3p22.1 and 10q22.3 Deletions Detected by Fluorescence In Situ Hybridization (FISH)

3p22.1 and 10q22.3 Deletions Detected by Fluorescence In SituHybridization (FISH):A Potential New Tool for Early Detection of Non-small Cell Lung Cancer (NSCLC)

Sai Yendamuri, MB, BS*, Ara A. Vaporciyan, MD†, Tanweer Zaidi, MD‡, Lei Feng, MS§,Ricardo Fernandez, BS‡, Nebiyou B. Bekele, PhD§, Wayne L. Hofstetter, MD†, Feng Jiang,MD, PhD||, Reza J. Mehran, MD†, David C. Rice, MD†, Margaret R. Spitz, MD, MPH¶, StephenG. Swisher, MD†, Garrett L. Walsh, MD†, Jack A. Roth, MD†, and Ruth L. Katz, MD‡

*Department of Thoracic Surgery, Roswell Park Cancer Institute, Buffalo, New York †Departmentof Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center,Houston, Texas ‡Department of Pathology, The University of Texas M. D. Anderson CancerCenter, Houston, Texas §Division of Quantitative Sciences, The University of Texas M. D.Anderson Cancer Center, Houston, Texas ||Department of Pathology, University of Maryland,Baltimore, Maryland ¶Department of Epidemiology, The University of Texas M. D. AndersonCancer Center, Houston, Texas

AbstractBackground—Our objective was to study the feasibility of detecting chromosomal deletions at3p22.1 and 10q22.3 by fluorescent in situ hybridization (FISH) and to examine their distributionin different areas of the airway in patients with non-small cell lung cancer.

Methods—Brush biopsies from the mainstem bronchus on the normal side contralateral to thetumor (NBB) and mainstem bronchus on the tumor side (TBB) were obtained from 122 patientswho underwent surgical resection. Touch preparations from the tumor (TTP), normal lungparenchyma, and bronchi adjacent to the tumor were also obtained. Two FISH assays using probescomplementary to 3p22.1 and 10q22.3 were used to detect deletions.

Results—NBB showed a relatively low deletion rate of 3p22.1 and 10q22.3 compared with TTP(p < 0.0001). TBB showed a significantly higher rate of deletions compared with NBB but lowerthan TTP from the tumor (p < 0.05) for both 3p22.1 and 10q22.3. A significantly higher deletionrate was seen at TTP compared with normal lung parenchyma at both the 3p22.1 and 10 q22.3 (p< 0.0001). Correlations were seen between the deletion rates of TTP and TBB at 3p22.1 (ρ = 0.61,p < 0.0001) and between TTP and bronchi adjacent to the tumor at 10q22.3 (ρ = 0.64, p < 0.0001).

Conclusion—Deletions of the 3p22.1 and 10q22.3 regions can be reliably detected by FISH. Asone progresses from the contralateral normal bronchus to the bronchus on the side of tumor andthe tumor itself, the percentage of chromosomal deletions increases in a statistically significantfashion. This suggests that, FISH analysis of bronchoscopic brushes may be useful for identifyingpatients at high risk for developing non-small cell lung cancer.

Copyright © 2008 by the International Association for the Study of Lung Cancer

Address for correspondence: Ruth L Katz, MD, The University of Texas M. D. Anderson Cancer Center, Department of Pathology,1515 Holcombe Blvd., Unit 53, Houston, Texas 77030. [email protected].

Disclosure: Drs. Katz and Jiang are holders of an issued USA patent (US Patent Appl. No. 20060078885) for FISH probes to 3p22.1and 10q22-23. The other authors declare no conflict of interest.

Presented at the 12th World Conference on Lung Cancer, Seoul, South Korea, September 2– 6, 2007.

NIH Public AccessAuthor ManuscriptJ Thorac Oncol. Author manuscript; available in PMC 2012 June 08.

Published in final edited form as:J Thorac Oncol. 2008 September ; 3(9): 979–984. doi:10.1097/JTO.0b013e3181834f3a.

