Tumor-Infiltrating Lymphocytes in Kidney Tumors and in the Surrounding Kidney Tissue PAOLA DAL CIN*, MAGDY S

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Proc. Natd. Acad. Sci. USA Vol. 89, pp. 9744-9748, October 1992 Genetics Trisomy 7 and trisomy 10 characterize subpopulations of tumor-infiltrating lymphocytes in kidney tumors and in the surrounding kidney tissue PAOLA DAL CIN*, MAGDY S. ALY*, JAN DELABIEt, JAN L. CEUPPENSt, STEFAAN VAN GOOLt, BOUDEWIJN VAN DAMMEt, Luc BAERT§, HEIN VAN POPPEL§, AND HERMAN VAN DEN BERGHE*¶ *Center for Human Genetics and Departments of tPathology, tPathophysiology, and fUrology, University of Leuven, B-3000 Leuven, Belgium Communicated by Peter C. Nowell, June 15, 1992

ABSTRACT We performed conventional cytogenetic anal- surrounding kidney tissue and to identify the cells carrying ysis and fluorescence in situ hybridization in short-term cul- this abnormality. FISH was used to complement CCA and in tures of normal and neoplastic kidney tissues. Cell populations situ hybridization (ISH) was also performed on frozen sec- carrying an extra chromosome 7 or an extra chromosome 10 as tions of kidney tissues. the only chromosome change could be identified in kidney tumors, mostly renal cell carcinomas, and in the surrounding MATERIALS AND kidney tissue, but not in nonneoplastic kidneys. To identify the METHODS type ofcells displaying these aneuploidies, we performed in situ Kidney Tissues. Tumor samples and a sample of nonma- hybridization (ISH) with probes specific for the centromeric lignant kidney tissue surrounding the resected tumor and region of chromosomes 7 and 10 on frozen kidney tissue microscopically free oftumor were obtained from 17 patients sections. Trisomy 7 and trisomy 10 were restricted to infiltrat- during surgery and were used for pathology and for short- ing inflammatory cells in the tumor as well as in the surround- term tissue culture for cytogenetic investigation (Table 1). ing tissue. Trisomy 7 and trisomy 10 were also found in Fresh tumor and surrounding kidney tissue from an addi- subpopulations of peripheral blood T cells of cancer patients tional 8 patients with RCC (Table 2) were used for isolation and of normal individuals, as well as in the thymus of five of viable lymphocytes. normal fetuses (21-29 weeks), but not in noninvaded reactive CCA on Short-Term Cultures ofKidney Tissue. Cytogenetic lymph node sections of patients without malicy. When analysis was performed on G-banded metaphase cells ob- lymphocytes were enriched from kidney tumors and surround- tained from short-term cultures (4-6 days) of overnight ing tissue by either Ficoll/Hypaque density gradient or immu- collagenase disaggregated malignant and nonmalignant kid- nomagnetic selection with anti-CD3, anti-CD4, or anti-CD8 ney tissue, according to the procedures described (8). Over monoclonal antibodies, it was confirmed that they contained a 100G-banded metaphases from each ofthe nonneoplastic and high percentage of trisomy 7 and trisomy 10 cells. Further normal kidney tissue preparations were analyzed. proof for T-lymphocyte origin of the trisomy 7 and trisomy 10 ISH in Short-Term Kidney Cell Cultures. FISH was per- cells was obtained by simultaneous stining of lymphocytes formed on unbanded slides of each kidney tissue sample and isolated from tumor tissue with anti-CD3, anti-CD4, and on conventional G-banded slides from short-term kidney anti-CD8 monoclonal antibodies and ISH. We condude that tumor cultures after destaining with fixative (methanol/ trbisomy 7 and trisomy 10, found in renal carcinomas and acetic acid; 3:1; vol/vol). Probe p7tl (9) and probe DiOZi surrounding kidney tissue, characterize subpopulations of tu- (10), specific for the centromeric region ofchromosome 7 and mor-infiltrating lymphocytes. The biologic sifcance of this of chromosome 10, respectively, were labeled by nick- phenomenon is unknown and further translation with bio-16-dUTP (Boehringer Mannheim). As a requires investigation. control to exclude triploidy in the short-term