Classic and Molecular Cytogenetic Analyses Reveal Chromosomal Gains and Losses Correlated with Survival in Head and Neck Cancer Patients

Vol. 11, 621–631, January 15, 2005 Clinical Cancer Research 621

Na´dia Aparecida Be´rgamo,1 that acquisition of monosomy 17 was a significant (P = Luciana Caricati da Silva Veiga,1 0.0012) factor for patients with a previous family history of Patricia Pintor dos Reis,4 Ineˆs Nobuko Nishimoto,3 cancer. Conclusions: The significant associations found in this Jose´ Magrin,3 Luiz Paulo Kowalski,3 4 2 study emphasize that alterations of distinct regions of the Jeremy A. Squire, and Sı´lvia Regina Rogatto genome may be genetic biomarkers for a poor prognosis. 1Department of Genetics, Institute of Biosciences and 2NeoGene Losses of chromosomes 17 and 22 can be associated with Laboratory, Department of Urology, Faculty of Medicine, Saõ Paulo a family history of cancer. State University; 3Department of Head and Neck Surgery and Otorhinolaryngology, AC Camargo Hospital, Saõ Paulo, Brazil and 4Department of Cellular and Molecular Biology, Princess Margaret INTRODUCTION Hospital, Ontario Cancer Institute, University of Toronto, Toronto, Carcinomas of the head and neck represent the sixth most Ontario, Canada frequent cancer worldwide and f90% to 95% are squamous cell carcinomas. Tobacco and alcohol consumption are the ABSTRACT most important nongenetic risk factors associated with the Purpose: Genetic biomarkers of head and neck tumors development of head and neck squamous cell carcinomas could be useful for distinguishing among patients with (HNSCC; ref. 1). Estimated age-standardized rates per similar clinical and histopathologic characteristics but 100,000 for 1990 showed 12.8 men and 3.7 women of oral having differential probabilities of survival. The purpose of cavity and pharynx cancers and 6.5 men and 1.1 women for this study was to investigate chromosomal alterations in head laryngeal cancer for Tropical South America, compared and neck carcinomas and to correlate the results with clinical with 12.1 and 5.7 men, respectively, to oral cancer and and epidemiologic variables. pharynx and laryngeal cancers for all areas in the world (2). Experimental Design: Cytogenetic analysis of short-term Tropical South America has one of the world’s highest age- cultures from 64 primary untreated head and neck squamous standardized rates of head and neck carcinomas (2, 3). cell carcinomas was used to determine the overall pattern of Abnormal karyotypes have been reported in >250 cultured chromosome aberrations. A representative subset of tumors HNSCC (4). Karyotypes are often complex, with many was analyzed in detail by spectral karyotyping and/or numerical and unbalanced structural aberrations. The most confirmatory fluorescence in situ hybridization analysis. frequent losses involve chromosome arms 3p, 7q, 8p, 9p, 11q, Results: Recurrent losses of chromosomes Y (26 cases) 13p, 14p, 15p, 16p, and 18q and gains at 1q, 3q, 8q, and 15q and and 19 (14 cases), and gains of chromosomes 22 (23 cases), band 11q13. Among recurrent structural alterations, the most 8 and 20 (11 cases each) were observed. The most frequent common are 8q isochromosomes, 3p deletions, and the presence structural aberration was del(22)(q13.1) followed by rear- of homogeneously staining regions at 11q13 (5–15). Previous rangements involving 6q and 12p. The presence of specific cytogenetic studies have primarily been observational and have cytogenetic aberrations was found to correlate significantly been drawn from predominantly European and North American with an unfavorable outcome. There was a significant cohorts. In this present study, we have examined cytogenetic association between survival and gains in chromosomes 10 aberrations in the context of accompanying clinical variables (P = 0.008) and 20 (P = 0.002) and losses of chromosomes 15 such as anatomic sites, histology, stage, grade, treatment, and (P = 0.005) and 22 (P = 0.021). Univariate analysis indicated outcome. Moreover, this study comprises a typical Brazilian patient cohort comprising multiracial ethnicity and the additional risk factors associated with South American lifestyle. This Received 4/22/04; revised 9/30/04; accepted 10/11/04. unique patient cohort has allowed us to associate genomic Grant support: Conselho Nacional de Pesquisa (Distrito Federal, alterations with familial history of cancer and other prognostic Brazil); Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı´vel Superior factors in 67 consecutive head and neck tumors arising in a (Distrito Federal, Brazil); Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (Centro de Pesquisa Inovaçaõ e Difusaõ, Saõ Paulo, Brazil); representative Brazilian patient population. and Canadian Cancer Society, National Cancer Institute of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked MATERIAL AND METHODS advertisement in accordance with 18 U.S.C. Section 1734 solely to Patients. Sixty-seven head and neck tumor samples were indicate this fact. obtained from the AC Camargo Hospital, Saõ Paulo, Brazil from Requests for reprints: Silvia Regina Rogatto, NeoGene Laboratory, 1994 to 1996. The patients were accrued consecutively and the Department of Urology, Faculty of Medicine, Saõ Paulo State University, CEP 18618-000, Botucatu, Saõ Paulo, Brazil. Phone: 55-14-38116271; inclusion criteria were no previous new histologic diagnosis of Fax: 55-14-38116271; E-mail: [email protected]. head and neck cancer and a tumor larger than 1 cm in size. Three D2005 American Association for Cancer Research. samples were excluded: an olfactory neuroblastoma (case 1),

