Cytogenetic Analysis of Head and Neck Carcinomas Ann-Leslie Zaslav, Judith Stamberg, Bettie M. Steinberg, Yue J. Lin, and Allen Abramson
ABSTRACT: Ten primary squamous cell carcinomas (SCC) of the head and neck were evaluated cytogenetically after 10-14 days of in vitro culture. Addition of 3% L-glutamine was essential for consistent epithelial growth of these carcinomas. Outgrowth of cells from tissue explants contained a mixture of chromosomally normal and abnormal cells; the abnormal cells had extensive changes including translocations, marker chromosomes, inversions, deletions, and duplications. In addition, all carcinomas contained cells with pulverization and double minute chromosomes (dmin). Chromosomes 11, 13, and 14 had "'hotspots" of rearrangements.
INTRODUCTION Acquired chromosome abnormalities are significant in the pathogenesis and treatment of various tumors. However, data on m a n y types of solid tumors are still scant, due to technical difficulties with their growth in culture and with their chromosomal evaluation. Little is k n o w n about the chromosome abnormalities occurring in primary head and neck tumors. A few recent reports describing the abnormalities of these tumors have been published [1-5], but most describe i n d i v i d u a l cases. The study presented here is an evaluation of a consecutive series of 10 solid primary malignant tumors of the head and neck region, in an attempt to u n d e r s t a n d the primary chromosomal events in head and neck carcinogenesis. All had abnormal metaphase spreads, with abnormal chromosomes and double m i n u t e s (dmin). Of these, seven had chromosomes of sufficient quality for karyotyping. Three could not be further analyzed. Two additional patients were eliminated from the study because only fibroblasts grew from their tumors.
MATERIALS AND METHODS Cells were cultured as previously described [6]. All specimens were washed 3 times in phosphate-buffered saline, m i n c e d with a sharp sterile scissors, placed in a 60-mm petri dish, and embedded i n 0.15 mg/ml collagen gel (Type I Collagen, Vitrogen Collagen Corp, CA) containing F12 m e d i u m , s u p p l e m e n t e d with 3% L-glutamine, Fromthe Departmentof BiologicalSciences,St. John's University,Jamaica, NY (A.-L.Z., Y. J. L.);Division of Human Genetics, Schneider Children's Hospital, Long Island Jewish Medical Center (A.-L. Z., J. S.); Department of Otolaryngology(B. M. S., A. A.), Long Island Jewish Medical Center, New Hyde Park, NY. Address reprint requests to: Dr. Ann-Leslie Zaslav, Division of Human Genetics, North Shore University Hospital, 300 Community Drive, Manhasset, N Y 11030. Received December 17, 1990; accepted April 2, 1991.
181 © 1991 ElsevierSciencePublishingCo., Inc. 655 Avenueof the Americas,New York, NY 10010
Cancer Genet Cytogenet56:181-187 (1991) 0165-4608/91/$03.50
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A.-L. Zaslav et al.
10% fetal calf serum, 1% penicillin-streptomycin, 0.5% gentamycin (Grand Island Biologics, NY), and 0.1% hydrocortisone (Sigma, St. Louis, MO). Specimens were cultured in a 5% CO 2 incubator and fed twice weekly with the above m e d i u m . W h e n the cells showed sufficient growth (between 10 and 14 days after initiation), they were harvested. Velban (Vinblastine sulfate, Lily, IN), (2.5% in F12 medium), was used as a mitotic spindle poison. After 1 hour of treatment, m e d i u m + velban were removed from the petri dish and collagenase (Sigma type IV, 2 mg/ml), was added for 1 hour to disrupt the collagen matrix. From this point the harvest followed standard cytogenetic techniques, i n c l u d i n g trypsinization, hypotonic solution, fixation, and trypsin-Giemsa b a n d i n g [7]. Four clinically normal biopsy specimens were used as controls: two pharyngeal biopsies of " n o r m a l " tissue outside the margins of carcinomas, one right vocal cord biopsy from a 3-year-old female with subglottic stenosis, and a right vocal cord biopsy from a 37-year-old female with bilateral vocal cord paralysis. Slides were scanned for well-spread analyzable metaphases. Microscopic analysis involved a detailed evaluation of metaphase spreads, looking for such changes as deletions, inversions, translocations, duplications, double minute chromosomes, homogeneous staining regions (HSRs), and abnormal banding regions (ABRs). The number oi chromosomes per cell was also counted to look for deviations from the normal number of 46. In many cases more than 20 cells were analyzed, and five or more karyotyped.