tissue cultures, probe pBR-12 (11), specific for the centromeric region of Trisomy 7 as the only chromosome change is a frequent chromosome 12, was labeled with digoxigenin ll-dUTP chromosome abnormality in renal cell carcinomas (for re- (Boehringer Mannheim) using the previously described con- view, see ref. 1), but it has also been found in other malignant ditions. Hybridization and subsequent detection were per- and nonmalignant tumors (for review, see ref. 2). It is less formed according to Speleman et al. (12). Three strong well frequently observed in lymphomas and is very infrequently delineated individualized signals in an interphase nucleus observed in leukemias. The finding of trisomy 7 in nonneo- after hybridization with the probes p7tl and DiOZi were plastic tissues from patients with lung, kidney, or brain tumor assumed to reflect the presence of three copies of chromo- by conventional cytogenetic analysis (CCA) on short-term somes 7 and 10, respectively. Unbanded slides from different cultures raised the question whether these cells carrying an types of short-term tissue cultures, such as fetal skin, amni- extra chromosome 7 reflect the presence of (pre)malignant otic fluid, chorionic villi, and phytohemagglutinin-stimulated cells, a culture-induced artefact, or, perhaps, an in vivo organ lymphocytes, used as normal controls, as well as cells from mosaicism (3-6). a week 13 miscarriage showing trisomy 7 as a constitutional Emanuel et al. (7) showed the presence of trisomy 7 and abnormality, were simultaneously hybridized with the same trisomy 10 by fluorescence in situ hybridization (FISH) in probes. Simultaneous double ISH experiments and bicolor normal kidney tissue of patients with renal cell carcinoma detection were performed according to the procedure out- (RCC) prior to short-term culture, confirming previous find- lined by Arnoldus et al. (13) with the following combinations: ings by Elfving et al. (6). p7tl (biotin)/DlOZ1 (digoxigenin), p7tl (biotin)/pBR12 The present study was undertaken to further analyze the (digoxigenin). occurrence of trisomy 7 and trisomy 10 in renal tumors and Abbreviations: mAb, monoclonal antibody; FISH, fluorescence in The publication costs of this article were defrayed in part by page charge situ hybridization; CCA, conventional cytogenetic analysis; ISH, in payment. This article must therefore be hereby marked "advertisement" situ hybridization; RCC, renal cell carcinoma. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 9744 Downloaded by guest on September 25, 2021 Genetics: Dal Cin et al. Proc. Natl. Acad. Sci. USA 89 (1992) 9745

Table 1. Data from 17 kidney tumors and their surrounding tissues analyzed karyotypically and by FISH to determine the presence of trisomy 7 and 10 in kidney tissue Tumor Surrounding kidney tissue Age, % trisomy 7 % trisomy 10 % Trisomy 7 % Trisomy 10 % Trisomy 7 % Trisomy 10 Patient* yr/sex Karyotype [no. of cells] by FISHt by FISHt by CCAt by CCAt by FISHt by FISHt 1 68/M 46,XY/45,X,-Y [20/4] 14 2.5 14.8 1.8 10.5 3 2 78/M 46,XY/46,X,-Y [80/8] 2 2.5 15.6 0.8 15.5 2 3 74/M Failure 10.5 1.8 10 4 70/M Failure 14 1 14 5 65/M Near triploid, -Y, 3q-, der(3)t(3;5)(p14;q23), ?i(12p), other clonal abnormalities [10] 2 12.7 2.9 11.5 6 65/F 44,XX,der(3)t(3;5)(p14;q25), 8p-, inv(9)(p21q12), -14, -14, -15, +t(14qlSq) [10] 4 8.5 0.9 9 7 58/M 45,X,-Y/46,XY [5/1] 8 8 2 9.5 8 67/M Failure - - 12.1 3.4 14 9 70/M Failure - - 11.4 4.3 10.5 10 69/M 45,X,-Y/46,X,-Y,+7/46,XY [19/2/3] 12 4 12 3 16 3.5 11 62/M 46,XY/45,X,-Y [17/1] 10 17.3 3.8 13.5 12 17/M 46,XY [15] 5.5 3.5 17.1 4.7 12 3 13 53/M 46,XY [15] 13 14.4 15.5 14 68/M 46,XY/45,XY, -3,der(6)t(3;6)(q21;q27) [3/3] 2.5 12.9 3.7 12 15 83/F Failure 12.6 3.3 11.5 16 64/M 46,XY/45,X,-Y/47,XY,+7 [14/1/1] 9 2 10.7 2.6 9.5 2 17 50/F 46,XX [10] 7 9.5 2.8 8.5 *RCCs (varying from grade I to IV) except for patients 11 (angioleiomyoma) and 12 (Ki 1 lymphoma). tData are expressed as percentage of nuclei with three positive signals (counted on 200 interphase nuclei). tData are expressed