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Distinct chromosome abnormalities in ataxia telangiectasia with chronic T-cell lymphocytic leukemia
Distinct Chromosome Abnormalities in Ataxia Telangiectasia with Chronic T-Cell Lymphocytic Leukemia Reinhard Becher and Carsten D ihrsen
ABSTRACT: We report chromosomal studies of a 27-year-old male patient with ataxia telangiectasia who developed a chronic T-cell lymphocytic leukemia. The leukemic cells grew spontaneously although a better yield of metaphases could be obtained after PHA stimulation. Chromosome analysis revealed a hypodiploid leukemic clone (44 chromosomes) and a single remaining normal metaphase. An isochromosome 8q was detected in a subclone at an early analysis, which was lost during clonal evolution. At the time of the last analysis, 6 months before the patient died, the diploid metaphases disappeared completely. The karyotype of the final chromosome study showed a monoclonal condition with the following abnormalities: 44,X,- Y , 4 q - , 6 p - , 1 4 q - , 1 9 p + ,20q + , - 2 0 , 2 2 q - . Loss of the Y chromosome was limited to the leukemic cells. Comparing our data with the chromosome abnormalities reported in the literature, breakpoints at band 14q11-12 (location of the gene for the alpha chain of the Tcell receptor), loss of a normal chromosome #20, as well as structural abnormalities of the remaining chromosome 20p seem to be nonrandom in T-CLL arising in patients with ataxia telangiectasia. A Philadelphia-like marker that seems to be a peculiar feature of the case described here, however, resembles a small marker chromosome qualified as unidentifiable in a similar case of T-CLL reported in the literature. INTRODUCTION According to a generally accepted hypothesis, cancer may originate from specific changes of the genome which express features typically related to neoplastic cell growth i n c l u d i n g metastatic spread. Therefore, syndromes presenting with chromosome instability and a high risk for malignancies apparently provide a good opportunity to study chromosome changes before and after neoplastic transformation. The occurrence of a higher than normal rate of various neoplasms in the condition of ataxia telangiectasia (A-T) has been well established [1]. Chromosomal instability has been shown to be a typical feature of A-T [2]. The instability is reflected by an elevated level of s p o n t a n e o u s chromosome breakage in peripheral lymphocytes and fibroblasts from some but not all affected i n d i v i d u a l s [3-7]. In addition, A-T cells have shown to be more sensitive to DNA damaging agents like bleomycin [8], various alkylating drugs [9], and x-rays [10-13]. This hypersensitivity has been interpreted as a result of deficient DNA repair. Clinically, A-T is an autosomal recessive
From the Innere Universit~itsklinik(Tumorforschung)West GermanTumor Center (R. B.), and the Medizinische Universit~tsklinikEssen, HaematologischeAbteilung(C. D.), Essen, Germany. Address requests for reprints to Dr. B. Becher, Inhere Universitdtstdinik [Tumofforschung), Westdeutsches Tumorzentrum, Hufelandgtrasse 55, D-4300 Essen 1, F.R.G. Received February 3, 1986; accepted March 28, 1986.
217 © 1987 ElsevierSciencePublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet26:217-225 (1987) 0165-4608/87/$03.50
218
R. Becher and C. Dfihrsen disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasias, and various degrees of expression of endocrine and immunologic disorders. Here we describe chromosomal abnormalities of a patient with A-T who developed a chronic T-cell lymphoproliferative disorder and discuss the significance of these changes for the classification of the disease.