RESULTS The clinical descriptions of the patients and histologic descriptions of the tumor biopsies are shown in Table 1. All patients were diagnosed by a pathologist as having poorly to extensively differentiated carcinomas. Tumors were outlined and staged Table 1 Case number
Clinical history of carcinoma patients Sex
Age
Site
Stage
1
F
66
Maxillary Sinus
III
2
M
71
Tonsil
IV
3
M
42
Larynx
IV
4
M
70
Larynx
IV
5
F
84
Larynx
III
6
M
69
Vocal cord
III
7
M
58
III
8 9
M M
56 60
Base Tongue Larynx Larynx
10A
F
65
Larynx
III
lOB °
F
65
Larynx
III
° Second biopsy from patient 10, taken 2 weeks later. Abbreviations:ca: carcinoma.F: female, M: male.
III lI
Histology Invasive, poorly differentiated sqnamons ca Infiltrating squamous ca Infiltrating sqnamons ca Infiltrating squamous ca Infiltrating sqnamous ca Poorly differentiated squamous ca Invasive squamous ca Squamous ca Infiltrating sqnamous ca Infiltrating squamous ca Infiltrating sqnamous ca
Carcinomas of the Head and Neck
183
according to anatomical sites involved, local extension, nodal involvement, and distance from metastases [8]. All patients were s y m p t o m a t i c several months prior to surgery. Ages of the patients ranged from 5 6 - 8 4 years, with the exception of patient 3, who was 42. Again with the exception of patient 3, all patients smoked and most were heavy alcohol drinkers. Patient 3 did not present with m a n y of the features of the rest of the patients in this study. This patient was younger than the rest and was diagnosed with ulcerative colitis at age 33, w h i c h eventually necessitated a colectomy in 1980. He had no family history of colitis or cancer. It is possible that his disease could be of a different etiology from the rest of the patients in the study, but the c h r o m o s o m a l abnormalities were like those seen in most of the patients. None of the patients investigated received any drug, chemotherapy, or r a d i o t h e r a p y prior to surgery, w h e n the biopsies were done. The head and neck carcinomas had a decreased proportion of d i v i d i n g cells compared to the normal control specimens. In addition, the overall quality of the metaphases from the carcinomas was poorer than that found in the controls. All cells analyzed from the control samples had normal karyotypes. The cells derived from all of the ten carcinomas were a mixture of epithelial cells and fibroblasts and a mixture of both normal and abnormal metaphases. There was no correlation between stage of disease and c h r o m o s o m e number. For example, Patient 4 had c h r o m o s o m e abnormalities in 94.6% of the cells analyzed, but the m o d a l chromosome n u m b e r in the abnormal cells was 46 (pseudodiploid). W i t h Patient 5, 50% of the metaphases analyzed were c h r o m o s o m a l l y abnormal and the m o d a l c h r o m o s o m e n u m b e r in the abnormal cells was a p p r o x i m a t e l y 75. Table 2 shows the range of c h r o m o s o m e number in abnormal cells. Most of the abnormal cells contained m u l t i p l e rearrangements, deletions, translocations, inversions, and marker chromosomes. Stage of disease did not appear to be correlated with structural aberrations, because c h r o m o s o m e abnormalities were observed in all stages. Chromosome abnormalities among the cases were therefore c o m p a r e d i n d e p e n d e n t of stage of disease. " H o t s p o t s " (specific c h r o m o s o m e s and regions of those chromosomes where abnormalities cluster) of rearrangement in these carcinomas were found on c h r o m o s o m e s 11, 13, and 14, with translocations being the most frequent types of structural abnormality. Figure 1 is an idiogram illustrating the clonal anomalies that occurred in the investigated cases. Abnormalities of c h r o m o s o m e 11 occurred in 50% of the cases studied. The breakpoints of c h r o m o s o m e 11 clustered in two areas, 11p15 and 11q23, w h i c h are at or near the ends of the long and short arms. Abnormalities of c h r o m o s o m e 13 were noted in 30% and abnormalities of chromosome 14 were noted in 70% of the cases studied. "Hotspots" of activity in these two c h r o m o s o m e s were clustered a r o u n d the p 1 1 - p 1 3 region of the chromosomes, close to the centromeres. The breakpoints were not the same in all of the rearrangements. However, patients 6 and 9 had abnormalities of chromosome 13 at the p l l region and patients 2, 5, 6, and 9 all had abnormalities of c h r o m o s o m e 14 involving the p l l region. Structural abnormalities were not the only changes seen. Pulverized c h r o m o s o m e s and d m i n were seen in all of the carcinomas analyzed, though not in all cells. Patient 10 was unique in having only one structural c h r o m o s o m e abnormality, an inv(14)(q13q23) (Fig. 2). Two biopsies obtained 2 weeks apart gave similar results, with the same inversion. The c h r o m o s o m a l l y abnormal metaphases were p s e u d o d i p loid, containing 46 c h r o m o s o m e s with DMs. DISCUSSION