as percentage of metaphases with trisomy 7 (counted on 100 metaphases). ISH in Frozen Kidney Tissue Sections. Patients were se- Serial 5-,tm sections were cut onto glass slides coated with lected in whom both normal and neoplastic kidney tissues aminopropyltriethoxysilane (Sigma), fixed in 4% paraformal- were available and for which the presence of trisomy 7 and dehyde, incubated in 0.2 M HCO solution, and dehydrated in trisomy 10 had been shown by CCA and FISH. As controls, an alcohol series. These slides were used for ISH with the kidney biopsies of 10 patients with functional kidney disease same biotin-labeled probes p7tl and DiOZ1 as outlined above and 3 reactive lymph node biopsies of patients without with the exception of immunohistochemical detection utiliz- tumors were used. Biopsies from fetal thymus, liver, and ing alkaline phosphatase-conjugated streptavidin-biotin kidney were analyzed in a similar fashion. The samples were complex (DAKO, Glostrup, Denmark) visualized with frozen in nitrogen-cooled isopentane and kept at -70°C until 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetra- used. zolium (Sigma).

Table 2. Trisomy 7 in leukocytes isolated from kidney tumors Tissue used % trisomy 7 h o i p % trisomy 7 for TIL in total Characteristics of isolated population in reciprocal Patient enrichment tissue Isolation method % CD45+ % T cells % monocytes % trisomy 7 population 18 Tumor 7 F/H gradient 74 44 (CD3+) 19 25 NA 19 Tumor ND F/H gradient 92 74 (CD3+) 14 16 NA 20 Tumor ND F/H gradient 87 74 (CD3+) 16 25 NA 21 Tumor ND Anti-CD3 and Dynabeads ND ND ND 78 4 22 Tumor ND Anti-CD3 and Dynabeads 68 60 (CD2+) 0 54 ND Surrounding tissue ND Anti-CD3 and Dynabeads 85 65 (CD2+) 20 60 ND 23 Tumor* ND Anti-CD3 and Dynabeads 85 68 (CD3+) 12 55 7 24 Tumor* 7 Anti-CD4-coated Dynabeads ND ND ND 57 6 25 Surrounding tissue 15 Anti-CD4-coated Dynabeads ND 83 (CD3+) <2 55 6 26 Tumor ND F/H gradient % 66 (CD3+) 10 35 NA Tumor Anti-CD4-coated Dynabeads ND ND ND 52 6 Surrounding tissue ND F/H gradient 39 ND 9 30 NA 27 Tumor ND F/H gradient 75 84 (CD3+) 15 39 NA F/H, Ficoll/Hypaque; ND, not done; NA, not applicable; TIL, tumor-infiltrating lymphocyte. Data for trisomy 7 are expressed as percentage of nuclei with three positive signals (counted on 100 interphase nuclei). *Cultured for 3 days in interleukin 2. Downloaded by guest on September 25, 2021 9746 Genetics: Dal Cin et al. Proc. Natl. Acad. Sci. USA 89 (1992) Isolation of Lymphocytes from Blood and Tumor Tissue. tumors in a percentage comparable to that obtained by CCA Peripheral blood mononuclear cells were isolated on Ficoll/ (Table 1). In contrast, short-term cultures of kidney tissue Hypaque gradients. T cells were purified with Lymphokwik from patients with end-stage functional nephropathy and (One Lambda, Los Angeles) and additional complement- other control tissues did not show trisomy 7 or trisomy 10. As fixing monoclonal antibody (mAb) to natural killer cells as a control for the technique, a week 13 miscarriage presenting reported (14). with constitutional trisomy 7 showed three spots in the Fresh tumor tissue was minced, passed through a metal majority of the cells. To exclude triploidy, double hybridiza- mesh, and cultured overnight. Enrichment for viable lym- tion with probes for the centromeric region of chromosomes phocytes was done either by centrifugation on a Ficoll/ 7 (or 10) and 12 was done. Only two signals for chromosome Hypaque gradient or by immunomagnetic selection with mAb 12 centromeres were present. to CD3 (OKT3, American Type Culture Collection) and goat ISH on Tissue Sections. Similar results were obtained with anti-mouse-coated Dynabeads (Dynal, Oslo) or anti-CD4- the p7tl and DMOZM probes. From 50%o to 701% of the nuclei coated or anti-CD8-coated Dynabeads (Dynal) (15). of cells in tissue sections showed hybridization signals with T-Cell Culture and Anti-CD3-Redirected Cytotoxicity. Cells p7tl. The nuclei of renal tubular epithelial cells and those of obtained from the tumor tissue were incubated in