Table 1 Description of cases according to age, gender, histopathological diagnosis, TNM status, clinical data and composite karyotype Case no. Sex/age Location Tumor grade TNM Affected relative/tumor site(s) Tobacco usage Nasal cavity 1 F/60 Nasal cavity 1 T1N0M0 Father/pancreas

Oral cavity 2y F/70 Maxillary antrum 1 T4N0M0 3 M/47 Maxillary antrum 1 T4N0M0 4y M/63 Oral mucosa 1 T4N0M0 + 5 M/52 Tongue 1 T2N0M0 Uncle/prostate and bowel + 6 M/68 Tongue 1 T3N0M0 Sister/breast + 7 M/39 Tongue 1 T3N0M0 8 F/87 Tongue 1 T3N1M0 Brother/lung 9 M/57 Tongue 1 T3N2aM0 + 10 M/64 Tongue 1 T4N0M0 11y M/61 Tongue 1 T4N0M0 Two sisters/kidney + 12 M/58 Tongue 1 T4N1M0 + 13 M/67 Tongue 1 T4N2bM0 + 14y F/40 Tongue 2 T1N1M0 + 15 M/53 Tongue 2 T2N0M0 16y M/35 Tongue 2 T4N0M0 Cousin/stomach 17 M/56 Tongue 2 T4N2aM0 + 18 M/54 Tongue 2 T4N2aM0 + 19y M/40 Tongue 4 T4N2aM0 Father/esophagus + 20 M/38 Gingiva 1 T4N0M0 + 21 M/64 Gingiva 1 T4N2M0 22 M/58 Gingiva 2 T4N0M0 + 23 M/49 Floor of mouth 1 T2N0M0 + 24y M/66 Floor of mouth 1 T4N0M0 + 25 M/46 Floor of mouth 1 T4N2cM0 + 26y F/74 Floor of mouth 2 T2N0M0 27y M/63 Floor of mouth 2 T2N0M0 Brother/US 28 F/50 Floor of mouth 2 T2N3M0 + 29 M/53 Floor of mouth 1 T3N0M0 + 30 M/66 Floor of mouth 3 T3N1M0 +

31 M/53 Retromolar 1 T3N1M0 Grandmother/stomach Uncle/larynx + 32 M/39 Retromolar 1 T4N2aM0 + 33 M/58 Retromolar 2 T3N0M0 + 34 F/61 Retromolar 2 T4N0M0 + 35* M/65 Retromolar 2 T4N2sM0 +

Pharygeal carcinomas 36 M/71 Tonsil 1 T4N0M0 + 37 M/42 Tonsil 1 T4N0M0 + 38* M/56 Tonsil 2 T3N2cM0 Mother/uterus Father/bowel + 39 M/62 Tonsil 2 T4N2aM0 ++ 40 M/55 Tonsil 3 T4N1M0 + 41 M/44 Oropharynx 1 T4N0M0 42 M/48 Oropharynx 3 T4N0M0 + 43 M/58 Pyriform sinus 2 T2N0M0 + 44 M/32 Pyriform sinus 2 T3N0M0 + 45 M/45 Pyriform sinus 2 T3N3M0 Sisters/breast Aunts/breast + 46 M/61 Pyriform sinus 2 T3N3M0 Grandmother/bowel + 47 M/61 Pyriform sinus 3 T4N3M0 Uncle/pharynx +

Laryngeal carcinomas 48z M/62 Epiglottis 1 T3N0M0 + 49y M/57 Epiglottis 1 T3N2cM0 + 50 M/50 Supraglottis 3 T4N1M0 + 51 M/67 Larynx 1 T3N0M0 + 52 M/67 Larynx 1 T3N0M0 53 M/69 Larynx 1 T3N0M0 + 54 M/70 Larynx 1 T3N2cM0 55 M/50 Larynx 1 T4N0M0 + 56 M/49 Larynx 1 T3N0M0 Mother/bowel Ca + 57* M/65 Larynx 1 T4N0M0 + 58y M/79 Larynx 2 T3N0M0 + (Continued on the next page)

DOD (16) 41-47,X,X,+8,+12,17,22,+mar [cp15]/46,XX[2]