It is very difficult to differentiate between primary aberrations (pathogenetically essential changes frequently found as the sole abnormality in a particular cancer), and secondary aberrations (changes that occur in a d d i t i o n to the p r i m a r y abnormality,
184 Table 2
A.-L. Z a s l a v et al
Cytogenetic data of investigated cases
% Abn cells with dmin
Range of chrom. number in abn cells (range)
84
18.5
60-100
59
36
33
50-70
87.5 5.4
12.5 94.6
4
50
50
50
75-100
6
39
46
54
52
50-100
7 8
17 42
65 69
35 31
83 100
50-70 67
9
17
42
53
53
100
10A a 10B
12 17
42 47
58 53
71 78
46 46
Number cells examined
% Norm cells
1
32
16
2
22
3 4
8 37
5
Case
% Abn cells
5.7
86 40-50
Clonal anomalies + X, - 3,del(4)(p14), + 5 , - 7 , del(8)(q24),der(12)t(12;?) (p13;?), + 11, + 11,der(11) (p13;q21) + 12, + 13, + 14, +15,+19 + 1, + 1,del(4)(p11),der(5)t(5;?) (q35;?), + del(8)(q22), der(13)t(13;?)(p13;?), der(13)t(13;?),(p13;?), + der(14)t(14;?)(p13;?), der(16)t(16;?)(q24;?), + der(16)t(16;?)(q24;?), + 17,+ 1 7 , + 1 7 , + 2 0 Too poor to interpret. del(3)(p11),del(6)(q12), der(7)t(7;?)(p22;?), der(11)t(11;?)(p15;?), der(12)t(12;?),der(14)t(14;?) (q24;?),der(15)t(15;15) (p11;p11),del(16)(q22), der(21)t(21;21)(p11;p11), der(21)t(21;21)(p11;p11), der(22)t(22;?)(q13;?) der(14)t(14;14)(p11;p11), der(5)t(5;?)(p15;?),del(5)(p13), der(6)t(6;?)(q23;?),i(11q), der(11)t(11;?)(q23;?), der(11)t(11;?)(q25;?) del(8)(q21),del(8)(q24),t(9;11) (p22;p13),t(13;14)(p11;p11) Too poor to interpret 12/13 abnormal cells contained only d m i n and pulverized chromosomes del(X)(p11),del(5)(p31), der(11)t(1;11)(p32;p15), der(11)t(1;11)(p32;p15), der(13)t(13;14)(p11;p11), der(13)t(12;?), + 20 inv(14)(q13q23) inv(14)(q13q23)
a Patient 12 biopsied twice, two weeks apart. Abbreviations: Norm: normal, Abn: abnormal, Chrom: chromosome, dmin: double minutes, del: deletion, der: derivative t: translocation, inv: inversion, i: isochromosome.
185
Carcinomas of the Head and Neck
CLONAL ABNORMALITIES
10
IN HEAD AND NECK CARCINOMAS
2
3
4
5
6
7
8
9
11
12
13
14
15
16
17
lS
X "~ANSLOCATION
ee
oe
19
20
• • -o
•
21
22
Y
iNVERSION Wtmle C ~ DUFIACATION V~ole C k m m o ~ DELETION ISO~HI~)MOCaOME
X
Figure I Idiogram showing clonal anomalies in the head and neck carcinomas. The inversion on chromosome 14 refers to the sole abnormality seen in patient 10 (inv(14)(p13q23)). rarely found as solitary changes in a particular cancer as described by Heim and Mitelman [9]). W h e n the same abnormality is observed in more than two cells in a single carcinoma it is considered a clone and is then considered to be a n o n r a n d o m aberration. W h e n the same abnormality is observed in other carcinomas from the same anatomic locations, then the abnormality is considered to be associated with that particular type of carcinoma. W h e n a sole abnormality is observed in a particular tumor and is seen in other tumors of the same type it is considered a primary aberration. Based on these distinctions, we conclude that abnormalities of chromosomes 11, 13, and 14 are n o n r a n d o m and may be involved in the primary tumorigenesis of head and neck carcinomas. In a report by Jin et al. [1] of a squamous cell carcinoma of the larynx, the only n o n r a n d o m abnormality similar to the ones in our study was an aberration involving the q23 region of chromosome 11 [10]. In a study by Wennerberg et al. [5] reported at a recent conference on head and neck oncology, abnormalities in 9 of 30 tumors were found to be clustered in the q13 region of chromosome 11. The abnormalities seen in m a n y tumors in our study were clustered around the p13 and q23 regions of chromosome 11. It is possible that these regions of chromosome 11 are involved in the primary
14
i n v (14) (q13q23)
Figure Z G-bandedpartialkaryotypeof a metaphase from Patient 10 showing the inverted chromosome 14,inv(14)(q13q23) on the right.