culture glomerular cells in the nonneoplastic kidney of the patients medium with 4% of a cytokine-rich supernatant of stimulated with and without kidney tumor showed one or two hybrid- lymphocytes containing =250 units of interleukin 2 per ml. ization signals (Fig. 1A). The nuclei of most cells in the Irradiated peripheral blood mononuclear cells were used as peritumoral interstitial inflammatory infiltrate of the patients feeder cells and added once a week. Cytotoxic activity ofthe with kidney tumor also showed one or two signals. However, cells was tested in an anti-CD3-redirected cytotoxicity assay, the nuclei of 5-10%o of mononuclear inflammatory cells with P815 cells (mouse mastocytoma cell line) as target cells displayed three hybridization signals (Fig. 1B). These inflam- and OKT3 as the anti-CD3 mAb (1 pug/ml) (16, 17). matory cells were present in the tumor but they were more FISH on Tumor-Infiltrating Lymphocytes or Peripheral numerous at the tumor pseudocapsule and the surrounding Blood T Lymphocytes. Lymphocytes enriched by the above- nonneoplastic kidney tissue (Fig. 1 C and D). Four signals mentioned methods were fixed (three times) with methanol/ were observed only in exceptional cases. A variable amount acetic acid (3:1; vol/vol), dropped onto glass slides, and dried of hybridization signals were seen in the nuclei of the tumor in an oven at 60'C overnight. FISH was performed as cells in the neoplastic kidney biopsies. The overall picture described above. obtained for chromosome 7 and chromosome 10 was very ISH Combined with Immunohistochemistry on Tumor- similar in appearance and tissue distribution, with that of Infiltrating Lymphocytes. Cells from the Ficoll/Hypaque chromosome 10 being somewhat less pronounced. In con- interphase were washed three times in phosphate-buffered trast, the lymphocytes in the reactive lymph node biopsies saline, fixed in 90% methanol/10%o acetone, and spread onto used as controls showed one or two hybridization signals. siliconized glass slides. A two-step immunohistochemical Detection of Trisomy 7 and Trisomy 10 by FISH in Lym- technique was performed. In the first step, the anti-CD3 mAb phocyte Populations Isolated from Neoplastic Kidney Tissue Leu 4 (Becton Dickinson, Erembodegem, Belgium), anti- and from Blood. Trisomy 7 was detected by FISH in 12-25% CD4 mAb OKT4 (Ortho Diagnostic System, Beerse, Bel- of the cells isolated from tumor tissue on Ficoll/Hypaque gium), and anti-CD8 mAb OKT8 (Ortho Diagnostic System) gradients and in 54-78% of the CD3+ T cells enriched by were used. Peroxidase-conjugated rabbit anti-mouse (Dako- immunomagnetic selection from minced tumor tissue. Simi- patts, Glostrup, Denmark) antibody was used in the second larly, 55-60%o of the cells removed from the tumor cell step. 3,3'-Diaminobenzidine and H202 were used as perox- suspension with anti-CD4-coated Dynabeads were found to idase substrates. After immunochemical staining, the same have three hybridization signals (Table 2). On one tumor ISH procedure used in the frozen kidney tissue section was sample (patient 19), isolated lymphocytes (>90%o CD3+) performed. were hybridized with the probes for chromosomes 7 and 10, and the percentages of positive cells were 16% and 10%1o, respectively. In three cases, CD3+ cells isolated from the RESULTS tumor (patients 18 and 24) or from surrounding kidney tissue Cytogenetic and FISH Investigations in Short-Term Kidney (patient 25) were separated into CD4+ and CD8+ populations. Cultures. Table 1 summarizes the data obtained in short-term It appeared that in CD4+ cells a majority showed trisomy 7, cultures of kidney tissues of 17 patients with a kidney tumor whereas in CD8+ only a minority showed three signals for initially investigated by CCA and by FISH using probes p7tl chromosome 7. CD8+ cells in contrast showed a mirror and DiOZi. Using the FISH technique, trisomy 7 cells were picture, with the cells having trisomy 10 rather than trisomy found to be present in the tumor in a frequency ranging from 7. Using an