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KeywordsLung; Cancer; Non-small cell; Screening; Fluorescent in situ hybridization; Bronchial brushes

Lung cancer is estimated to affect 213,380 patients and result in 160,390 deaths in theUnited States in 2007 alone (www.lungusa.org), making it the most lethal cancer, bothamong men and women. Although early stage lung cancer is eminently curable by surgery,1

most lung cancer is detected at an advanced stage. This makes the overall outlook of lungcancer dismal. This underscores the importance of developing methods of early detection forlung cancer. Several approaches to early detection have been evaluated. High resolutioncomputed tomography scanning is one such approach. Another approach is to use sputumcytology2 and various modes of bronchoscopy including white light bronchoscopy andautofluorescence bronchoscopy.3 These techniques have their own limitations.Bronchoscopy has limited utility in the detection of peripheral lesions (which are forming anincreasing proportion of non small cell lung cancer [NSCLC]). Sputum cytology has a lowsensitivity in the detection of lung cancer. One of the ways that the sensitivity of sputumcytology has been improved is to study genetic changes in bronchial cells seen in sputumthat predate morphologic changes detected by cytology.4 Several methods have been used todetect early changes, including chromosomal changes. One of these ways is fluorescent insitu hybridization (FISH) technology.5,6

FISH uses segments of DNA labeled with a fluorescent dye that can be detected atprespecified wavelengths. These segments of DNA are hybridized to cells on slides andcompared with their corresponding centromeric region to detect gain or loss of thechromosomal segment in question. This enables the detection of a few abnormal cells in abackground of predominantly normal cells, which is typically found in sputum cytologicspecimens. This method has been found to be useful in bladder cancer. A similar set ofprobes has been used in the detection of abnormal cells in sputum cytology based on thedetection of chromosomal gain in the 5p, 7p, and 8q regions7 These probes have been shownto improve the sensitivity of sputum cytology. We have developed in-house probes for thedetection of chromosomal deletions at the 3p22.1 and 10q22.3 regions. These regions havebeen previously shown to be chromosomally deleted by CGH array data.8 In this study,these probes have been used to measure the deletion rate of these chromosomal regions inbronchoscopic brush biopsies. We show that this technique is feasible and shows instructivedata on the distribution of these deletions.

PATIENTS AND METHODSPatient Population and Specimen Collection

Patients presenting to the thoracic surgery clinic at our institution for potential resectionwere consented for acquisition of bronchoscopic brush biopsies according to an IRBapproved protocol. Patients were entered into the study only if they were deemed resectableand did not receive any preoperative radiation or chemotherapy. Biopsies were performed atthe time of their preoperative bronchoscopy immediately before the proposed resection.Clinical and pathologic data was extracted from the prospective thoracic surgery databasemaintained in our institution. Brush biopsies were obtained from bronchoscopically normalmainstem bronchus both on the side of the lesion (TBB) and the opposite side (NBB). Oncethe tumor was resected and sent for pathologic analysis, touch preps were made frombronchus adjacent to the tumor (TAB), from the tumor itself (TTP) and from normal lungparenchyma away from the tumor (NTP), depending on the amount of tissue remaining afterclinical pathologic use (Figure 1).

Yendamuri et al. Page 2

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Specimen PreparationThe brush biopsies were received in saline. Cytospins were prepared for cytology and FISHanalysis. The cytology slides were fixed in Carnoy’s solution and stained with Papanicolaoustain, whereas the air dried slides were stained with Diff-Quik (Baxter Scientific, Deerfield,IL). The cytologic features of all the specimens were assessed. Both the cytospins and thetouch imprints were fixed for FISH in methanol and acetic acid at 3:1 ratio before labeling.FISH was performed with two probe sets.

FISH TechniqueTwo probe sets were used. The first probe set included centromeric 3 (CEP3; Vysis) and thelocus specific probe 3p22.1, which was labeled in-house. The second probe set consisted ofcentromeric 10 (CEP10; Vysis) and the locus specific probe 10q22.3 which was alsodeveloped in-house. These probes were mixed with blocking DNA, enriched in repetitivesequences and hybridized to the specimens. Spectrum Green was used to label the locusspecific probes while the centromeric probes came labeled with Spectrum Orange.