186
A.-L. Zaslav et al.
tumorigenesis of head and neck carcinomas. Interestingly, similar abnormalities of chromosome 11 were found in approximately one third of the bladder carcinomas investigated [11, 12]. These aberrations were deletions of the short arm of the chromosome, where the 11p15 region was missing. In the head and neck carcinomas investigated in our study m a n y of the structural aberrations involving chromosome 11 also clustered around the 11p15 region of the chromosome. To our knowledge structural aberrations of chromosome 13 or 14 have not often been reported in carcinomas. In a report by Jin et al. [4] only one of five squamous cell carcinomas of the larynx had an abnormality involving band 13p11, with a translocation between chromosomes 1 and 13: der(13)t(1;13)(p11;p11). In our specimens, aberrations were clustered in 1 3 p l l and 14p11, the centromeric regions of both chromosomes. In general, the centromeric regions of chromosomes break and recombine readily, which makes this region more susceptible to translocation events. Antioncogenes and oncogenes have been associated with chromosomes 11 and 14. The c-ets-1 oncogene has been mapped to the 11q23 region [10] and H-ras is located at 11p15 [13] the major chromosome breakpoints seen in our tumors. Chromosome 11 as well as 14 have also been implicated in tumor suppression. In fusion studies of malignant HeLa cells with normal h u m a n fibroblasts, the loss of one copy of either chromosomes 11 or 14 was associated with reexpression of tumor-forming ability [141. The chromosomal analysis of early outgrowth of solid tumors is a formidable task, but one of the first steps in determining which chromosome(s) and thus which gene(s) are involved in the pathogenesis of the disease in question. We have applied this approach to the study of the pathogenesis of head and neck carcinomas. This research was carried out in partial fulfillment of the requirements of a Ph.D. degree from St. John's University, Jamaica, N.Y. This research was supported in part by grant 5 PO1 DC00203 from the National Institute on Deafness and other Communication Disorders, and by a grant from the Morris S. and Florence H. Bender Foundation Inc., New York. We would like to acknowledge Teresa DiLorenzo for her assistance throughout this project. REFERENCES 1. Jin YSM, Mandahl N, Heim S, Biorklund A, Wennerberg J, Mitelman F (1988A): Unique karyotypic abnormalities in a squamous cell carcinoma of the larynx, Cancer Genet Cytogenet 30:177-179. 2. Jin Y, Heims S, Mandahl N, Biorklund A, Wennerberg J, Mitelman F (1988B): Inversion inv(4)(p15q26) in a squamous cell carcinoma of the hypopharynx. Cancer Genet Cytogenet 36:233-234. 3. Jin Y, Mandahl N, Heim S, Biorklund A, Wennerberg J, Mitelman F (1988C): t(6;7)(q23;p22) as the sole chromosomal anomaly in a vocal cord carcinoma. Cancer Genet Cytogenet 32:305-307. 4. Jin Y, Heim S, Mandahl N, Biorklund A, Wennergerg J, Mitelman F (1990): Multiple clonal chromosome aberrations in squamous cell carcinomas of the larynx. Cancer Genet Cytogenet 44:209-216. 5. Wennerberg J, Helm S, Jin Y, Higashi K, Mandahl N, Biorklund A, Mitelman F (1990): Rearrangements involving chromosome bands lp22 and 11q13 in squamous cell carcinomas of the head and neck. Third International Head and Neck Oncology Research Conference: 9.2 (Abstract). 6. Steinberg BM, Abramson AL, Meade RP (1982): Culture of human laryngeal papilloma cells in vitro. Otolaryn Head Neck Surg 1982; 90;728-735. 7. Seabright M (1971): A rapid banding technique for human chromosomes. Lancet 2:971-972. 8. American Joint Committee for Cancer Staging and End-Result Reporting, Manual for Staging (1978). Chicago, IL.
C a r c i n o m a s of t h e H e a d and N e c k
18 7
9. Heim S, Mitelman F (1987): Cancer Cytogenetics. Liss, New York. 10. Watson DK, Sacchi N, McWilliams-Smith MJ, O'Brien SJ, Papas TS (1986): The avian and mammalian ets genes: Molecular characterization, chromosome mapping, and implication in human leukemia. Anticancer Res 6:631-636. 11. Gibas Z, Prout GR, Pontes JE, Connolly JG, Sandberg AA (1984): Nonrandom chromosomal changes in transitional cell carcinoma of the bladder. Cancer Res 44:1257-1264. 12. Gibas Z, Prout GR, Pontes JE, Connolly JG, Sandberg AA (1986): A possible specific chromosome change in transitional cell carcinoma of the bladder. Cancer Genet Cytogenet 19:229-238. 13. Ryan J, Barker PE, Shimizu K, Wigler M, Ruddle FH (1983): Chromosomal assignment of a family of human oncogenes. Proc Natl Acad Sci USA 80:4460-4463. 14. Stanbridge EJ, Flandermeyer RR, Daniels DW, Nelson-Rees WA (1981): Specific chromosome loss associated with the expression of tumorigenicity in human cell hybrids. Somatic Cell Genet 7:699-712.