DOD (18) 43-49,XX,+5,+8,+17,19,20,del(22)(q13.1),+mar[cp14]/46,XX [13] Alive (24), lost to follow-up 41-47,XY,+6,+17,20 [cp6]/46,XY[2] + Alive (15), lost to follow-up 41-48,X,Y,+9,add(9)(p24),+13,18,19,+22[cp21]/46,XY [8] + ANR (47) 46,XY[7] + ANR (36) 40-46,X,Y,22[cp9]/46,XY[4] AWC (41) 44-48,XY,+8,+22,+mar[cp4]/46,XY[5] AWC (33) 43-48,X,Y,3,+5,+7,+8,19,20,21,22 [cp12]/ 46,XY[4] + DOC (9) 44-47,XY,+10,del(11)(q23.3),del(12)(p12),+18,+20,21,+22 [cp12]/46,XY [10] DOD (29) 41-48,X,Y, 2,+8,10,14,+17,22,+22 [cp18]/ 46,XY[5] + DOD (36) 42-48,XY,10,del(10)(p13),+11,+18,+20,+del(22)(q13.1)[cp19]/ 46,XY[3] + DOD (12) 42-48,XY,5,13,+22[cp10]/46,XY[10] + DOD (3) 67-69,XXY,inc[28] ANR (44) 43-47,X,X,21,+del(22)(q13.1)[cp9] /46,XX[20] ANR (52) 41-47,X,X,i(6p),+6,+7,+9,11,14,+?del(22)(q13.1),+mar[cp15]/ 46,XX[4] + DOD (6) 43-48,XY,9,+10,17,+22[cp15] /46,XY[21] DOD (37) 42-49,X,Y,9,+10,+12,+17,22,+mar[cp11]/ 46,XY[5] + DOD (18) 43-49,XY,del(6)(q24),+7,9,10,16,+18,del(18)(p11.2),+20,+21, +22,+mar[cp17] /46,XY[8] + DOD (31) 44-48,X,Y,+10,+20,+22[cp10]/46,XY[17] + ANR (47) 44-46,X,Y,+12,+22[cp7]/46,XY[4] ANR (49) 43-47,XY,13,+22,+mar[cp6]/46,XY[2] + DOD (52) 46,XY[7] + ANR (48) 40-49,X,Y,+8,16,+mar[cp7]/46,XY[4] + DOC (1) 41-45,X,Y,+17,20,+del(22)(q13.1) [cp19]/46,XY[5] + DOD (18) 44-49,X,Y,del(5)(q15q23),+8,9,13,19,21,+22,+mar[cp10]/ 46,XY[10] ANR (42) 41-47,X,X,del(6)(q23),+8,10,13,del(17)(p11.2),+22[cp14]/ 46,XX[8] + ANR (48) 42-48,X,Y,3,9,14,16,18,19,+22, +mar[cp16]/ 46,XY[9] DOD (26) 42-47,X,X,+8[cp5] + DOD (13) 42-46,X,Y,15,20,+mar[cp11]/46,XY[2] + DOC (38) 40-47,X,Y,del(12)(p12),+17,19,20,+22 [cp16]/46,XY[14] + DOD (46) 41-46,XY,6,+8,9,11,12,21,22,+mar[cp12]/ 83-92, XXYY,1,2,4, 6,7,10,11,del(11)(p11.2),12,16,17,18,+18,19,+mar[cp13]/ 46,XY[3] + DOD (15) 46,XY[5] + ANR (12), lost to follow-up 48,XY,+?del(22)(q13.1),+mar[2]/46,XY[8] ANR (47) 47,XY,+mar[2]/46,XY[8] + AWC (45) 43-47,X,Y,del(1)(q21),9,10,inv(12)(p13.3q12),18,21,+22,+mar [cp21]/ 46,XY[17]

+ ANR (36) 46,XY[5] + DOD (19) 42-48,XY,16,20,22,+22,+mar[cp12] + ANR (30) 44-47,X,Y,+7,19,20,+22,+mar [cp17]/46,XY[1] + DOD (8) 42-47,X,Y,3,15,+15,17,19,22 [cp17]/46,XY[17] + ANR (42) 42-47,X,Y,+mar[cp8]/46,XY[8] + Alive (4), lost to follow-up 42-47,XY,5,+6,9,13,14,18,+18,19,+20,+21,+22,+mar[cp20]/ 46,XY[7] + ANR (63) 40-48,X,Y,+2,+4,+6,7,9,14,18,20,+mar [cp19]/46,XY[6] + DOD (28) 46,XY[6] + DOD (31) 42-47,XY,+8,12,16,19,22,+22,+mar[cp14] /46,XY[1] + DOD (7) 43-48,X,del(X)(p11.4),Y,+4,6,+8,11,+13,16,17,+20,22[cp20]/ 46,XY[5] + DOC (30) 40-47,X,Y,del(6)(q21),+7,22,+22,+mar[cp12] /46,XY[10] + DOC (32), bladder Ca 44-48,XY,Y,+20,+22[cp10]/47,XX[7]