The technique for making the probes has been previously described6,9. Briefly, DNA waslabeled by a commercial nick translation kit. A gel was run to confirm that the size of theDNA fragments was 500 to 2000 base pairs. The probe was then placed in a 65°C water bathfor 15 minutes and then stored at −20°C.

Slides were immersed in 2X standard sodium citrate (SSC) for 3 minutes at 77°C and then ina protease solution (50 mL of 1 X phosphate-buffered saline [PBS], pH 2.0, and 25 mg ofprotease). Protease digestion was then performed (5– 6 minutes for cytospins and7– 8minutes for touch preparations). Slides were then washed in 1 X PBS for 5 minutes andsubsequently fixed in a 1% formaldehyde solution for 5 minutes. Later, the slides were againwashed with 1 X PBS for 5 minutes and dehydrated in sequential 70%, 85%, and 100%ethanol solutions. After a brief period of drying, the probe mixture was applied to the targetareas on each slide, covered with a cover slip, and sealed with rubber cement. The slideswere then kept in a hybridization machine (Vysis) for ≥20 hours at 37°C. After the rubbercement and cover slips were removed, the slides were washed in a 50% formamide solution(pH 7.45) in 3 separate jars for 10 minutes each at 45°C. Next, the slides were washed in 2X SSC for 10 minutes at 45°C and then in 2 X SSC/0.1% ethoxylated octyl phenol (NP-40)for 10 minutes. The slides were then dried. Slides were counterstained with 4′ 6-Diamidine-2-phenylin-dole dihydrochloride (10 μl per slide), cover slipped, and viewedunder a fluorescent microscope (Leica DWRXA or Leica DMLB; Leica Microsystems, Inc.,Buffalo, NY) with the appropriate filters for the probes.

The hybridized slides were counted with the appropriate filter sets for visualizing SpectrumGreen or Spectrum Orange and 4′ 6-Diamidine-2-phenylindole dihydrochloridecounterstain. The nuclei of individual cells that did not overlap were chosen for analysis.Slides were analyzed only if 80% of the cells were interpretable in the field of view and thebrightness of the signals was >2+ on a scale of 0 to 3+. Each cell was scored individually forthe number of green (3p22.1 or 10q22.3) and orange signals (CEP3 or CEP10, respectively)that were used as internal controls. Cells with fewer green signals than orange signals werepositive, reflecting deletion. The ratio of green to orange signals was interpreted as thepercentage of deleted cells. To avoid misinterpretation due to insufficient hybridization,cells were counted only if at least one bright orange and one bright green signal werepresent. Split signals were counted as one if the space between them was less than thediameter of a single signal. A control specimen was used for each batch. Figure 2 showsrepresentative slides of the FISH technique used. From the NBB, TBB, TAB, and NTP sites,deletions were scored in 100 morphologically intact bronchial epithelial cells at each site. In



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tumor touch preparations, at least 100 tumor cells were evaluated for deletions of 3p22.1 and10q22.3 relative to the internal centromeric probes. The accuracy of manual scoring wasconfirmed by a random sample check performed independently by a second cytogenetictechnologist. This score showed a strong correlation with the first score.

Analysis of DataDeletions rates at different locations were tabulated in percentages. To compare the deletionrates at different sites, these were normalized to their technical controls before comparison.The Wilcoxon signed rank test was used for comparison of deletion rates at different sites.Correlations of FISH measurements between the brush biopsies (TBB and TAB) and thetumor touch preps (TTP) were evaluated by Spearman correlation coefficients. Kaplan-Meier curves were plotted for estimating time to death distributions. The log-rank test wasused to compare the difference in survival and recurrence free survival between the FISHmeasurement high score and low score groups, defined by the median deletion rate. All testsare two-sided, and p-values less than 0.05 are considered statistically significant. Analyseswere performed using the SPSS software version 13.0 (SPSS Inc., Chicago, IL).