ABSTRACT: Ten primary squamous cell carcinomas (SCC) of the head and neck were evaluated cytogenetically after 10-14 days of in vitro culture. Addition of 3% L-glutamine was essential for consistent epithelial growth of these carcinomas. Outgrowth of cells from tissue explants contained a mixture of chromosomally normal and abnormal cells; the abnormal cells had extensive changes including translocations, marker chromosomes, inversions, deletions, and duplications. In addition, all carcinomas contained cells with pulverization and double minute chromosomes (dmin). Chromosomes 11, 13, and 14 had "'hotspots" of rearrangements.
INTRODUCTION Acquired chromosome abnormalities are significant in the pathogenesis and treatment of various tumors. However, data on m a n y types of solid tumors are still scant, due to technical difficulties with their growth in culture and with their chromosomal evaluation. Little is k n o w n about the chromosome abnormalities occurring in primary head and neck tumors. A few recent reports describing the abnormalities of these tumors have been published [1-5], but most describe i n d i v i d u a l cases. The study presented here is an evaluation of a consecutive series of 10 solid primary malignant tumors of the head and neck region, in an attempt to u n d e r s t a n d the primary chromosomal events in head and neck carcinogenesis. All had abnormal metaphase spreads, with abnormal chromosomes and double m i n u t e s (dmin). Of these, seven had chromosomes of sufficient quality for karyotyping. Three could not be further analyzed. Two additional patients were eliminated from the study because only fibroblasts grew from their tumors.
MATERIALS AND METHODS Cells were cultured as previously described [6]. All specimens were washed 3 times in phosphate-buffered saline, m i n c e d with a sharp sterile scissors, placed in a 60-mm petri dish, and embedded i n 0.15 mg/ml collagen gel (Type I Collagen, Vitrogen Collagen Corp, CA) containing F12 m e d i u m , s u p p l e m e n t e d with 3% L-glutamine, Fromthe Departmentof BiologicalSciences,St. John's University,Jamaica, NY (A.-L.Z., Y. J. L.);Division of Human Genetics, Schneider Children's Hospital, Long Island Jewish Medical Center (A.-L. Z., J. S.); Department of Otolaryngology(B. M. S., A. A.), Long Island Jewish Medical Center, New Hyde Park, NY. Address reprint requests to: Dr. Ann-Leslie Zaslav, Division of Human Genetics, North Shore University Hospital, 300 Community Drive, Manhasset, N Y 11030. Received December 17, 1990; accepted April 2, 1991.
181 © 1991 ElsevierSciencePublishingCo., Inc. 655 Avenueof the Americas,New York, NY 10010
Cancer Genet Cytogenet56:181-187 (1991) 0165-4608/91/$03.50
182
A.-L. Zaslav et al.
10% fetal calf serum, 1% penicillin-streptomycin, 0.5% gentamycin (Grand Island Biologics, NY), and 0.1% hydrocortisone (Sigma, St. Louis, MO). Specimens were cultured in a 5% CO 2 incubator and fed twice weekly with the above m e d i u m . W h e n the cells showed sufficient growth (between 10 and 14 days after initiation), they were harvested. Velban (Vinblastine sulfate, Lily, IN), (2.5% in F12 medium), was used as a mitotic spindle poison. After 1 hour of treatment, m e d i u m + velban were removed from the petri dish and collagenase (Sigma type IV, 2 mg/ml), was added for 1 hour to disrupt the collagen matrix. From this point the harvest followed standard cytogenetic techniques, i n c l u d i n g trypsinization, hypotonic solution, fixation, and trypsin-Giemsa b a n d i n g [7]. Four clinically normal biopsy specimens were used as controls: two pharyngeal biopsies of " n o r m a l " tissue outside the margins of carcinomas, one right vocal cord biopsy from a 3-year-old female with subglottic stenosis, and a right vocal cord biopsy from a 37-year-old female with bilateral vocal cord paralysis. Slides were scanned for well-spread analyzable metaphases. Microscopic analysis involved a detailed evaluation of metaphase spreads, looking for such changes as deletions, inversions, translocations, duplications, double minute chromosomes, homogeneous staining regions (HSRs), and abnormal banding regions (ABRs). The number oi chromosomes per cell was also counted to look for deviations from the normal number of 46. In many cases more than 20 cells were analyzed, and five or more karyotyped.