+ Alive (46), lost to follow-up 43-48,X,Y,+4,7,9,+12,13,+18,19,+del(22)(q13.1)[cp14]/ 46,XY[8] + DOD (22) 41-48,X,Y,+6,+del(6)(q22),11,12,15,16,19,22,+del(22) (q11.2),+mar[cp18]/46,XY[6] + DOD (17) 46,XY,del(6)(p11.2p21.3)/46,XY[6] + ANR (59) 47,XY,+2[3] + ANR (46) 45-47,XY,+8,+mar[cp5]/46,XY[12] + Alive, lost to follow-up 46-54,XY,inc[8] + DOD (12) 44-48,X,Y,add(3)(q29),+del(3)(q12),+12,15,+20,+mar[cp23]/ 46,XY[7] + ANR (55) 46,XY[5] + ANR (40) 43-47,XY,add(2)(q37),+mar[cp9]/46,XY[5] + ANR (45) 44-49,XY,del(1)(q41),?add(4q),+20,del(22)(q13.1),+mar[cp11]/46,XY[13] + DOD (42) 45-54,XY,15,21,+del(22)(q13.1)[cp5]/46,XY[11] (Continued on the next page)

Table 1 Description of cases according to age, gender, histopathological diagnosis, TNM status, clinical data and composite karyotype (Cont’d) Case no. Sex/age Location Tumor grade TNM Affected relative/tumor site(s) Tobacco usage

59y,z M/64 Larynx 2 T3N0M0 60 M/69 Larynx 2 T3N0M0 + 61 M/77 Larynx 2 T3N0M0 Grandson/sarcoma Granddaughter/brain 62y M/54 Larynx 2 T3N0M0 + 63 F/43 Larynx 2 T3N2bM0 + 64 M/64 Larynx 2 T3N2aM0 + 65 M/60 Larynx 2 T4N0M0 +

Other oral tumors 66 M/32 Maxillary antrum adenocarcinoma 3 T4N0M0 67y F/44 Floor of the mouth liposarcoma 2 T4N2aM0 Brother/gastric (US) + Abbreviations: () negative history; (+) positive history; US, unspecified site on medical records; ANR, alive, nonrecurrence; DOD, died of disease; DOC, died of other causes; AWC, alive with cancer; TNM, tumor-node-metastasis. *From surgery until December 2003. Survival time since the surgery is given in parenthesis. yAnalyzed by FISH. zAnalyzed by SKY.

a maxillary antrum adenocarcinoma (case 66), and an oral cavity months (median, 35.9 months). The median age of the patients liposarcoma (case 67). A total of 64 squamous cell carcinomas was 58.0 years (range, 32-87 years), the male-to-female ratio were eligible to be included in this study, and the tumor sites was 9.7:1; 79.7% (51 of 64) were tobacco consumers and 76.6% were the following: oral cavity (34 cases), larynx (18 cases), and (49 of 64) were alcohol users (Table 1). pharynx (12 cases). None of the patients had received Cytogenetic Study. Fresh tumor samples were obtained radiotherapy or chemotherapy before surgery and sample under sterile conditions and immediately processed. Chromo- collection for cytogenetic analysis. Selected patients with oral, somal preparations and cytogenetic analysis were carried out by oropharynx, hypopharynx, and supraglotic carcinomas were standard techniques following short-term harvesting of primary submitted to elective selective neck dissection. All patients with cultures (9-12 days) as previously described (15). Metaphase positive margins, tumors with perineural infiltration, or vascular chromosomes were subjected to GTG banding (18). The mean embolization as well as those with metastatic lymph nodes number of metaphases analyzed in each sample was 20 (range, confirmed by histologic examination of the surgical specimens 7-35 cells). The karyotype description and the requirements for were submitted to postoperative radiotherapy. None received clonality followed the International System for Human Cytoge- adjuvant chemotherapy. All of the patients were advised of the netic Nomenclature criteria (19). A further requirement for procedures and provided written informed consent. This study clonality was that cytogenetic changes had to be found in at least was approved by the Brazilian Ethics Committee-CONEP 813/ two culture flasks. 2000. The medical records of all patients were examined to Fluorescence In situ Hybridization. Slides stored at obtain detailed clinical and histopathologic data, including 20jC were used for fluorescence in situ hybridization (FISH) information about consumption of alcohol beverages and and spectral karyotyping analyses. Probe labeling, hybridiza- tobacco, cancer family history, and other demographic data. tion, suppression hybridization, detection, fluorescence micros- Alcohol users were defined as the weekly consumption of 7 to copy, and microphotography were done as previously described 10 drinks of alcohol for >1 year anytime. Types of alcoholic (20, 21). The PAC134J15 (110-kb insert) was isolated from a beverages (wine, beer, or spirit) were converted into drink screen with probe D22S543 (22). The DNA probe was labeled equivalents, and each was given a value of 13.6 g of absolute with biotin-14-dUTP (Life Technologies, Carlsbad, CA) by nick alcohol. Tobacco use was defined as patient responding translation (21). FISH was done in 17 cases. An average of 100 positively to being a current or regular smoker for >1 year in nonoverlapping interphase cells with intact morphology and 10 their lifetime (unless smoking stopped >20 years ago). metaphase cells based on 4,6-diamidino-2-phenylindole coun- Information concerning the extent of usage, such as number of terstaining were scored to determine the number of hybridiza- pack-years, was not obtained. For the family history of cancer, tion signals for the chromosome 22 probe. Normal controls first-degree and second-degree relatives with cancer were were phytohemagglutinin-stimulated normal male lymphocytes. considered as positive, and whenever possible, the evidence of A case was considered to have a numerical chromosome cancer was based on assessment of medical records or abnormality when the percentage of cells showing an abnormal ascertained from the death certificate. The histopathologic number of hybridization signals was higher than the mean value classification was based on the WHO International Classification plus 2 SDs obtained for the same chromosome in the normal of Diseases for Oncology (16). The clinical staging was control. The results were verified independently by a second determined using the tumor-node-metastasis staging system observer. (17). For statistical analysis, the patients were grouped in two Spectral Karyotyping. A commercially available spec- classes because of the small number of cases in each tumor stage tral karyotyping kit from Applied Spectral Imaging (Carlsbad, [i.e., (T1-T2) versus (T3-T4) and (N0) versus (N1,N2a,N2b,N3c)]. CA) was used to analyze 12 metaphases from two cases. Slide The patients were followed prospectively from surgery date to treatment, post-hybridization detection, and washes were done December 2003 and the follow-up time range was 1 to 103.5 according to published protocols and the manufacturer’s

