DWI at 3T. In simple logistic regression PSA, Gleason score, greatest percentage of biopsy core, percentage of positive cores, ADC of index lesion in prostate gland and MRI T stage were all independent predictors of regional lymph node metastasis. In multiple analysis only MRI T stage was significant. This finding is similar to study IV since MRI-T-stage is a strong predictor in this study. A limitation of the study by Park et al is that only 9 patients had lymph node metastasis, whereas 92 patient did not, this makes the logistic regression model unbalanced.

Another recent study by Batra et al (179) investigated predictive factors for lymph node metastases in 100 patients undergoing ePLND. Variables examined in simple logistic regression analysis were PSA, Gleason score, clinical stage, D’Amico risk category and features of locally advanced dis-ease on MRI (defined as extra prostatic extension, seminal vesicle invasion

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and enlarged pelvic lymph nodes). Clinical stage and features of locally ad-vanced disease were predictive of lymph node metastases. In multiple lo-gistic regression, clinical stage only was predictive of lymph node metasta-ses.

These results are not directly comparable to ours since all but 2 patients in the study by Batra et al had clinically localized disease, whereas the majority of patients in study IV (77.4%) had T3 disease. Further the definition of locally advanced disease included findings of enlarged pelvic lymph nodes, while in study IV MRI-T-stage was defined by T stage on MRI. A disad-vantage of the study by Batra et al is that only 17% of the included patients had N1 disease.

The ROC curve analysis optimal cutoff for lymph node size short axis di-ameter was 7.9 mm in study IV, this is similar to the cutoff of 8 mm for lymph node size that has been reported in two previous studies of pelvic nodes in PCa (118, 120). Regarding optimal cutoff for lymph node SUVmax in 11C Acetate PET/CT, there are no previous publications to compare with.

Interestingly we could show that lymph nodes with round shape were predictive of metastases, which is confirming its position in general interpre-tation criteria of CT and MRI imaging in PCa. Regarding the multiple lo-gistic regression analysis, one can argue that the combination of LN-shape and MRI-T-stage (model eight) had AUC and pseudo R2 close to model three (LN-ADCmean and MRI-T-stage) and even higher accuracy 0.78 vs. 0.71 compared to model three. However only LN-shape in mode eight ap-peared as independent predictor. LN-ADCmean and LN-SUVmax were in-dependent predictors in model one as were LN-ADCmean in combination with LN-shape in model two, however not reaching the results in model three.

It should be noted that all of the predictive factors in study IV except LN-SUVmax can be obtained from non contrast enhanced MRI DWI, this is of relevance since 11C Acetate PET/CT is associated with high cost and limited availability.

MRI-targeted biopsies in PCa The importance of targeted biopsies compared to conventional biopsies is currently the focus of a large number of publications (131, 180-183). In study III, MRI-TB alone detected fewer significant cancers than SB, 20 (38%) compared to 32 (60%), these findings differs diametrically against a recent meta-analysis by Schoots el al (130), were they concluded the oppo-site, targeted biopsies had a higher detection rate of significant prostate can-cer compared with conventional biopsy. A probable reason for this is that Schoots et al only included studies that targeted suspicious MRI lesions.

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Furthermore, Schoots et al included studies with different inclusion criteria; biopsy naïve patients and patients with previous negative biopsies.

A recent study by Siddiqui et al published in JAMA a cohort of 1003 pa-tients, showed that conventional biopsy is clearly inferior compared to MRI targeted biopsy because it overlooks aggressive tumors, targeted biopsy alone increased the detection of Gleason >= 4+3 cancers by 30% compared to conventional biopsy (131). These findings are in contrast to study III as targeted biopsy missed six Gleason ≥ 4+3 cancers compared with conven-tional biopsies, whereas conventional biopsy missed two Gleason 4+3 can-cers compared with the targeted approach (Table 4). Furthermore Siddiqui et al found 17 (1.7%) additional ≥ high volume Gleason 3+4 cancers by adding targeted biopsy to conventional biopsy, however direct comparison with results in study III is hampered by a (unknown) number (n of 86) of low volume Gleason 3+4 cancers not included in the analysis. Moreover, only patients with suspicious lesions on MRI were included and almost half of the patients had a prior negative biopsy and the other half were biopsy naïve or had a prior positive biopsy.

