Role of Elevated PHIP Copy Number as a Prognostic and … · 1 Role of Elevated PHIP Copy Number as...

1

Role of Elevated PHIP Copy Number as a Prognostic and Progression Marker for

Cutaneous Melanoma

Vladimir Bezrookove 1, Mehdi Nosrati 1, James R. Miller III 1, David De Semir 1, Altaf

A. Dar 1, Elham Vosoughi 1, Edith Vaquero 1, Antje Sucker 3, Alexander J. Lazar 2,

Jeffrey E. Gershenwald 2, Michael A. Davies 2, Dirk Schadendorf 3, Mohammed

Kashani-Sabet 1*

1 Center for Melanoma Research and Treatment, California Pacific Medical Center

(CPMC) Research Institute, San Francisco, CA; 2 Departments of Pathology, Genomic

Medicine, Dermatology & Translational Molecular Pathology, The University of Texas

MD Anderson Cancer Center, Houston, TX; Department of Dermatology, 3 University of

Duisburg-Essen, Essen, Germany, and German Cancer Consortium, Heidelberg,

Germany.

Running title: PHIP copy number in melanoma progression

* Corresponding Author: Mohammed Kashani-Sabet, M.D., California Pacific Medical Center Research Institute, 475 Brannan Street, Suite 130, San Francisco, CA, 94107. Phone: (415) 600-3166, Fax: (415) 600-1719; Email: [email protected] Conflicts of interest: J.R.M. has ownership interests (including patents) at MDMS LLC. J.E.G. has served on advisory boards for Merck, Syndax, and Castle Biosciences. M.A.D. has served on advisory boards for Bristol-Myers Squibb, Roche/Genentech, Novartis, Sanofi-Aventis, Syndax, and Vaccinex, as a consultant for Nanostring (without compensation), and has received grant support from Bristol-Myers Squibb, Roche/Genentech, GSK, Sanofi-Aventis, Oncothyreon, Merck, and Astra Zeneca. D.S. has served on advisory boards for and received honoraria from Bristol-Myers Squibb, Roche, Merck, Incyte, Amgen, Novartis, and EMD. M.K.S. has ownership interest in Melanoma Diagnostics, has received honoraria from Cepheid, and has received grant support from Merck. All other authors declare no conflicts.

Research. on February 11, 2020. © 2018 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 18, 2018; DOI: 10.1158/1078-0432.CCR-18-0791

http://clincancerres.aacrjournals.org/

2

Translational Relevance This manuscript presents a comprehensive analysis of the role of PHIP copy number in

sequential stages of melanoma progression. We confirm that elevated PHIP copy number

is an independent prognostic marker for primary cutaneous melanoma. In addition, we

identify distinct molecular subtypes of melanoma in which PHIP is enriched. Finally, we

show that the percentage of melanoma cells with elevated PHIP copy number is

increased in the progression from primary to metastatic melanoma. Taken together, these

studies describe a key role for PHIP in melanoma progression, confirming it as a

promising target for therapy.




3

Abstract

Purpose: Previous studies have indicated an important role for pleckstrin homology

domain-interacting protein (PHIP) as a marker and mediator of melanoma metastasis.

Here we aimed to confirm the role of PHIP copy number in successive stages of

melanoma progression.

Experimental Design: PHIP copy number was examined using fluorescence in situ

hybridization (FISH) in three independent cohorts by recording the percentage of cells

harboring > 3 copies of PHIP. The impact of PHIP copy number on survival was

assessed using Cox regression analysis. The enrichment of PHIP was assessed in various

molecular melanoma subtypes. PHIP expression was analyzed in The Cancer Genome

Atlas (TCGA) melanoma cohort.

Results: Elevated PHIP copy number was significantly predictive of reduced distant

metastasis-free survival (DMFS) and disease-specific survival (DSS), and increased

prevalence of ulceration in primary melanoma (cohort #1). By multivariate analysis,

PHIP FISH scores were independently predictive of DMFS and DSS. PHIP copy number

was enriched in metastatic melanomas harboring mutant NRAS or expressing PTEN

protein (cohort #2). PHIP copy number was significantly elevated in metastatic

melanomas when compared with matched primary tumors from the same patient (cohort

#3). Several of these associations were replicated using TCGA cohort analysis.