Table 1 Description of cases according to age, gender, histopathological diagnosis, TNM status, clinical data and composite karyotype (Cont’d) Alcohol usage Follow-up* (mo) Composite karyotype based on ISCN guidelines (1995) DOD (42) 44-48,X,-Y,+7,10,21,+22,+del(22)(q13.1)[cp20]/46,XY[9] + DOD (16) 43-47,XY,Y,8,+10,+20,22,+22[cp11]/46,XY[[8] Alive (23), lost to follow-up 45,XY,del(1)(p22),+4[4]/46,XY[4] + ANR (60) 43-47,XY,+1,9,10,+11,11,13,16,17,+22,+mar[cp14] ANR (48) 44-47,XX,del(12)(p12),del(17)(q11.2),19,+?del(22)(q13.1)[cp12]/ 46,XX[10] + DOD (18) 47,XY,+20[2]/46,XY[6] + DOD (30) 45-48,XY,del(2)(q32),t(5;12)(q35;p12)[cp10]/46,XY[2]

+ DOD (20) 43-47,X,Y,del(6)(q21q23),+10,+22[cp11]/46,XY[6] ANR (52) 42-50,X,X,2,10,del(12)(p12.3),+15,19,20,+22,+mar[cp24]/ 46,XX[8]

instructions (23). Spectral images were acquired and analyzed followed up and the interval in months between surgery and with a SD 200 spectral bio-imaging system (Applied Spectral death or until the last date of follow-up in December 2003 was Imaging Ltd., MigdalHaemek, Israel) attached to a Zeiss calculated. microscope (Axioplan 2) by means of a C-mount consisting of an optical head with a special Fourier-transformed spectropho- tometer (SAGNAC common path interferometer) to measure the RESULTS spectrum, and a cooled CCD camera for imaging. The images Cytogenetic Analysis. The karyotypes of the 67 tumors were stored in a computer for further analysis using the after GTG banding are presented in Table 1. Chromosome SKYVIEW (ASI, Carlsbad, CA) software. Based on the banding analysis showed complex karyotypes with multiple measurement of the spectrum for each chromosome, a spectral numerical and structural alterations in almost all cases. Six cases classification algorithm was applied. 4,6-Diamidino-2-phenyl- showed normal karyotypes. Although in low frequency, normal indole images were acquired from all metaphases analyzed using cells were detected in all cases, except in five samples. a 4,6-diamidino-2-phenylindole–specific optical filter. In the 64 cases of HNSCC, numerical changes most Statistical Analysis. Associations with the monosomy frequently involved the loss of chromosomes Y (26 cases), 19 and trisomy groups and risk factors were verified with 5% of (14 cases), 22 (12 cases), 9 (11 cases), 16 and 20 (9 cases each), significance level using the Fisher’s exact test. The tobacco and 10 and 21 (8 cases each), and 13 (7 cases). Gains most frequently alcohol consumption were determined using criteria described affected the chromosomes 22 (23 cases), 8 and 20 (11 cases above. The Kaplan-Meier method was applied to obtain survival each), 17 and 18 (6 cases each). In the oral cavity, laryngeal, and curves and to calculate the actuarial estimators of survival (24). pharyngeal carcinomas, the most common numerical chromo- The log-rank test was used to compare survival curves of the some alterations were trisomy 22 (44.1% of cases), trisomy 20 different categories of the same variable. The patients were (20.2% of cases), and trisomy 22 (41.7% of cases), respectively.