RESULTSTable 1 shows the demographic and clinical data that describes this cohort of 122 patientsincluded in the study. The high percentage of women (52.5%) and adenocarcinomatoushistology (56.5%) is reflective of changing epidemiologic trends in NSCLC. Very few ofthese patients have well differentiated tumors (9.8%) and most patients are in the earlierstages of the disease, as would be expected in a patient group deemed resectable withoutneoadjuvant therapy.

Table 2 demonstrates the success rate of data acquisition at different locations. Although ahigh percentage of the bronchial brushes could be analyzed successfully with FISH, lowernumbers of tumor and normal lung parenchyma could be analyzed. A large part of thisdifference is attributable to the sequence of specimen acquisition. Although bronchialbrushes were obtained on all patients first, a subset of these patients underwentmediastinoscopy before surgical resection. If N2 or N3 nodes were positive atmediastinoscopy, surgical resection was abandoned at the time, in keeping with currentpractice at our institution. Specimens at TTP, NTP, and TAB could therefore not be obtainedfrom these patients. Furthermore, some patients did not have an area suitable for obtainingTAB specimens after clinical pathologic use, further limiting the specimens available fromthis area. The mean deletion rate at different locations for both 3p22.1 and 10q22.3 are alsotabulated here. The highest deletion rate is obtained from TTP at both loci. The next highestdeletion rate is obtained from bronchi adjacent to the tumor (TAB) or on the same side(TBB). The lowest deletion rate is from areas farthest away from the tumor which includenormal lung parenchyma (NTP) and mainstem bronchi on the nontumor side (NBB) (Figure3).

The deletion rate at different areas was compared with the deletion rate at TTP using theWilcoxon signed rank test. The deletion rate at TTP was statistically significantly higherthan each of the other areas. There is also a statistically significant difference in the deletionrate between the main-stem bronchi on the tumor side and the nontumor side (TBB andNBB). This holds true at both loci- 3p22.1 and 10q22.3.

Correlations between areas with a low deletion rate and TTP were also determined. A strongstatistically significant correlation is demonstrated between the deletion rate at TBB and thedeletion rate at TTP at the 3p22.1 locus (Figure 4A; spearman correlation coefficient =0.61). A similar statistically significant correlation is seen between the deletion rate at TAB



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and the deletion rate at TTP at the 10q22.3 locus (Figure 4B; spearman correlationcoefficient = 0.64). The deletion rate of 3p22.1 at different locations was not predictive ofsurvival (data not shown). The median deletion rate at 10q22.3 both at TBB and TTPseparated two groups that demonstrated differing survival trends. This difference was notstatistically significant (Figure 5).

DISCUSSIONEarly detection of lung cancer is of paramount importance due to the vast gap between theoverall survival of patients diagnosed with lung cancer and the survival of patients withearly lung cancer.10 Improvement in imaging has renewed interest in image based screeningand early diagnostic efforts11 after earlier studies using chest radiograph screening werenegative, presumably due to low sensitivity of the screening test.12 Similarly,methodological improvement in the detection of chromosomal alterations in sputum andbronchoscopic specimens has led to a renewed interest in their use in the screening of lungcancer. Sputum and bronchoscopic cytologic analysis has been limited by low sensitivity.Fluorescent in situ hybridization increases the sensitivity of cytologic analysis13 as thistechnique is better at the detection of a small number of abnormal cells in a background ofnormal cells.

A number of molecular changes have been described in lung cancer. These include both lossof tumor suppressor genes and activation of oncogenes.14,15 The 3p region is presumed to bethe site of several tumor suppressor genes including FHIT, RASSF1A, and FUS1.16 –18 It isone of the earliest genetic losses in lung tumorigenesis. Therefore, using a probe in thisregion is a logical choice for early detection. The 10q22.3 region that is used as a probe inour study includes the gene encoding the surfactant associated protein A (SP-A). Pulmonarysurfactant is important for normal lung physiology and alterations in surfactant have beenassociated with a number of lung diseases in children and adults. Of the 4 surfactantproteins, SP-A is the most abundant. The SP-A gene locus consists of 2 homologous genes,each about 4.5 kb in length separated by an 59 kb area.19 Alterations in SP-A have beenlooked at using various techniques including reverse transcription-polymerase chainreaction, immunohistochemistry, immunoblot analysis, and enzyme-linked immunosorbentassay, with inconsistent results.20,21 In a cDNA microarray analysis, we have shown thisarea to be one of the more commonly deleted regions in lung cancer.8 Loss of this region hasalso been demonstrated to portend a poorer prognosis in patients with early stage lungcancer.22