Conclusions: These results underscore the important role of PHIP copy number

elevation in melanoma progression, and identify molecular subtypes of melanoma in

which PHIP is enriched. Finally, as elevated PHIP copy number appears to be selected




4

for during the progression of primary to metastatic melanoma, these results confirm PHIP

as a promising therapeutic target for melanoma.




5

Introduction

Melanoma represents the fifth most common malignancy in the United States (1).

As the clinical behavior of melanoma is unpredictable, the development of prognostic

markers has represented an important area of research. Histological prognostic factors are

the mainstay of prognostic assessment for primary cutaneous melanoma (2). While many

factors may play a role in refining melanoma prognosis, tumor thickness and ulceration

are the only factors currently incorporated within the melanoma staging classification for

primary melanoma (3).

Molecular factors represent the next frontier in prognostic factor research. The

development of molecular prognostic factors for melanoma has been hampered largely by

the lack of sufficient validation of promising markers in independent tissue sets. In

addition, the role of a putative marker in melanoma progression has rarely been explored

in the context of the same patient. Finally, given the identification of dominant

mutational drivers of melanoma, information regarding the role of a given marker in the

context of the known mutational landscape of melanoma would be important (4). As a

result, no molecular markers are routinely utilized in the prognostic assessment of

melanoma patients, and none are incorporated into its staging classification.

Our previous studies identified both a functional and a biomarker role for PHIP in

melanoma progression, and as a molecular prognostic factor for melanoma. PHIP was

identified as the top gene overexpressed in melanoma metastases when compared with

primary tumors using cDNA microarray analysis (5). Subsequent studies showed an

independent prognostic role for PHIP in primary melanoma (6,7). Functional studies

demonstrated that increasing PHIP expression enhanced the distant metastatic potential




6

of melanoma (6,7). Here, we aimed to confirm the prognostic role of PHIP in primary

melanoma, to identify the molecular subtypes of melanoma in which PHIP is enriched,

and to determine whether PHIP is selected for during the progression from primary to

metastatic melanoma.




7

Materials and Methods

Study population

We developed three non-overlapping patient cohorts to assess PHIP’s biomarker role: (i)

cohort #1- unstained primary melanoma tissue sections obtained from 204 stage I/II

patients in the German Cancer Consortium (see Table 1 for characteristics); (ii) cohort

#2- tissue microarrays (TMA) from a previously described cohort of 130 stage III

melanoma patients in the MD Anderson Cancer Center (8); and (iii) cohort #3- 37

matched primary and metastatic melanomas obtained from the same patient (N=15) who

developed both regional lymph node and distant metastasis, identified from the CPMC

Center for Melanoma Research and Treatment archives (see Supplemental Table 1 for

characteristics). In cohort #3, 21 samples were from 7 patients with triple-matched

primary melanoma, regional lymph node and distant metastasis, and 16 samples were

from 8 patients where the primary melanoma was matched either with a regional lymph

node or distant metastasis. The mean follow up of cohort #1 was 4.58 years. Information

regarding mitotic rate was not available in cohort #1. These analyses were complemented

by analysis of PHIP expression levels obtained from TCGA (4), available from 66

primary tumors and 264 metastases. The investigations described here were conducted in

accordance with recognized ethical guidelines (i.e., Declaration of Helsinki), were

approved by the Sutter Health Institutional Review Board, and informed consent was

obtained from each subject.

FISH analysis

FISH for PHIP copy number was performed as previously described (6,7) using bacterial

artificial chromosome (BAC) clones RP11-767O1and CTD-2297E14 to detect the PHIP




8

locus and clones RP11-26M18 and RP11-136K2 to detect 6q11.1 and 6p11.1,

respectively (interpreted as chromosome 6 centromere). FISH for PTEN copy number

was performed using BAC clones RP11-79A15 and RP11-813O3. For a chromosome 10-

specific probe, BAC clone RP11-96F8 was used to detect 10p11.1 (all BAC clones were

obtained from the Children's Hospital Oakland Research Institute). The quality and

mapping of all probes were verified by hybridization to normal metaphase spreads in

combination with a commercially available centromeric probe for chromosomes 6 and 10

(Open Biosystems, Lafayette, CO) before tissue analysis. Z-stacked images were

acquired using a Zeiss Axio Image Z2 microscope controlled by AxioVision software

(Zeiss, Jena, Germany). At least 30 nuclei from each case were evaluated, and the signals

were interpreted according to guidelines described previously (9). Signals from BAC

clones detecting 6q11.1, 6p11.1 and 10p11.1 were interpreted as the centromeric signal

for chromosome 6 and 10, respectively.