CASE HISTORY
The patient, a 27-year-old male was first admitted to the medical department of the University of Essen, in July 1983. Evaluation of his past history revealed a moderate but generalized muscular weakness during the first year of his life. Then he developed a slowly progressive incoordination of gait and a dysarthric speech leading to the diagnosis of cerebellar ataxia. At the age of 6 years he was confined to a wheelchair. In 1966 he was operated for an acute appendicitis with perforation. At the age of 20 years, first ocular and then cutaneous telangiectasias developed. He suffered from herpes zoster of the trigeminal nerve in 1981. On admission he presented with an increased peripheral leukocyte count (52,200/V3). Physical examination showed a slightly underweight patient (55 kg, height 165 cm). Besides oculocutaneous telangiectasias his skin was covered with depigmented vitiligo-like areas of variable size. In addition, he had a mild bilateral gynecomastia. The right testicle was not palpable and the size of the left one in the lower normal range. There were no lymph nodes palpable and there was no hepatosplenomegaly. Neurologic examination showed severe cerebellar ataxia, ocular dysmetria, nystagmus, dysarthric speech, intentional tremor, dysdiadochokinesis, and inability to stand or move without help; tendon reflexes were absent. He had a sock-like hypanesthesia with reduction of the vibration sense. Laboratory studies showed a decrease of IgA (<42 mg/dl) and igE (<5 mU/ml) and an increase of IgM (702 mg/dl). Alpha-l-fetoprotein was markedly increased (359 ng/ml). The serum testosterone level was in the lower normal range (491 ng/ dl). Raised basal serum concentration of LH (13.3 mU/ml) and FS-H (19.4 mU/ml) with further increase after stimulation with LH-RH (45.3 mU/ml) and 28.9 mU/ml, respectively) were compatible with primary hypogonadism. The hemoglobin was 13.7 g/dl, the leukocyte count 52,500/V~1 with 73% atypical, medium sized lymphocytes with irregular, partly indented nuclei. The bone marrow showed an infiltration with lymphocytes of the same type. The platelet count was normal (436,000/ ~1). Immunologic studies indicated a chronic T-cell leukemia (CLL). Eighty-nine percent of the mononuclear peripheral blood cells (PBMC) rosetted with sheep erythrocytes. About 98% of them reacted strongly with pan-T and suppressor T-cell reagents. In approximately 60% of the cells a weak reaction was found with anti-helper T-cell antibodies. 95% of PBMC were positive for Leu-2a, 55% for Leu-3a. Whereas all ceils staining for Leu-3a simultaneously reacted with Leu-2a, about one-third of the cells only exhibited positively for Leu-2a. A panel of monoclonal antibodies failed to demonstrate an increased expression of antigens associated with thymocytes, B-lymphocytes, or monocytes. The leukemic lymphocytes responded normally to stimulation with concanavalin A and slightly weaker to PHA and pokeweed mitogen (PWM). After stable disease for 8 months, the leukocyte count increased up to 230,000/p,1 and the patient was subsequently treated with chemotherapy consisting of chlorambucil and prednisone, which proved to be ineffective. The intensification of chemotherapy by cyclophosphamide (100-150 mg/day) induced a reduction of leukocyte count but was followed by a severe hemorrhagic cystitis. After discontinuation of cyclophosphamide, leukocyte counts increased again, exceeding 80,000/p.1. A certain reduction was accomplished by intermittent leuko-
T-CLL in A-T
2 19
pheresis performed at intervals of 15-30 days and led to i m p r o v e m e n t of the patient's general condition. The patient finally died from recurrent severe urinary bleeding on June 2, 1985; autopsy was refused by his relatives. Evaluation of the family history revealed no other cases of A-T, however, there was a high frequency of various other neoplasias in the paternal line of the patient's family.