A number of previous studies have used FISH detected chromosomal gain but not loss todetect abnormal cells to improve detection.5,7,13 A pilot study performed by our groupdemonstrated the utility of FISH detected chromosomal loss in the diagnosis of lungcancer.9 Here, we present results of FISH detected deletions at the 3p22.1 and 10q22.3 locusin a larger cohort of patients.

From the data presented, several conclusions can be reached. The first is that FISH analysiswith these probes is technically feasible and reproducible enough for routine clinicalapplication. Good quality results can be obtained from minimally invasive bronchoscopicmethods like bronchial brushes. The second conclusion is that there is a field effect that canbe demonstrated in a quantifiable fashion. The pattern of this effect is intuitive, with thegreatest deletion rate being seen in tissue closest to the tumor and the lowest deletion rateseen in tissue farthest away from the tumor. This pattern is easily recognized whencomparing the deletion rate between the mainstem bronchus on the normal side (NBB) andthe tumor side (TBB), raising the possibility of utilizing this data in the early detection oflesions. Of note, it is not necessary for the tumor to have squamous histology or be a central



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tumor to exhibit these changes. The strong correlation of deletion rates between thebronchoscopic brushes and the TTP not only increases the biologic validity of the data, butalso raises the possibility of using the deletion rates in bronchoscopic specimens in lieu ofTTP. This is particularly important given the trend in survival shown by patients with lowdeletion rates compared with patients with high deletion rates at TBB and TTP. Thissuggests that we can potentially use bronchoscopically acquired deletion rates forprognostication. The difference in survival in these groups corresponds well with earlierpublished data of the ability of SP-A expression to separate out patients with a goodprognosis from patients with a bad prognosis.22 This data also raises the possibility ofstudying these deletions in sputum samples and this has been a focus of further investigationby our group.23

CONCLUSIONChromosomal deletions of the 3p22.1 and 10q22.3 regions can be reliably detected by FISHin brush biopsies and touch preparations. As one progresses from the contralateral normalbronchus to the bronchus on the side of tumor and the tumor itself, the percentage ofchromosomal deletions increases in a statistically significant fashion. This suggests thatFISH analysis of bronchoscopic brushes may be useful for identifying patients at high riskfor developing NSCLC.

AcknowledgmentsSupported by Specialized Program of Research Excellence in Lung Cancer grant P50CA70907, National CancerInstitute, Bethesda, MD-Roth, Spitz, Katz; (1) National Cancer Institute, NIH, Department of Health and HumanServices (to M.R.S.) grant CA 55769; (2) M. Keck Center for Gene Therapy Award (to F.J.); (3) U.T. M. D.Anderson Cancer Center Institutional Research Grant (to F.J.); (4) U.T. M. D. Anderson Cancer Center TobaccoSettlement Fund (to F.J.).

The authors would like to acknowledge the assistance of Jinping Zhang and Hua-Zhong Zhang in collection of thedata.