Statistical Analysis

The impact of elevated PHIP copy number on melanoma progression was analyzed in

several ways. In cohort #1, consistent with our previous analysis (6), elevated PHIP copy

number was measured by recording the percentage of cells harboring 3 or more copies of

the PHIP locus. The cut-point utilized to define high PHIP copy number was that which

maximized the average of sensitivity and specificity for predicting DSS (determined to be

greater than or equal to 19%). The same cut-point was uniformly applied to analyses of

DMFS and ulceration status. Subsequently, a previously identified cut-point for PHIP

copy number (greater than or equal to 16%) (6) was applied to the analysis of DMFS,

DSS, and ulceration in this cohort. The association between elevated PHIP copy number




9

and ulceration status was determined using logistic regression analysis. The association

between elevated PHIP copy number and survival outcomes (i.e., DMFS and DSS) was

determined using Kaplan-Meier analysis, and univariate and multivariate Cox regression

analyses. In cohort #2, the enrichment of PHIP in various molecular subtypes of

melanoma was assessed using the T test or the one-way analysis of variance (ANOVA).

In cohort #3, for the analysis of 7 triple-matched tissue specimens, the significance of

monotonically increasing PHIP copy number in melanoma progression was assessed

using the Friedman two-way analysis of variance by ranks test. The significance of

increasing PHIP copy number between primary and metastatic melanoma in the 8

double-matched specimens was assessed using the binomial sign test. As a control,

identical analyses were performed for PTEN copy number in the same 15-patient

matched cohort. Except for the directional analyses just described, all P values reported

are two-sided.




10

Results

In this study, we aimed to perform a comprehensive analysis of the role of PHIP

levels in distinct, sequential stages of melanoma progression, and to confirm its role as a

prognostic marker for primary melanoma. Initially, we aimed to replicate the prognostic

role of elevated PHIP copy number (assessed as percentage of cells harboring 3 or more

copies of PHIP) in primary melanoma in cohort #1. We assessed the potential association

between PHIP copy number and survival using univariate Cox regression. Increasing

PHIP FISH scores (as a continuous variable, using the entire scale) were significantly

predictive of distant metastasis-free survival (DMFS, P<0.03) and disease-specific

survival (DSS, P=0.025). By Kaplan-Meier analysis, high PHIP scores (assessed as a

dichotomous variable) were also significantly predictive of DMFS (P=0.0017, Fig. 2A)

and DSS (P=0.0002, Fig. 2B). In addition, we assessed the association between PHIP

FISH scores and both tumor thickness and ulceration status (6). Increasing PHIP copy

number (using the entire scale) was associated separately with increased tumor thickness

(P=0.0001) and with a higher prevalence of ulceration (P<0.0001) by univariate logistic

regression analysis. High PHIP FISH scores (assessed as a dichotomous variable) were

significantly associated with increased prevalence of ulceration (P<0.00005).

We then assessed the independent impact of elevated PHIP copy number on

survival associated with melanoma using multivariate stepwise Cox regression analysis

with backward elimination. We included in the model the following covariates included

in the legacy prognostic analyses performed by the AJCC melanoma committee (10):

tumor thickness (assessed by T category), ulceration status, Clark level, patient age,

patient gender, and tumor site. In the analysis of DMFS, elevated PHIP copy number (as




11

a dichotomous variable) emerged as significantly predictive of survival (P=0.025), along

with tumor thickness and gender (Table 2). In the analysis of DSS, PHIP FISH scores

were also significantly predictive of survival (P=0.01), along with tumor thickness (Table

2). Thus, elevated PHIP copy number was independently predictive of both DMFS and

DSS, replicating our original analysis (6).