RESULTS The clinical descriptions of the patients and histologic descriptions of the tumor biopsies are shown in Table 1. All patients were diagnosed by a pathologist as having poorly to extensively differentiated carcinomas. Tumors were outlined and staged Table 1 Case number
Clinical history of carcinoma patients Sex
Age
Site
Stage
1
F
66
Maxillary Sinus
III
2
M
71
Tonsil
IV
3
M
42
Larynx
IV
4
M
70
Larynx
IV
5
F
84
Larynx
III
6
M
69
Vocal cord
III
7
M
58
III
8 9
M M
56 60
Base Tongue Larynx Larynx
10A
F
65
Larynx
III
lOB °
F
65
Larynx
III
° Second biopsy from patient 10, taken 2 weeks later. Abbreviations:ca: carcinoma.F: female, M: male.
III lI
Histology Invasive, poorly differentiated sqnamons ca Infiltrating squamous ca Infiltrating sqnamons ca Infiltrating squamous ca Infiltrating sqnamous ca Poorly differentiated squamous ca Invasive squamous ca Squamous ca Infiltrating sqnamous ca Infiltrating squamous ca Infiltrating sqnamous ca
Carcinomas of the Head and Neck
183
according to anatomical sites involved, local extension, nodal involvement, and distance from metastases [8]. All patients were s y m p t o m a t i c several months prior to surgery. Ages of the patients ranged from 5 6 - 8 4 years, with the exception of patient 3, who was 42. Again with the exception of patient 3, all patients smoked and most were heavy alcohol drinkers. Patient 3 did not present with m a n y of the features of the rest of the patients in this study. This patient was younger than the rest and was diagnosed with ulcerative colitis at age 33, w h i c h eventually necessitated a colectomy in 1980. He had no family history of colitis or cancer. It is possible that his disease could be of a different etiology from the rest of the patients in the study, but the c h r o m o s o m a l abnormalities were like those seen in most of the patients. None of the patients investigated received any drug, chemotherapy, or r a d i o t h e r a p y prior to surgery, w h e n the biopsies were done. The head and neck carcinomas had a decreased proportion of d i v i d i n g cells compared to the normal control specimens. In addition, the overall quality of the metaphases from the carcinomas was poorer than that found in the controls. All cells analyzed from the control samples had normal karyotypes. The cells derived from all of the ten carcinomas were a mixture of epithelial cells and fibroblasts and a mixture of both normal and abnormal metaphases. There was no correlation between stage of disease and c h r o m o s o m e number. For example, Patient 4 had c h r o m o s o m e abnormalities in 94.6% of the cells analyzed, but the m o d a l chromosome n u m b e r in the abnormal cells was 46 (pseudodiploid). W i t h Patient 5, 50% of the metaphases analyzed were c h r o m o s o m a l l y abnormal and the m o d a l c h r o m o s o m e n u m b e r in the abnormal cells was a p p r o x i m a t e l y 75. Table 2 shows the range of c h r o m o s o m e number in abnormal cells. Most of the abnormal cells contained m u l t i p l e rearrangements, deletions, translocations, inversions, and marker chromosomes. Stage of disease did not appear to be correlated with structural aberrations, because c h r o m o s o m e abnormalities were observed in all stages. Chromosome abnormalities among the cases were therefore c o m p a r e d i n d e p e n d e n t of stage of disease. " H o t s p o t s " (specific c h r o m o s o m e s and regions of those chromosomes where abnormalities cluster) of rearrangement in these carcinomas were found on c h r o m o s o m e s 11, 13, and 14, with translocations being the most frequent types of structural abnormality. Figure 1 is an idiogram illustrating the clonal anomalies that occurred in the investigated cases. Abnormalities of c h r o m o s o m e 11 occurred in 50% of the cases studied. The breakpoints of c h r o m o s o m e 11 clustered in two areas, 11p15 and 11q23, w h i c h are at or near the ends of the long and short arms. Abnormalities of c h r o m o s o m e 13 were noted in 30% and abnormalities of chromosome 14 were noted in 70% of the cases studied. "Hotspots" of activity in these two c h r o m o s o m e s were clustered a r o u n d the p 1 1 - p 1 3 region of the chromosomes, close to the centromeres. The breakpoints were not the same in all of the rearrangements. However, patients 6 and 9 had abnormalities of chromosome 13 at the p l l region and patients 2, 5, 6, and 9 all had abnormalities of c h r o m o s o m e 14 involving the p l l region. Structural abnormalities were not the only changes seen. Pulverized c h r o m o s o m e s and d m i n were seen in all of the carcinomas analyzed, though not in all cells. Patient 10 was unique in having only one structural c h r o m o s o m e abnormality, an inv(14)(q13q23) (Fig. 2). Two biopsies obtained 2 weeks apart gave similar results, with the same inversion. The c h r o m o s o m a l l y abnormal metaphases were p s e u d o d i p loid, containing 46 c h r o m o s o m e s with DMs. DISCUSSION