Fig. 1 Ideogram of DNA copy number changes identified by G-banding in 67 head and neck carcinomas. Left bars, chromosome losses; right bars, chromosome gains.

Fig. 2 A, G-banded metaphase from a squamous cell carcinoma of pyriform sinus (case 46) with karyotype 46,XY,del(5)(p15.2),20, +?del(22)(q13.1). Partial trisomy 22 was seen in case 59 after G-banding (box). B, partial metaphase cells after FISH analysis using PAC134J15 (isolated from D22S543) in cases 4 and 16 with trisomy 22. Interphasic cells of cases 57 and 62 with three signals (arrowheads). C, spectral karyotyping analysis showing the metaphase spread depicted in the classified colors (top), pseudocolors (right), and the inverted 4,6-diamidino-2- phenylindole banding image (left) in the primary cancer of pyriform sinus (case 45) showing monosomies of the chromosomes 20 and 22 and chromosome Y loss.

Table 2 Distribution of patients according to selected demographic factors and monosomies 15, 17, and 22 and trisomies 10 and 20 Variables Categories Trisomy 10 Monosomy 15 Monosomy 17 Trisomy 20 Monosomy 22 No Yes P*NoYes P*NoYes P*NoYes P*NoYes P* Sex Female 6 0 0.99 6 0 0.99 6 0 0.99 6 0 0.58 6 0 0.99 Male 53 5 53 5 53 5 47 11 46 12 Age (y) V55 24 2 0.99 25 1 0.64 22 4 0.15 22 4 0.99 22 4 0.75 >55 35 3 34 4 37 1 31 7 30 8 Tumor site Larynx 17 1 na 15 3 na 17 1 na 14 4 na 16 2 na Oral cavity 30 4 33 1 32 2 30 4 29 5 Pharynx 12 0 11 1 10 2 9 3 7 5 Tumor stage 1, 2 9 0 0.99 9 0 0.99 9 0 0.99 9 0 0.34 9 0 0.19 3, 4 50 5 50 5 50 5 44 11 43 12 Necrosis stage N0 36 2 0.39 36 2 0.39 36 2 0.39 34 4 0.10 33 5 0.20 N+ 23 3 23 3 23 3 19 7 19 7 Family history No 45 3 0.59 45 3 0.59 47 1 0.01 41 7 0.44 42 6 0.06 Yes 14 2 14 2 12 4 12 4 10 6 Tobacco usage No 12 1 0.99 13 0 0.57 12 1 0.99 12 1 0.44 11 2 0.99 Yes 47 4 46 5 47 4 41 10 41 10 Alcohol usage No 14 1 0.99 15 0 0.33 15 0 0.33 15 0 0.05 12 3 0.99 Yes 45 4 44 5 44 5 38 11 40 9 Death Noy 25 0 0.15 25 0 0.15 24 1 0.64 24 1 0.04 24 1 0.02 Yes 34 5 34 5 35 4 29 10 28 11 Survival probability (5 y) 42.5 0.0 42.5 0.0 41.0 13.3 45.5 9.1 46.6 8.3 z 0.008 0.005 0.079 0.002 0.021 Abbreviation: na, not available. *P obtained Fisher’s Exact test with 5% level of significance. yNo death: patients alive or lost to follow up. zLog–rank test P value for survival curves comparison.

Loss of chromosome Y in male patients was detected in 14 of 29 The most frequent structural changes detected in the oral cavity tumors, 7 of 12 pharyngeal, and 5 of 17 laryngeal 64 HNSCC involved chromosomes 22 (12 cases), 6 (6 cases), carcinomas. Numerical chromosomal alterations identified in all and 12 (5 cases). Overall, 42 out of 64 cases showed samples are showed in Fig. 1. clonal chromosome aberrations involving chromosome 22.

Fig. 3 Kaplan-Meier survival curves for monosomies of chromosomes 15 (A) and 22 (B) and trisomies of chromosomes 10 (C) and 20 (D).