In addition, given that the cut-point identified in this analysis (19%) was very

close to that identified in our original analysis (16%) (6), we examined whether use of the

previously-identified cut-point would be predictive of outcome in cohort #1. High PHIP

scores using the original cut-point were also significantly predictive of DMFS (P=0.03),

DSS (P=0.0069), and ulceration status (P<0.00005) by univariate analysis.

Next, we assessed the role of PHIP in melanoma nodal metastases in cohort #2.

We initially assessed the potential prognostic role of elevated PHIP copy number in this

cohort. There was no significant association between PHIP copy number (assessed as a

continuous variable) in metastatic tumors and survival in stage III melanoma. The dataset

from this cohort contained information regarding three important molecular markers in

melanoma: BRAF and NRAS mutation status, and PTEN expression level (8). Our

analysis identified a significant association between elevated PHIP copy number and

NRAS mutation status in melanoma. The mean percentage of melanoma cells harboring >

3 copies of PHIP was 45.7% in NRAS-mutant melanomas, compared with 19.3% in

BRAF-mutant melanomas, and 15.4% in BRAF/NRAS-wild-type melanomas to

(P<0.00005, ANOVA, Fig. 3A). Separately, our analysis indicated that PHIP copy

number was significantly higher in melanomas with intact PTEN protein expression

versus those with PTEN loss. Specifically, the mean percentage of melanoma cells




12

harboring > 3 copies of PHIP increased from 13.5% in melanomas with PTEN loss to

22.6% in PTEN-expressing melanomas (P=0.004, T test, Fig. 3B). In addition, in BRAF-

mutant melanomas, there was a trend toward increased PHIP copy number in PTEN-

expressing melanomas vs. those with PTEN loss (P=0.086, T test, Fig. 3C). Finally, cases

with a mean PHIP copy number of > 3 were present in each molecular subtype of

melanoma investigated, including in wild-type BRAF/wild-type NRAS/PTEN-expressing

melanomas. Taken together, these results indicate that PHIP levels are enriched in NRAS-

mutant and PTEN-expressing melanoma, and that PHIP copy number elevations are

observed in both BRAF-mutant and BRAF wild-type/ NRAS wild-type/PTEN-expressing

melanomas.

Subsequently, we assessed the role of PHIP as a progression marker in

melanoma. We performed FISH analysis of PHIP copy number on a matched cohort of

15 patients from whom the primary tumor and at least one metastatic tumor were

available (cohort #3, Supplemental Table 1). Among the 7 triple-matched patients (in

whom the primary tumor, a lymph node metastasis, and a distant metastasis were

available for analysis), PHIP copy number increased monotonically in the transition from

primary to lymph node to distant metastasis in 4 patients (Fig. 4). In two patients, the

lymph node metastasis had a lower copy number than the primary tumor, but the distant

metastasis had a higher copy number than both the primary tumor and the lymph node

metastasis. In one patient, the lymph node metastasis had a slightly higher copy number

than the distant metastasis, but both the lymph node and distant metastasis had higher

copy numbers than the primary tumor. There was a significant increase in PHIP copy

number in the transition from primary tumor to lymph node to distant metastasis




13

(P=0.008, Friedman test). The mean percentage of cells with elevated PHIP copy number

increased monotonically from 13.5% in the primary tumor to 21.6% in the lymph node

metastases, and to 55.5% in the distant metastases. In the matched-pair analysis, for each

of the 8 cases, PHIP copy number was higher in the metastatic lesion when compared to

the corresponding primary tumor (P=0.0039, binomial sign test, Fig. 4). To determine

whether this systematic change was unique to PHIP, we performed an identical analysis

of PTEN copy number on the same matched-specimen cohort. There was no significant

change in PTEN copy number in the transition from primary tumors to either lymph node

or distant metastases or both (Supplemental Fig. 1). By comparison, the mean percentage

of cells with elevated PTEN copy number was 9.1% in the primary tumor, 12.7% in the

lymph node metastases, and 11.8% in the distant metastases (P=0.19, Friedman test

applied to the same 7 matched triplets).