Methods for targeting MRI lesions The two most common methods for targeting MRI lesions are cognitive bi-opsy, used in the study III (184), and software-based registration of MRI and trans-rectal ultrasound - MRI/TRUS fusion biopsy (185). Cognitive biopsies are straightforward and do not depend upon new hardware or training. On the other hand there is a possibility for human error (186) and discrepancy in image orientation between axial MR imaging and trans-rectal ultrasound oblique projection (187). MRI/TRUS fusion biopsy gives the operator a vis-ual feedback of the MRI target, however prone to targeting and fusion inac-curacy and a learning curve. Several studies have compared cognitive biop-sies with MRI/TRUS fusion biopsies regarding detection of clinically signif-icant PCa (188-191) with conflicting results. Delongscamps et al. showed that MRI/TRUS fusion detected significantly more high Gleason score ≥7 cancers than SB, while cognitive biopsies did not. Conversely, Puech et al. found no statistically significant difference between the biopsy methods and neither did Wysock et al. Oberlin et al. found a borderline significant (P=0.07) higher detection of Gleason≥ 7 for MRI/TRUS fusion compared with the cognitive approach.

A recent study by Costa et al. (192) reported superior sensitivity for the detection of PCa with endorectal coil at 3T mpMRI compared to an exami-nation without the endorectal coil. However the endorectal coil leads to de-formity in the prostate contour (193-195) and the anatomic distortion result-ing from it can potentially cause difficulties with MRI targeted biopsies.

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Although our group did not experience any difficulties targeting cognitive biopsies.

Likert and PIRADS The fact that the biopsy results did not correspond well to the Likert score may be attributed to pitfalls that may confound the interpretation of mpMRI (196). For example overlap in appearance between cancer and benign pro-cesses exist. Further the biopsies may have missed the index lesion on mpMRI leading to misclassification of the Gleason score (197).

Cancer detection using imaging has traditionally been interpreted as being either positive or negative sometimes with the addition of a third indetermi-nate score or statement as part of the formal written report in the clinical setting. The five-grade (Likert-type) scoring system has also been adopted (198) (199) (200), the most known being Breast Imaging Reporting and Data System (BI-RADS) with following five assessment categories: BI-RADS 1: Negative; BI-RADS 2: Benign; BI-RADS 3: Probably benign; BI-RADS 4: Suspicious; BI-RADS 5: Highly suggestive of malignancy.

The Prostate Diagnostic Imaging Consensus Meeting (PREDICT) (155) published their panel report from a European consensus meeting aimed at standardizing mpMRI for PCa detection, localization, and characterization in April 2011. In the area of reporting, the consensus was to use a subjective Likert-type five-grade scoring scale to communicate the probability of PCa. Likert score 1: Clinically significant disease is highly unlikely to be present; Likert score 2: Clinically significant disease is unlikely to be present; Likert score 3: The presence of clinically significant cancer is equivocal; Likert score 4: Clinically significant cancer is likely to be present; Likert score 5: Clinically significant cancer is highly likely to be present.

In February 2012, an ESUR consensus meeting proposed the use of the MR Prostate Imaging Reporting and Data System (PI-RADS) (122). In this scoring system every sequence, T2W, DWI, DCE and MRSI, is scored on a five-point scale. The total PI-RADS score is a summation of each of these scores resulting in a score range of 3–15 without MRSI and 4–20 with MRSI. In addition individual lesions is given an overall score. In a retrospec-tive study published in Radiology August 2014, the Likert and PI-RADS score were compared in 215 patients (201). Likert score provided a more accurate evaluation of the likelihood of malignancy of prostate lesions found at MR imaging, compared with the PI-RADS score. Interobserver agreement was more accurate with the Likert score than with the PI-RADS score. A few other studies (202, 203) have also shown only moderate interobserver agreement of the PI-RADS score.