Chromosome 22 was rearranged in 6 of 34 oral cavity tumors chromosomal alterations were younger than 50 years in 25% and in 6 of 18 laryngeal carcinomas. Losses of chromosome 22 of cases (7 of 28 cases), 14.3% (4 of 28 cases) were nonusers were observed in 5 of 12 pharyngeal, 3 of 34 oral cavity, and 2 of of alcohol and tobacco, 21.4% (6 of 28 cases) were 18 laryngeal carcinomas. nontobacco users, and 42.8% (12 of 28 cases) died of disease In addition, FISH was applied in 14 cases to evaluate the before 24 months. Taken together, the data presented indicates frequency of aneuploidies of chromosome 22. Interphase FISH that the presence of cytogenetic aberrations of chromosomes yielded one signal (i.e., monosomy) in 12 cases and all the 14 10, 15, 20, and 22 were a significant determinant of poor outcome in head and neck carcinomas. cases showed three or more signals, with frequencies higher than the control (5% and 6%, respectively). The frequency of monosomy 22 ranged from 10% to 40% and the trisomy 22 DISCUSSION from 9% to 27%. A small submetacentric chromosome was Cytogenetic analysis of head and neck tumors has re- detected and was interpreted as del(22)(q13.1) (Fig. 2A). Using vealed extensive genetic heterogeneity and karyotype com- the probe PAC134J15, we confirmed the marker as a chromo- plexity. The pattern of chromosomal alterations observed in some 22 derivative in eight cases (Fig. 2B). our G-banding study and the identification of the most To determine whether a cryptic chromosomal rearrange- frequently aberrant chromosomes were consistent with other ment had led to misclassification using conventional cytogenetic reports on the cytogenetics for head and neck carcinomas (4, methods, we did spectral karyotyping analysis. Spectral 10, 14, 25, 26). karyotyping analysis was done in two cytogenetic preparations Although head and neck carcinomas have been inten- (cases 48 and 59). The analysis of 12 cells from case 48 revealed sively studied using classic cytogenetic techniques (4) and clonal losses involving chromosomes Y, 13, 19 and a gain of comparative genomic hybridization (27, 28), the correlation chromosome 22. Monosomies 7 and 9 and trisomies 4 and 12 between specific chromosomal abnormalities and clinical were nonclonal. Thirteen metaphase cells from case 59 revealed outcome, tumoral staging, anatomic sites, treatment, and losses of chromosomes Y and 10 and gains of chromosome 22. family history of cancer is still poorly understood. In this Figure 2C shows a cell from case 48 analyzed using the study, we found different profiles of chromosome gains and chromosome-painting probe. losses depending on the tumor location. In 1995, Jin et al. (7) Statistical Analysis. The mean age of the subjects was had already suggested that there might be karyotypic 57 years (range, 32-87 years). Of the 64 patients, 85.9% were variations among HNSCC from different anatomic sites. coded T3 or T4 and 40.6% had more than one clinically Subsequently, the same group reported clonal chromosome positive lymph node. Around 78% of the patients were aberrations in 56 laryngeal tumors; rearrangements involving alcohol and/or tobacco users and only 25% had a positive 22p11-q11 were detected in 20% of the cases (29). We found family history of cancer. The absolute and relative frequencies similar chromosome alterations in 18 laryngeal tumors, of some selected clinical and demographic variables are shown including rearrangements involving 22q in six cases (33%). in Table 2. The association test was accomplished for all However, a small submetacentric marker del(22)(q13.1) was chromosome alterations (data not shown). No significant also observed in oral carcinomas (17% of cases) but not in associations were observed in univariate and survival analyses pharyngeal tumors. A previous analysis by our group using (data not shown) despite the high frequencies of trisomies and microsatellite markers revealed a higher frequency of allelic monosomies observed. losses on 22q12.1-13.3 in laryngeal tumors as compared with In spite of a small sample size, a significant association oral tumors and other HNSCC (30). (P = 0.012) was observed between a family history of cancer and Changes involving chromosome 22 were the most frequent monosomy 17. Monosomy 22 and family history of cancer was alteration detected by G-banding and confirmed by FISH and marginally significant (P = 0.058). Borderline significance spectral karyotyping analyses: gains were detected in 25 cases, (P = 0.054) was detected between alcohol consumption and losses in 13 cases, and structural rearrangements in 12 cases. trisomy 20. Univariate analysis revealed a possible association Abnormalities affecting 22q are common in squamous cell between the presence of all monosomies and trisomies and the carcinomas of larynx (11, 29, 31, 32), oral cavity (6, 7, 13, 31, occurrence of death, but only monosomy 22 and trisomy 20 33), and HNSCC from several anatomic sites (34–37). In showed significant associations (Table 2). agreement with our G-banding results, comparative genomic The impact of the numerical chromosomal alterations on hybridization data have revealed copy number changes in >50% the overall survival probability was assessed by the log-rank of the analyzed cases showing deletions and, more frequently, test. The survival probability was worse in the presence of gains of chromosome 22. Recently, we studied 40 primary oral alterations in chromosomes 10, 15, 20, and 22, and the tumors by quantitative real-time PCR and identified a deletion difference between the survival curves was significant of the DIA1 gene (mapped on 22q13) and D22S274 (P < 0.05; Fig. 3). Although monosomy 22 was associated (22q13.31) sequences in 25% of the cases; these losses were with a significant reduction on the survival expectancy significantly correlated with family history of cancer and a (P = 0.021), in the analysis by T-stage, a nonsignificant reduced probability of survival (38). As shown here, there was association of survival rates (P = 0.061) and T3 -T4 staged a significant association (P = 0.012) between a familial cases was verified. T3 and T4 tumors with trisomy 10 history of cancer and the loss of chromosome 17, whereas this (P = 0.020), monosomy 15 (P = 0.016), and trisomy 20 association was only marginally significant (P = 0.058) for (P = 0.008) were significantly associated with survival rates chromosome 22 loss. Loss of heterozygosity, deletions, and decreased (data not shown). Patients with at least one of these other rearrangements involving chromosome 17, particularly