anti-CD3-redirected cytotoxicity assay, we could 2% to 14%, while trisomy 10 was less frequently present show that the total T-cell population had a strong cytotoxic (Table 1). potential. CD3+ cells isolated from the tumor of patient 23 Karyotypic findings in the nonneoplastic kidney tissues and cultured in vitro on a feeding layer and with various surrounding the tumors from the same patients showed that mitogens, including interleukin 2, showed a low proliferation trisomy 7 was always present, in a frequency ranging be- rate. tween 8% and 17.3%. Sixteen patients also showed an In peripheral blood cells, three chromosome 7 signals were additional population with trisomy 10 as the only abnormal- detected by FISH in 7-8% of CD3+ peripheral blood T cells ity, and in six cases trisomy 7 and trisomy 10 occurred in the freshly isolated from patients with kidney tumors (n = 3) but same cells. Loss of the Y chromosome occurred in a variable also in a similar percentage of T cells isolated from blood of proportion of the cells in all male patients. In contrast, two healthy controls (5% and 6%, respectively). Trisomy 10 short-term culture (4-6 days) of kidney biopsy tissue from in peripheral blood T cells was 6% for patient 18 and 5% for patients with end-stage functional nephropathy did not show two controls. any clonal chromosome abnormality. Combined ISH and Immunohistochemistry in Lymphocyte The results of CCA, with regard to trisomy 7 and trisomy Populations Isolated from Neoplastic Kidney Tissue. This 10, were confirmed by FISH. The signal-per-nucleus distri- analysis was done on cells freshly isolated from tumor tissue bution in interphase nuclei with the probes p7tl and DiOZi on Ficoll/Hypaque gradients. Trisomy 7 and trisomy 10 were showed that trisomy 7 and trisomy 10, respectively, were seen in a subpopulation of CD31 lymphocytes. Other CD3+ present in nonneoplastic kidney tissue surrounding kidney lymphocytes and the CD3- cells showed a normal chromo- Downloaded by guest on September 25, 2021 Genetics: Dal Cin et al. Proc. Natl. Acad. Sci. USA 89 (1992) 9747

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FIG. 1. ISH with probe p7tl on kidney tissue sections. (A) Tubular epithelial cells show two hybridization signals. (B) Mononuclear inflammatory cell (arrowhead) infiltrating among large RCCs showing three hybridization signals, reflecting trisomy 7. (x600.) (C) Kidney tissue section showing renal carcinoma (RC), tumor pseudocapsule (P), and surrounding nonneoplastic kidney tissue (KT). Mononuclear inflammatory cells are revealed by immunohistochemical staining for CD45. Inflammatory cells are most numerous at the tumor pseudocapsule and surrounding nonneoplastic kidney tissue. (x50.) (D) ISH with a centromeric chromosome 7 probe on a serial kidney tissue section, showing trisomy 7 in a subpopulation of mononuclear inflammatory cells (arrowheads), located at the tumor pseudocapsule. (x600.) some 7 and chromosome 10 complement. The cells displaying and 12-20%o presented three signals for trisomy 10. Double trisomy 7 were shown to be mostly CD41 T cells (Fig. 2), hybridization for trisomy 7 and trisomy 10 showed trisomy 7 whereas those with trisomy 10 were predominantly CD81 T only in 12-19%o, trisomy 10 only in 7-9.5%, and combined cells (Fig. 3). Although the vast majority of CD4+ cells and trisomy 7 and 10 in 4.5-9.5%. of CD8+ cells predominantly showed trisomy 7 and trisomy These probes, when applied to liver and kidney from the 10, respectively, some CD4+ cells showed trisomy 10 and same fetuses, failed to show the existence of trisomic sub- some CD8+ cells showed trisomy 7, a situation similar to that populations. found in the FISH studies mentioned above. Trisomy 7, Trisomy 10, and Combined Trisomy 7 and 10 as DISCUSSION Detected by FISH in Fetal Thymus and Other Tissues. In five fetuses (21-29 weeks) between 17% and 30%o of isolated In the present study, trisomy 7 and trisomy 10 have been thymocytes were found to present three signals for trisomy 7, identified in a substantial number of mononuclear inflammatory cells infiltrating kidney tumors and surrounding tissue. These inflammatory cells, characterized by trisomy 7 and