Finally, we examined several of these associations by analyzing PHIP transcript

levels in the TCGA cutaneous melanoma cohort (4). Increasing PHIP expression

(assessed as a continuous variable) in the primary tumor was associated with significantly

reduced relapse-free (RFS, P=0.02) and overall survival (OS, P=0.018) by univariate Cox

regression analysis. By multivariate analysis, PHIP expression levels remained

significantly predictive of RFS (P<0.05) and OS (P=0.015), even with the inclusion of

AJCC stage. Similar to the FISH analysis, there was no significant association between

increasing PHIP expression levels in metastatic tumors and survival. In addition, mean

PHIP expression levels were significantly (4.3-fold) higher in NRAS-mutant melanomas

when compared with NRAS-wild-type cases (P=0.02, Mann-Whitney test; Supplemental

Fig. 2A). Finally, mean PHIP transcript levels were significantly (3.7-fold) higher in




14

melanoma metastases when compared to primary tumors (P<0.00005; Supplemental Fig.

2B).




15

Discussion

We have performed a rigorous analysis to elucidate the role of PHIP copy

number elevation in sequential stages of melanoma progression, and as a prognostic

marker for primary melanoma. We analyzed the role of PHIP copy number in three

independent patient cohorts from different centers. In cohort #1, we show that elevated

PHIP copy number is an independent prognostic marker for primary cutaneous

melanoma in a cohort of patients derived from a distinct population in Germany, and

confirm a significant association with ulceration status, an important prognostic factor

incorporated into the AJCC melanoma staging system. In cohort #2, we identify distinct

molecular subtypes of melanoma in which PHIP is enriched. In cohort #3, we determine

that the degree of PHIP copy number elevation is uniformly increased in the progression

from primary to metastatic melanoma. Several of the key findings from these three

cohorts were replicated using TCGA cohort analysis.

The independent prognostic role of PHIP levels in primary cutaneous melanoma

has now been shown using FISH analysis in two distinct cohorts, replicated at the protein

level using immunohistochemical analysis (7), and replicated at the RNA level using

TCGA cohort analysis. In addition, our prior studies demonstrated a significant

correlation between PHIP copy number and expression in primary melanoma (6). There

are few (if any) molecular markers whose prognostic impact has been demonstrated both

in distinct cohorts of melanoma patients and using several distinct platforms. These

studies advance PHIP as an important molecular marker of melanoma outcome, though

additional studies in larger cohorts will be required in order for it be routinely used as a

prognostic factor.




16

Beyond its independent prognostic role, our studies of cohort #1 confirmed a

powerful association between PHIP copy number and ulceration. While the prognostic

significance of ulceration is well appreciated, the biologic basis for ulceration

development in melanoma has been poorly understood. Our previous studies showed that

PHIP mediates some of its effects on melanoma metastasis by virtue of activation of the

IGF1R-PI3K pathway. PHIP is a potent activator of AKT (7), which drives both glucose

metabolism and angiogenesis (11,12). Our studies in melanoma cell lines showed that

PHIP gene silencing results in decreased glycolysis (including reduced LDH expression)

in vitro and decreased angiogenesis in vivo (6). These results are consistent with a model

in which PHIP activation, in part through elevated copy number, promotes glycolysis and

angiogenesis, resulting in increased risk of development of ulceration and distant

metastasis. Intriguingly, this model suggests that a common signaling pathway may

underlie the development of and explain the biological basis of ulceration, a key

prognostic marker in stage I-III melanoma, and LDH levels, a prognostic marker for

stage IV disease.

In addition, our analyses of cohort #2 identified the molecular subtypes of

melanoma in which PHIP copy number elevations are present, and in which they are

specifically enriched. Elevated PHIP copy number was present in each molecular subtype

of melanoma examined, including melanomas with BRAF mutation, NRAS mutation, as

well as in melanomas without any alterations in these markers. Moreover, we found that

PHIP copy number was significantly higher in melanomas with NRAS mutation or with

intact PTEN expression. The observation that PHIP participates in the IGF1R-PI3K

signal transduction pathway in which PTEN also operates provides a rational basis for




17

these results, suggesting that either molecular alteration (PHIP copy number gain or