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small cell lung cancer 1 cm or less in size. J Thorac Cardiovasc Surg. 2006; 132:1382–1399.[PubMed: 17140961]

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4. Romeo MS, Sokolova IA, Morrison LE, et al. Chromosomal abnormalities in non-small cell lungcarcinomas and in bronchial epithelia of high-risk smokers detected by multi-target interphasefluorescence in situ hybridization. J Mol Diagn. 2003; 5:103–112. [PubMed: 12707375]

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8. Jiang F, Yin Z, Caraway NP, Li R, Katz RL. Genomic profiles in stage I primary non small cell lungcancer using comparative genomic hybridization analysis of cDNA microarrays. Neoplasia. 2004;6:623– 635. [PubMed: 15548372]



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16. Zabarovsky ER, Lerman MI, Minna JD. Tumor suppressor genes on chromosome 3p involved inthe pathogenesis of lung and other cancers. Oncogene. 2002; 21:6915– 6935. [PubMed:12362274]

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FIGURE 1.Schematic showing the areas of biopsy. NBB, brush biopsy of the mainstem bronchus on theside opposite the tumor; TBB, brush biopsy of the mainstem bronchus on the side of thetumor; TAB, touch prep of the bronchus adjacent to the tumor; NTP, touch prep of normallung parenchyma; TTP, touch prep of the tumor.



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FIGURE 2.Sample slides of the FISH analysis. Orange signals are centromeric signals. Green signalsindicate 3p22.1 and 10q22.3 probes. The cells with fewer green signals than orange signalsshow cells that have deletions at either the 3p22.1 (A) and 10q22.3 (B) loci.



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FIGURE 3.The deletion rate at 3p22.1 and 10q22.3 increases systematically as one progresses fromtissue away from the tumor towards the tumor itself. The deletion rate is significantly greaterin brush biopsies from mainstem bronchi on the tumor side (TBB) compared with thenormal side (NBB) (p < 0.05). Similarly, the deletion rate at the tumor itself (TTP) issignificantly greater than TBB itself (p < 0.05).



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FIGURE 4.A, 3p22.1 deletions at TBB correlate with 3p22.1 deletions at TTP. Spearman coeff = 0.61,p < 0.00001. B, 10q22.3 deletions at TAB correlate with 10q22.3 deletions at TTP.Spearman coeff = 0.64, p < 0.00001.



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FIGURE 5.Survival curves for patients separated by median deletion rate at the 10 q locus at (A) TTP(p = 0.176) (B) TBB (p = 0.153).



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TABLE 1

Clinical Characteristics of the Study Population

Demographics n = 122

Age

Mean 66

Range 32–84

Gender

Male 47.5%

Female 52.5%

Histology

Adenocarcinoma 69 (56.5%)

Squamous cell cancer 37 (30.3%)

Adenosquamous 3 (2.4%)

NSCLC (not specified) 7 (5.8%)

Other neoplasm 6 (4.8%)

Grade

Well differentiated 12 (9.8%)

Moderately differentiated 52 (42.6%)

Poorly/undifferentiated 49 (40.2%)

Grade not specified 9 (7.2%)

Pathologic stage

IA 29 (23.8%)

IB 40 (32.8%)

IIA 3 (2.5%)

IIB 13 (10.7%)

IIIA 21 (17.2%)

IIIB 10 (8.2%)

IV 6 (4.9%)


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TAB

LE 2

Nor

mal

ized

Del

etio

n R

ate

Det

ecte

d by

FIS

H in

Dif

fere

nt R

egio

ns o

f th

e A

irw

ay

3p22

.1N

%M

ean

Del

etio

n R

ate

± SE

10q2

2.3

N%

Mea

n D

elet

ion

Rat

e ±

SE

3p22

.1N

BB

119

97.5

1.14

± 0

.12

10q2

2.3N

BB

119

97.5

0.53

± 0

.06

3p22

.1T

BB

120

98.4

2.80

± 0

.26

10q2

2.3T

BB

119

97.5

1.61

± 0

.09

3p22

.1T

AB

7057

.41.

93 ±

0.2

10q2

2.3T

AB

6956

.61.

76 ±

0.1

5

3p22

.1N

TP

9577

.91.

46 ±

0.1

410

q22.

3NT

P93

76.2

0.88

± 0

.09

3p22

.1T

TP

9678

.77.

83 ±

0.5

910

q22.

3TT

P96

784.

76 ±

0.3

4


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3p22.1 and 10q22.3 Deletions Detected by Fluorescence In Situ Hybridization (FISH)

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