FIG. 2. Combined immunochemistry and ISH on Ficoll-isolated leukocytes from a renal carcinoma sample. Tumor-infiltrating lym- FIG. 3. Combined immunochemistry and ISH on Ficoll-isolated phocyte expressing CD4 and displaying three hybridization signals leukocytes from a renal carcinoma sample. CD8+ lymphocyte show- with the p7tl. ing three hybridization signals with the DlOZ1 probe. Downloaded by guest on September 25, 2021 9748 Genetics: Dal Cin et al. Proc. Nadl. Acad. Sci. USA 89 (1992) trisomy 10, were weakly enriched in cell populations isolated epithelial tumors) cells, then their apparent in vivo recruit- by passing minced tumor tissue over a Ficoll/Hypaque ment in the kidney with RCC, as well as their division in the density gradient. As the percentage of cells with trisomy 7 presence of living kidney tumor cells in short-term in vitro was highest in cells isolated from tumor tissue by immuno- culture could be explained. magnetic selection with anti-CD3 and anti-CD4 antibodies, Our findings could open the way for lines of research in an the predominant cell population with an abnormal chromo- attempt to find out whether and how these aneuploidies some 7 complement is characterized as T-helper/inducer characterizing subsets of T cells may be involved in success lymphocytes. Trisomy 10 was highest in cells isolated with or failure of immune surveillance mechanisms. The aneu- immunomagnetic selection with anti-CD8 antibody, which ploidization mechanism itself, which may originate in the were therefore characterized as T-suppressor/cytotoxic lym- fetal thymus, also constitutes a major challenge to further phocytes. These findings were confirmed by combined im- research. munohistochemistry and ISH on isolated tumor-infiltrating inflammatory cells. We thank Carl Hilliker for supervision of the FISH experiments; Our findings could explain the frequent presence oftrisomy Chris De Wolf-Peeters for the helpful discussion; Lut Meekers, 7 and trisomy 10 in short-term cultures of kidney tumors, Magda Dehaen, Christel Van den Broeck, and Frieda Van Vaeck for although we could not yet positively identify the nature ofthe technical assistance; and Rita Logist for clerical assistance. We are cells with aneuploid metaphases in these short-term cultures. indebted to Dr. J. J. Sotto (Centre Hospitalier Regional et Univer- In addition, the finding of trisomy 7 and trisomy 10 in sitaire, Grenoble, France) and Dr. P. Petit for providing us with the short-term cultures ofnonneoplastic kidney tissue ofpatients cell lines of tumor-infiltrating lymphocyte clones and of the week 13 was confirmed in the present miscarriage with trisomy 7, respectively. This work was supported with a kidney tumor, which by the Inter-University Network for Fundamental Research spon- study, may be ascribed to tumor-infiltrating lymphocytes. sored by the Belgium Government (1991-1995); the European Com- Indeed, T cells with trisomy 7 and trisomy 10 are not only munity Concerted Action "Molecular Cytogenetics of Solid Tu- present in the tumor but are also numerous or sometimes mours" (MR4*-0224-B); Grant 3.0037.87 of the Belgian National more numerous in the tumor-surrounding tissue, as shown by Fund for Medical Research; and the private fund "Action Against ISH on tissue sections. It is tempting to speculate that the Cancer." J.D. is a research assistant ofthe Belgian National Fund for presence of tumor-infiltrating lymphocytes is responsible for Scientific Research. J.L.C. is recipient of a special mandate from trisomy 7 and trisomy 10 observed in a range of other tumors. Belgian National Fund for Scientific Research for fundamental We studied a series of cancers, including epithelial tumors clinical research. originating in the lung, breast, thyroid, prostate, colon, and bladder and mesenchymal tumors such as lipoma, myxoid 1. Sandberg, A. A. (1990) The Chromosomes in Human Cancer