PTEN loss) may be sufficient to activate this pathway in melanoma. Given the

importance of PTEN loss specifically in BRAF-mutant melanomas (4), our analysis also

identified a trend toward higher PHIP copy number in PTEN-expressing BRAF-mutant

tumors, introducing PHIP as a novel potential driver of this molecular subtype of

melanoma. An intriguing and unexpected finding was the enrichment of PHIP copy

number in NRAS-mutant melanoma. Given that NRAS mutations are thought to activate

both the MAPK and PI3K pathways, our results suggest the intriguing possibility of

novel biochemical functions of PHIP beyond its participation in the IGF1R-PI3K axis.

However, our results are consistent with the observation that PTEN loss is essentially

mutually exclusive with NRAS mutation. In addition, they suggest PHIP targeting as a

potential strategy for the treatment of NRAS-mutant melanoma, which has represented an

important therapeutic challenge given the modest activity of MEK inhibitors (13). Recent

studies describing the development of a small molecular inhibitor of PHIP (14)

demonstrate its druggability, thereby highlighting its role as a potential therapeutic target.

Our studies of cohort #3 analyzed the role of PHIP in the progression cascade of

melanoma by comparing copy number changes in matched primary versus metastatic

tumors from the same patient. Remarkably, the matched-specimen analysis revealed

elevated PHIP copy number in at least one metastatic lesion in every case analyzed when

compared with its corresponding primary tumor. In many instances, the copy number in

the metastasis was extremely high, with three or more copies of PHIP observed in greater

than 50% of cells in 40% of the metastases, including 56% of distant metastases

examined. In addition, in the majority of cases where we had access to the primary tumor,




18

and both lymph node and distant metastases, there was a monotonic increase in PHIP

copy number elevation in the transition from primary melanoma to lymph node

metastasis, and then to distant metastasis. These results strongly suggest that elevated

PHIP copy number is selected for in melanoma progression, supporting our original

cDNA microarray analysis in which PHIP was identified, and which was further

supported by TCGA cohort analysis, showing significantly increased PHIP expression in

metastatic versus (unmatched) primary melanomas. We are unaware of any similar

studies analyzing the role of a given molecular marker in successive stages of melanoma

progression using tissues from the same patient. Similarly, a recent analysis of colon

cancer assessed the somatic variants in hypermutable DNA regions in a cohort of 19

patients with primary colon cancer and matched lymph node and distant metastasis,

enabling a conclusion that two-thirds of distant metastases have a profile distinct from

that of the lymph node metastasis (15). Our analysis underscores the potency of these

matched-tissue comparisons to determine the role of a given gene in cancer progression

within individual patients.

In conclusion, our studies to date have shown that increased PHIP levels are

predictive of melanoma distant metastasis, promote distant metastasis in mouse models of

melanoma, and are enriched in melanoma metastases. The presence of elevated PHIP

copy number in distant melanoma metastases as shown in this study indicates that this

molecular target is present and enriched in the setting of advanced melanoma. Taken

together with functional studies demonstrating an important role for PHIP in promoting

melanoma progression, these studies describe an unprecedented role for PHIP in

melanoma progression, suggesting it as a promising target for therapy.




19

Acknowledgments

This work by supported by a grant from the National Institutes of Health

(R01CA175768) to MKS. The collection of the tissue set at MD Anderson was supported

by the following: The Melanoma Specialized Program of Research Excellence of the

University of Texas MD Anderson Cancer Center (P50 CA093459); and philanthropic

contributions from the Dr. Miriam and Sheldon G. Adelson Medical Research

Foundation, the AIM at Melanoma Foundation, and the MD Anderson Melanoma Moon

Shot Program.




20

References

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin 2018;68(1):7-30 doi 10.3322/caac.21442.

2. Zettersten E, Shaikh L, Ramirez R, Kashani-Sabet M. Prognostic factors in primary cutaneous melanoma. Surg Clin North Am 2003;83(1):61-75.

3. Gershenwald J, Scolyer R, Hess K, Thompson J, Long G, Ross M. Melanoma of the skin. AJCC Cancer Staging Manual2017.

4. Cancer Genome Atlas N. Genomic Classification of Cutaneous Melanoma. Cell 2015;161(7):1681-96 doi 10.1016/j.cell.2015.05.044.