the TP53 gene, are the most common mutations described in have reported a loss of Y chromosome in leukemia, lymphoma, HNSCC (reviewed in refs. 25, 39). TP53 mutations have been and solid tumors (4). In HNSCC, Jin et al. (6) reported loss of the correlated with exposure to alcohol and tobacco and seem to Y chromosome in 20 out of 21 cases, 14 of which showed be associated with a short recurrence interval (40). A novel aneusomy Y as the only chromosomal alteration. Most studies of candidate cancer susceptibility gene located on chromosome hematologic diseases have shown a loss of the Y chromosome in 17p is ELAC2. Carriers of mutations in ELAC2 have a higher both normal and malignant marrows, with the frequency risk of developing prostate cancer (41). showing an age-dependent increase (52). According to univariate analysis, overall poor survival Although there is no evidence for the existence of a tumor probabilities were observed for the monosomies 15 and 22 suppressor gene on the Y chromosome, several genes mapped to and trisomies 10 and 20 (P < 0.05). Although monosomy this chromosome have been implicated in disease progression 22 had a significant log-rank test P value, it was not significant (53–55). Recent studies have shown that the Y chromosome (P = 0.061) in T3-to-T4 stages of the disease. The small contains more functioning genes than previously thought sample size of our study precluded the use of multivariate (56, 57). As expected, older patients (>50 years) were most analysis to determine whether monosomy 22 was a possible frequently affected in our study (44 out of 58 male patients). independent prognostic factor. Overall, these results indicated Chromosome Y aneusomy was absent in 23 patients ages >50 that the loss of 22 was correlated with a worse outcome. In years. We detected a loss of chromosome Y in 26 out of 58 agreement with our results, Ashman et al. (32), using (45%) head and neck carcinomas, 24 of which were T3-T4; 14 of comparative genomic hybridization analysis, showed that the 26 cases showed involvement of the lymph nodes and 16 of 26 deletion of 22q was associated with a reduced survival in head died of the disease. Five patients ages <50 years showed loss of and neck cancer patients, suggesting that this abnormality is an chromosome Y; four of them were T4 stage and two died of the independent prognostic marker. disease. Although these data were not statistically significant, We also assessed the impact of the chromosomal they showed a possible association between loss of the alterations on the overall survival rates. The probability of Y chromosome and a poor outcome. survival decreased with trisomy 10 (P = 0.008), trisomy 20 Chromosome 8 overrepresentation, the second most com- (P = 0.002), monosomy 15 (P = 0.005), and monosomy 22 mon copy number gain, was detected in 11 cases, including (P = 0.021). Trisomy 10 has been most commonly observed pharyngeal, laryngeal, and oral carcinomas. A gain of chromo- as a nonrandom abnormality in hematologic diseases, such as some 8 is a frequent alteration in neoplasia and has been detected acute myeloid leukemia (42, 43) and less frequently in some in a wide variety of tumor types, particularly HNSCC (4). Huang human solid tumors (4). In our series, all the five patients et al. (58) reported that +8q2 was the second most important early with trisomy 10 showed recurrence and/or metastasis and died chromosomal event in all subtypes of HNSCC. Recently, Silva of cancer. This finding suggests that trisomy 10 is associated Veiga et al. (15) detected gains and/or amplifications at 8q23.2 in with a more aggressive behavior in head neck tumors. The 19/19 HNSCC cases by FISH and suggested that an increase in analysis of the distribution of this abnormality in the different copy number at 8q23.2 was a potential early marker in HNSCC. anatomic sites showed that trisomy 10 was present in four In summary, we found significant associations between cases of oral cavity tumors and in one laryngeal carcinoma. trisomies of chromosomes 10 and 20 and monosomies of An increase in the chromosome 20 copy number has been chromosomes 15 and 22 suggesting that these abnormalities may reported as a frequent aberration in several solid tumors, be genetic biomarkers for a poor prognosis and an increased risk including oral carcinomas (44, 45). Trisomy 20 was found in of death in these patients. These genetic markers could be useful all anatomic sites and 10 out of 11 patients died of the disease for distinguishing among patients with similar clinical and after 7 to 36 months. Amplification of 20q13 was considered a histopathologic characteristics, but distinct probabilities of predictor of poor survival in breast cancer (46). Two candidate survival. In addition, chromosome 8 gains could be involved genes, the ZNF217 putative oncogene and CYP24 (encoding in early stages of the disease. Our findings support the idea that vitamin D 24 hydroxylase) are mapped at 20q13.2. Gains on karyotypic variations occur among HNSCC arising from chromosomes 8q and 20 were reported to be predictors of a poor different anatomic sites. Comprehensive molecular profiling outcome in hepatoblastomas (47). 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Nádia Aparecida Bérgamo, Luciana Caricati da Silva Veiga, Patricia Pintor dos Reis, et al.

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