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A revised version of PI-RADS, called PI-RADS 2 was made public early 2015 (204). The summary of the changes in the new version compared to PI-RADS 1 are: Introduction of a dominant sequence; DWI is the most im-portant sequence in the peripheral zone and T2W in the transition zone. DCE has been assigned a smaller role, and the 3-15 score is abandoned in favor of an overall 5-point score identical to the Likert score.

Definition of clinically significant cancer The definition of clinical significant PCa varies across studies evaluating targeted biopsies (131, 188, 189, 205, 206) from Gleason score only to Gleason score in combination with number of positive cores and/or the pro-portion of core/s involved with cancer. According to START consensus cri-teria (205) definitions of clinical significance in MRI targeted biopsy studies, should be limited to histologic definitions only, i.e. not include PSA or MRI findings, and that a new definition for targeted biopsy was required. MRI targeted biopsies tend to oversample areas of suspicion, resulting in longer cancer core lengths (198), compared to standard biopsies, this can lead to a difference in risk stratification between TB and SB if proportion of cores is used in the definition. Furthermore several studies (207-210) have shown that tumor stage and Gleason grade are more important prognostic factors than tumor volume.

In a large review (211) analyzing the concept of significant versus insig-nificant PCa, the authors concluded that the literature fails to highlight a single predictive model as gold standard. In light of above findings our group, like many others, decided to use only Gleason score in the definition of significant and insignificant PCa in study III.

Limitations A limitation of studies I and II, is the fact that there is not a 100% match between the imaging regions (external, obturatoral and internal) and the sur-gical regions. For example lymph nodes close to the bifurcation of the com-mon iliac vessel can be located in any of the regions of an extended pelvic lymph node dissection. This is a source of error impacting sensitivity and specificity of the two imaging methods´ ability to localize lymph nodes. The majority of patients where at high risk, only 4 out of 40 patients where at intermediate risk, which is important when interpreting the results. Another weakness is that no interobserver agreement was performed in study II.

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Surgeons were deliberately not masked to the results of the MRI DWI and 11C Acetate PET/CT in order to gain an appreciation of the clinical utility of the technology.

A limitation of study IV is that the number of observations did not allow for more than two variables in multiple logistic regression analysis, which prevented us from exploring the true diagnostic performance of a large mod-el with all predictors included.

Another limitation in study IV is that the ADC measurements in lymph nodes smaller than 6 mm could be hampered by partial volume effect. Limitations in study III; targeted biopsies taken after standard biopsies may result in non-uniform prostate swelling and biopsy bleeding which could hamper the accuracy of targeted biopsies. Cognitive biopsies may be subject to targeting error and potentially have inferior detection of significant cancer compared to MRI/TRUS fusion biopsy, as discussed above.

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Conclusions

11C Acetate PET/CT has a high specificity but low over all sensitivity in detecting lymph node metastasis in primary, predominantly high-risk pros-tate cancer. The PET/CT true-positive patients had a considerably higher tumor burden of lymph node metastases compared to false-negative patients. (Paper I) MRI DWI lymph node staging of primary, predominantly high-risk prostate cancer, exhibited a high specificity but a low sensitivity. The MRI DWI true-positive patients had a considerably higher tumor burden of lymph node metastases compared to false-negative patients. (Paper II) Detection rate of clinically significant prostate cancer increased by adding MRI targeted biopsy to standard trans-rectal ultrasound guided biopsy, in this study where majority of included men were scheduled for curative ther-apy. The biopsy results did not correspond well to the Likert score. (Paper III) Several quantitative and qualitative lymph node and primary tumor parame-ters from DWI MRI and 11C Acetate PET/CT are predictive of regional lymph node metastasis in intermediate- and high-risk prostate cancer. By combining lymph node ADCmean and MRI T-stage the predictive value was increased compared to that of each parameter alone. (Paper IV)

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Acknowledgements

I wish to express my sincere appreciation to everyone who in any way have contributed to make this thesis possible.

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1248

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A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

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