5. Haqq C, Nosrati M, Sudilovsky D, Crothers J, Khodabakhsh D, Pulliam BL, et al. The gene expression signatures of melanoma progression. Proc Natl Acad Sci U S A 2005;102(17):6092-7 doi 10.1073/pnas.0501564102.

6. Bezrookove V, De Semir D, Nosrati M, Tong S, Wu C, Thummala S, et al. Prognostic impact of PHIP copy number in melanoma: linkage to ulceration. J Invest Dermatol 2014;134(3):783-90 doi 10.1038/jid.2013.369.

7. De Semir D, Nosrati M, Bezrookove V, Dar AA, Federman S, Bienvenu G, et al. Pleckstrin homology domain-interacting protein (PHIP) as a marker and mediator of melanoma metastasis. Proc Natl Acad Sci U S A 2012;109(18):7067-72 doi 10.1073/pnas.1119949109.

8. Bucheit AD, Chen G, Siroy A, Tetzlaff M, Broaddus R, Milton D, et al. Complete loss of PTEN protein expression correlates with shorter time to brain metastasis and survival in stage IIIB/C melanoma patients with BRAFV600 mutations. Clin Cancer Res 2014;20(21):5527-36 doi 10.1158/1078-0432.CCR-14-1027.

9. Bayani J, Squire J. Multi-color FISH techniques. Curr Protoc Cell Biol 2004;Chapter 22:Unit 22 5 doi 10.1002/0471143030.cb2205s24.

10. Balch CM, Soong SJ, Gershenwald JE, Thompson JF, Reintgen DS, Cascinelli N, et al. Prognostic factors analysis of 17,600 melanoma patients: validation of the American Joint Committee on Cancer melanoma staging system. J Clin Oncol 2001;19(16):3622-34 doi 10.1200/JCO.2001.19.16.3622.

11. Arsham AM, Plas DR, Thompson CB, Simon MC. Akt and hypoxia-inducible factor-1 independently enhance tumor growth and angiogenesis. Cancer Res 2004;64(10):3500-7 doi 10.1158/0008-5472.CAN-03-2239.

12. Elstrom RL, Bauer DE, Buzzai M, Karnauskas R, Harris MH, Plas DR, et al. Akt stimulates aerobic glycolysis in cancer cells. Cancer Res 2004;64(11):3892-9 doi 10.1158/0008-5472.CAN-03-2904.

13. Dummer R, Schadendorf D, Ascierto PA, Arance A, Dutriaux C, Di Giacomo AM, et al. Binimetinib versus dacarbazine in patients with advanced NRAS-mutant melanoma (NEMO): a multicentre, open-label, randomised, phase 3 trial. Lancet Oncol 2017;18(4):435-45 doi 10.1016/S1470-2045(17)30180-8.

14. Cox OB, Krojer T, Collins P, Monteiro O, Talon R, Bradley A, et al. A poised fragment library enables rapid synthetic expansion yielding the first reported inhibitors of PHIP(2), an atypical bromodomain. Chemical Science 2016;7(3):2322-30 doi 10.1039/C5SC03115J.




21

15. Naxerova K, Reiter JG, Brachtel E, Lennerz JK, van de Wetering M, Rowan A, et al. Origins of lymphatic and distant metastases in human colorectal cancer. Science 2017;357(6346):55-60 doi 10.1126/science.aai8515.




22

Figure Legends

Fig. 1. PHIP copy number analysis by FISH. Representative images of melanoma

samples with normal (panel A) versus elevated (panel B) PHIP copy number as detected

using probes for PHIP (red) and centromere of chromosome 6 (green). Scale bars, 20 μm.

Fig. 2. Kaplan-Meier analysis of the prognostic significance of PHIP copy number in

cohort #1. Analysis of DMFS (panel A) and DSS (panel B) in patients with low PHIP

copy number (curve 1) versus patients with high PHIP copy number (curve 2).