It is very difficult to differentiate between primary aberrations (pathogenetically essential changes frequently found as the sole abnormality in a particular cancer), and secondary aberrations (changes that occur in a d d i t i o n to the p r i m a r y abnormality,
184 Table 2
A.-L. Z a s l a v et al
Cytogenetic data of investigated cases
% Abn cells with dmin
Range of chrom. number in abn cells (range)
84
18.5
60-100
59
36
33
50-70
87.5 5.4
12.5 94.6
4
50
50
50
75-100
6
39
46
54
52
50-100
7 8
17 42
65 69
35 31
83 100
50-70 67
9
17
42
53
53
100
10A a 10B
12 17
42 47
58 53
71 78
46 46
Number cells examined
% Norm cells
1
32
16
2
22
3 4
8 37
5
Case
% Abn cells
5.7
86 40-50
Clonal anomalies + X, - 3,del(4)(p14), + 5 , - 7 , del(8)(q24),der(12)t(12;?) (p13;?), + 11, + 11,der(11) (p13;q21) + 12, + 13, + 14, +15,+19 + 1, + 1,del(4)(p11),der(5)t(5;?) (q35;?), + del(8)(q22), der(13)t(13;?)(p13;?), der(13)t(13;?),(p13;?), + der(14)t(14;?)(p13;?), der(16)t(16;?)(q24;?), + der(16)t(16;?)(q24;?), + 17,+ 1 7 , + 1 7 , + 2 0 Too poor to interpret. del(3)(p11),del(6)(q12), der(7)t(7;?)(p22;?), der(11)t(11;?)(p15;?), der(12)t(12;?),der(14)t(14;?) (q24;?),der(15)t(15;15) (p11;p11),del(16)(q22), der(21)t(21;21)(p11;p11), der(21)t(21;21)(p11;p11), der(22)t(22;?)(q13;?) der(14)t(14;14)(p11;p11), der(5)t(5;?)(p15;?),del(5)(p13), der(6)t(6;?)(q23;?),i(11q), der(11)t(11;?)(q23;?), der(11)t(11;?)(q25;?) del(8)(q21),del(8)(q24),t(9;11) (p22;p13),t(13;14)(p11;p11) Too poor to interpret 12/13 abnormal cells contained only d m i n and pulverized chromosomes del(X)(p11),del(5)(p31), der(11)t(1;11)(p32;p15), der(11)t(1;11)(p32;p15), der(13)t(13;14)(p11;p11), der(13)t(12;?), + 20 inv(14)(q13q23) inv(14)(q13q23)
a Patient 12 biopsied twice, two weeks apart. Abbreviations: Norm: normal, Abn: abnormal, Chrom: chromosome, dmin: double minutes, del: deletion, der: derivative t: translocation, inv: inversion, i: isochromosome.
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Carcinomas of the Head and Neck
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Figure I Idiogram showing clonal anomalies in the head and neck carcinomas. The inversion on chromosome 14 refers to the sole abnormality seen in patient 10 (inv(14)(p13q23)). rarely found as solitary changes in a particular cancer as described by Heim and Mitelman [9]). W h e n the same abnormality is observed in more than two cells in a single carcinoma it is considered a clone and is then considered to be a n o n r a n d o m aberration. W h e n the same abnormality is observed in other carcinomas from the same anatomic locations, then the abnormality is considered to be associated with that particular type of carcinoma. W h e n a sole abnormality is observed in a particular tumor and is seen in other tumors of the same type it is considered a primary aberration. Based on these distinctions, we conclude that abnormalities of chromosomes 11, 13, and 14 are n o n r a n d o m and may be involved in the primary tumorigenesis of head and neck carcinomas. In a report by Jin et al. [1] of a squamous cell carcinoma of the larynx, the only n o n r a n d o m abnormality similar to the ones in our study was an aberration involving the q23 region of chromosome 11 [10]. In a study by Wennerberg et al. [5] reported at a recent conference on head and neck oncology, abnormalities in 9 of 30 tumors were found to be clustered in the q13 region of chromosome 11. The abnormalities seen in m a n y tumors in our study were clustered around the p13 and q23 regions of chromosome 11. It is possible that these regions of chromosome 11 are involved in the primary
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Figure Z G-bandedpartialkaryotypeof a metaphase from Patient 10 showing the inverted chromosome 14,inv(14)(q13q23) on the right.