liposarcoma, synovial sarcoma, neurofibrosarcoma, leiomy- and Leukemia (Elsevier, New York), 2nd Ed. and uterine leio- 2. Mitelman, F. (1991) Catalog of Chromosome Aberrations in osarcoma, malignant fibrous histiocytoma, Cancer (Wiley-Liss, New York), 4th Ed. myoma (data not shown) and found that trisomy 7 is almost 3. Lee, J. S., Pathak, S., Hopwood, V., Tomasovic, B., Mullins, exclusively found in connection with tumors of epithelial T. D., Baker, F. L., Spitzer, G. & Neidhart, J. (1987) Cancer origin. Studies on trisomy 10 remain to be done. Res. 47, 6349-6352. No explanation for the occurrence of the trisomies can be 4. Kovacs, G. & Brusa, P. (1989) Cancer Genet. Cytogenet. 37, provided at this moment. One possibility could be that the 289-290. cells with trisomy 7 and trisomy 10 could represent normally 5. Heim, S., Mandahl, N., Jin, Y., Stromblad, S., Lindstrbm, E., occurring subpopulations of T cells. Strong support for this Salford, L. G. & Mitelman, F. (1989) Cytogenet. Cell Genet. hypothesis comes from our finding of aneuploidy for chro- 52, 136-138. in a considerable 6. Elfving, P., Cigudosa, J. C., Lundgren, R., Limon, J., Man- mosomes 7 and 10 as shown by FISH dahal, N., Kristoffersson, U., Heim, S. & Mitelman, F. (1990) percentage of thymocytes. These subpopulations could be Cytogenet. Cell Genet. 53, 123-125. the precursors of the trisomy 7 and trisomy 10 cells detected 7. Emanuel, A., Szucs, S., Weier, H. U. G. & Kovacs, G. (1992) in, respectively, 5-6% and 5% of isolated T cells from the Genes Chromosomes Cancer 4, 75-77. peripheral blood of normal adults and of patients with kidney 8. Limon, J., Dal Cin, P. & Sandberg, A. A. (1986) Cancer Genet. cancer. Alternatively, the presence of trisomy 7 and trisomy Cytogenet. 23, 305-313. 10 could also represent an abnormality occurring in previ- 9. Waye, J. S., England, S. B. & Willard, H. F. (1987) Mol. Cell. ously diploid T cells and preceding or accompanying T-cell Biol. 7, 349-356. inactivation or even T-cell death. This may be an explanation 10. Devilee, P., Kievits, T., Waye, J. S., Pearson, P. L. & Willard, of 7 and 10 cells H. F. (1988) Genomics 3, 1-7. for the finding of a high percentage trisomy 11. Baldini, A., Rocchi, M., Archidiacono, N., Miller, 0. J. & in the thymus, where thymic selection leads to apoptosis of Miller, D. A. (1990) Am. J. Hum. Genet. 46, 784-788. a large percentage of differentiating thymocytes. Trisomy 7 12. Speleman, F., Mangelschots, K., Vercruyssen, M., Dal Cin, P., and 10 cells in and surrounding the tumor would then be a Aventin, A., Offner, F., Laureys, G., Van den Berghe, H. & manifestation of a failing defense mechanism, in which tumor Leroy, J. (1991) Cytogenet. Cell Genet. 56, 14-17. growth itself, secretion of toxic substances, or antigen rec- 13. Arnoldus, E. P. J., Wiegant, J., Noordermeer, I., Wessels, ognition in the absence of the appropriate accessory signals J. W., Beverstock, G. C., Grosveld, G. C., van der Ploeg, M. could play an essential role. Trisomy 7 T cells apparently do & Raap, A. K. (1990) Cytogenet. Cell Genet. 54, 491-502. not grow well in vitro. They are insensitive to interleukin 2 14. Ceuppens, J. L., Baroja, M. L., Lorr6, K., Van Damme, J. & T-cell This may Billiau, A. (1988) J. Immunol. 141, 3868-3875. and a series of classical mitogens. explain 15. Lea, T., Vartdal, F., Davies, C. & Ugelstad, J. (1985) Scand. why they were almost never found in phytohemagglutinin- J. Immunol. 22, 207-216. stimulated cultures in the past and why trisomy 7 was not 16. Leeuwenberg, J. F. M., Spits, H., Tax, W. J. M. & Capel, found in long-term clonally proliferating tumor-infiltrating P. J. A. (1985) J. Immunol. 134, 3770-3775. lymphocytes from a different origin (unpublished observa- 17. Vanham, G., Kestens, L., Gigase, P., Colebunders, R., Van- tion). If, however, they would be sensitive to putative denbruaene, M., Brijs, L. & Ceuppens, J. L. (1990) Clin. Exp. mitogenic signals from neoplastic kidney (and perhaps other Immunol. 82, 3-9. Downloaded by guest on September 25, 2021