Fig. 3. Comparison of PHIP copy number in different molecular subgroups of melanoma

in cohort #2. (A) Box plots showing PHIP copy number in melanomas with wild-type

BRAF/NRAS vs. mutant BRAF or mutant NRAS. (B) Box plots showing PHIP copy

number in melanomas expressing PTEN protein versus melanomas with PTEN loss. (C)

Box plots showing PHIP copy number in BRAF-mutant melanomas expressing PTEN

protein versus melanomas with PTEN loss.

Fig. 4. PHIP copy number in a matched cohort of 15 patients, including the primary and

at least one metastatic tumor (cohort #3). Bar graphs present the percentage of cells with

at least 3 copies of PHIP.




23

Table 1. Characteristics of cohort #1 (N=204) Male gender 111 (54.4%)

Age over 50 (years) 155 (76.0%)

Ulceration present 53 (26.0%)

T category

T1 56 (27.5%)

T2 51 (25.0%)

T3 59 (28.9%)

T4 30 (14.7%)

Unknown 8 (3.9%)

Median thickness 1.85 mm

Clark level

II/III 84 (41.2%)

IV/V 100 (49.0%)

Unknown 20 (9.8%)

Tumor site

Trunk 100 (49.0%)

Extremity 74 (36.3%)

Head and neck 24 (11.8%)

Unknown 6 (2.9%)




24

Table 2. Results of stepwise multivariate Cox regression analysis of the impact of various

factors on DMFS and DSS

DMFS

Covariate Risk ratio P value

Tumor thickness 2.88 < 0.0001

Gender 3.19 0.0045

PHIP copy number 1.51 0.025

DSS

Covariate Risk ratio P value

Tumor thickness 2.61 < 0.0001

PHIP copy number 1.68 0.01




A

B

Figure 1




A

B

Group 1Group 2

0.5 1 2.5 510665

10162

8545

4420

Number at risk by group

0 5 10 15 20

21

50

100

Surv

ival

Fra

ctio

n (%

)

0

Group 1Group 2

0.5 1 2 410259

9851

8744

6321

Elapsed Time (Years) Number at risk by group 8

278

0 5 10 15 20

21

50

100

Surv

ival

Fra

ctio

n (%

)

0p = 0.0017

p = 0.0002

Figure 2

Elapsed Time (Years)

Years

Years




Figure 3

A

5

10

15

0

55

20

25

30

35

40

45

50

60

65

PTEN lossPTEN present

B

% c

ells

with

≥3

copi

es

WT

5

10

15

0

55

20

25

30

35

40

45

50

65

60

% c

ells

with

≥3

copi

es

BRAF MutantNRAS Mutant

C

5

10

15

0

55

20

25

30

35

40

45

50

60

65

PTEN lossPTEN present

% c

ells

with

≥3

copi

es




0102030405060708090

100

1 a

1 b

1 c

2 a

2 b

2 c

3 a

3 b

3 c

4 a

4 b

4 c

5 a

5 b

5 c

6 a

6 b

6 c

7 a

7 b

7 c

8 a

8 b

9 a

9 b

10 a

10 b

11 a

11 b

12 a

12 b

13 a

13 b

14 a

14 b

15 a

15 b

Figure 4

Primary tumorRegional lymph node metastasisDistant metastasis

Matched triplets Matched pairs

% c

ells

with

≥3

copi

es




Published OnlineFirst May 18, 2018.Clin Cancer Res Vladimir Bezrookove, Mehdi Nosrati, James R. Miller, et al. Progression Marker for Cutaneous MelanomaRole of Elevated PHIP Copy Number as a Prognostic and

Updated version

10.1158/1078-0432.CCR-18-0791doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2018/05/18/1078-0432.CCR-18-0791.DC1

Access the most recent supplemental material at:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/early/2018/05/18/1078-0432.CCR-18-0791To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-18-0791

http://clincancerres.aacrjournals.org/content/suppl/2018/05/18/1078-0432.CCR-18-0791.DC1

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/early/2018/05/18/1078-0432.CCR-18-0791


Date post:	27-Jan-2020
Category:	Documents
Upload:	others
View:	3 times
Download:	0 times

Role of Elevated PHIP Copy Number as a Prognostic and … · 1 Role of Elevated PHIP Copy Number as...

Documents