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A.-L. Zaslav et al.
tumorigenesis of head and neck carcinomas. Interestingly, similar abnormalities of chromosome 11 were found in approximately one third of the bladder carcinomas investigated [11, 12]. These aberrations were deletions of the short arm of the chromosome, where the 11p15 region was missing. In the head and neck carcinomas investigated in our study m a n y of the structural aberrations involving chromosome 11 also clustered around the 11p15 region of the chromosome. To our knowledge structural aberrations of chromosome 13 or 14 have not often been reported in carcinomas. In a report by Jin et al. [4] only one of five squamous cell carcinomas of the larynx had an abnormality involving band 13p11, with a translocation between chromosomes 1 and 13: der(13)t(1;13)(p11;p11). In our specimens, aberrations were clustered in 1 3 p l l and 14p11, the centromeric regions of both chromosomes. In general, the centromeric regions of chromosomes break and recombine readily, which makes this region more susceptible to translocation events. Antioncogenes and oncogenes have been associated with chromosomes 11 and 14. The c-ets-1 oncogene has been mapped to the 11q23 region [10] and H-ras is located at 11p15 [13] the major chromosome breakpoints seen in our tumors. Chromosome 11 as well as 14 have also been implicated in tumor suppression. In fusion studies of malignant HeLa cells with normal h u m a n fibroblasts, the loss of one copy of either chromosomes 11 or 14 was associated with reexpression of tumor-forming ability [141. The chromosomal analysis of early outgrowth of solid tumors is a formidable task, but one of the first steps in determining which chromosome(s) and thus which gene(s) are involved in the pathogenesis of the disease in question. We have applied this approach to the study of the pathogenesis of head and neck carcinomas. This research was carried out in partial fulfillment of the requirements of a Ph.D. degree from St. John's University, Jamaica, N.Y. This research was supported in part by grant 5 PO1 DC00203 from the National Institute on Deafness and other Communication Disorders, and by a grant from the Morris S. and Florence H. Bender Foundation Inc., New York. We would like to acknowledge Teresa DiLorenzo for her assistance throughout this project. REFERENCES 1. Jin YSM, Mandahl N, Heim S, Biorklund A, Wennerberg J, Mitelman F (1988A): Unique karyotypic abnormalities in a squamous cell carcinoma of the larynx, Cancer Genet Cytogenet 30:177-179. 2. Jin Y, Heims S, Mandahl N, Biorklund A, Wennerberg J, Mitelman F (1988B): Inversion inv(4)(p15q26) in a squamous cell carcinoma of the hypopharynx. Cancer Genet Cytogenet 36:233-234. 3. Jin Y, Mandahl N, Heim S, Biorklund A, Wennerberg J, Mitelman F (1988C): t(6;7)(q23;p22) as the sole chromosomal anomaly in a vocal cord carcinoma. Cancer Genet Cytogenet 32:305-307. 4. Jin Y, Heim S, Mandahl N, Biorklund A, Wennergerg J, Mitelman F (1990): Multiple clonal chromosome aberrations in squamous cell carcinomas of the larynx. Cancer Genet Cytogenet 44:209-216. 5. Wennerberg J, Helm S, Jin Y, Higashi K, Mandahl N, Biorklund A, Mitelman F (1990): Rearrangements involving chromosome bands lp22 and 11q13 in squamous cell carcinomas of the head and neck. Third International Head and Neck Oncology Research Conference: 9.2 (Abstract). 6. Steinberg BM, Abramson AL, Meade RP (1982): Culture of human laryngeal papilloma cells in vitro. Otolaryn Head Neck Surg 1982; 90;728-735. 7. Seabright M (1971): A rapid banding technique for human chromosomes. Lancet 2:971-972. 8. American Joint Committee for Cancer Staging and End-Result Reporting, Manual for Staging (1978). Chicago, IL.
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9. Heim S, Mitelman F (1987): Cancer Cytogenetics. Liss, New York. 10. Watson DK, Sacchi N, McWilliams-Smith MJ, O'Brien SJ, Papas TS (1986): The avian and mammalian ets genes: Molecular characterization, chromosome mapping, and implication in human leukemia. Anticancer Res 6:631-636. 11. Gibas Z, Prout GR, Pontes JE, Connolly JG, Sandberg AA (1984): Nonrandom chromosomal changes in transitional cell carcinoma of the bladder. Cancer Res 44:1257-1264. 12. Gibas Z, Prout GR, Pontes JE, Connolly JG, Sandberg AA (1986): A possible specific chromosome change in transitional cell carcinoma of the bladder. Cancer Genet Cytogenet 19:229-238. 13. Ryan J, Barker PE, Shimizu K, Wigler M, Ruddle FH (1983): Chromosomal assignment of a family of human oncogenes. Proc Natl Acad Sci USA 80:4460-4463. 14. Stanbridge EJ, Flandermeyer RR, Daniels DW, Nelson-Rees WA (1981): Specific chromosome loss associated with the expression of tumorigenicity in human cell hybrids. Somatic Cell Genet 7:699-712.