CHROMOSOME STUDIES Cytogenetic studies were performed from u n s t i m u l a t e d bone marrow and PHA stimulated lymphocytes cultured in RPMI 1640 medium. Standard G- and C-banding methods were utilized for chromosome analyses, e.g., short-term trypsin treatment for G-banding and alkaline treatment for C-banding [14]. Dates and results of our chromosome studies are summarized in Table 1. Abnormal cells were observed in u n s t i m u l a t e d cells after i n c u b a t i o n overnight for approximately 16 hours and after PHA stimulation during a culture time of 72 hours. The quality, as well as the number, of metaphases clearly improved after PHA stimulation. Besides the leukemic cells, normal cells (46,XY) were detected on two occasions (during the first and second study) which, in contrast to the leukemic ones, still contained the Y chromosome. The leukemic cells had a hypodiploid modal n u m b e r of 44 chromosomes. A representative G-banded karyotype is shown in Figure 1. None of the examined metaphases contained a t(14;14) marker. The following numerical and structural abnormalities were found in all metaphases: - Y , 4 q - , 6 p - , 1 4 q - , 19p +, 2 0 q + , - 2 0 , 2 2 q - . The structural changes were interpreted in the following way: rcp t(4;20)(q13;q12), interstitial del(14)(q21q24). Region 14ql of the deleted chromosome #14 appeared atypically condensed due to a small interstitial deletion of part of region ql, designed as del(14)(qllq13). The 19p + marker is composed of a translocation of 6p on the p arm of chromosome #19. The additional light stained chromatin between 6p and 19q could most probably constitute the deleted part of the q arm of chromosome #22, resulting in a t(6;19;22)(p11;p13;q13) as shown in Figure 2. C-banding studies revealed that the 19p+ marker contained only one centromere. In addition, no i n v o l v e m e n t of the Y chromosome in the complex rearrangements could be detected by C-banding. One G-banded metaphase was missing the 2 2 q - marker, which was interpreted as a random loss because no second identical
Table 1 Date
Chromosomal findings in u n s t i m u l a t e d and PHA stimulated cultures Total number of metaphases Mode a
5/7/84
8b
7/2/84
14c
12/14/84
35c
44(7) 46(1) 42(1) 44(11) 46(2)
Karyotypes
The morphology of metaphases allowed no banding (1) 46,XY , (3) 44,X, - Y4q- ,6p - ,14q - ,19p + ,20q +, - 20,22q(1) 44,X, - Y4q- ,6p - ,i(8q),14q- ,19p + ,20q +, - 20,22q(1) 42,X,-Y4q- , - 5,6p- ,i(8q),- 13,- 14,19p + ,20q+ ,22q43(2) (5) 44,X, - Y 4 q - ,6p - ,14q- ,19p + ,20q+ , - 20,22q44(32) (1) 43,X, - Y4q - ,6p - ,14q - ,19p + ,20q +, - 20, - 21,22q 45(1) (1) 43,X, - Y4q- ,6p - ,14q - , - 15,19p + ,2Oq-, - 20,22q-
aNumber of cells in parentheses. bUnstimulatedcells. CPHAstimulation.
220
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T-CLL in A-T t(6,19,22)(p12,p13,q13)
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Figure 2 Illustration of the complex rearrangement t(6;19;22) demonstrating the breakpoints involved. cell was found. At the time of the second study a subclone with an i(8q) and corresponding loss of one normal chromosome #8 was observed which, however, disappeared in subsequent analyses. DISCUSSION Most patients with a diagnosis of A-T are characterized by poor prognosis and short survival [15]. Our patient, as well as the other cases with A-T and T-CLL reached a relatively high age, ranging from 25 to 47 years, as shown in Table 2 [16-18]. This observation may support the hypothesis that a certain age might be a prerequisite for the manifestation of the disease, due to long generation times of CLL cells. Cytogenetic changes of malignant clones of CLL in A-T patients are of specific interest because "normal cells" of these patients are cancer prone, possibly due to a defect related to DNA repair [19]. Our chromosomal findings, compared with those reported in the literature, are summarized in Table 2. The leukemic clones of the published cases including ours were all hypodiploid with modal numbers of 41--44 chromosomes. The patient presented here, to our knowledge is the first male individual with this condition. Therefore, the behavior of the Y chromosome could be studied in benign and malignant cells. From various types of leukemias it is well known [20] that loss of the Y chromosome may occur during clonal evolution. This phenomenon was also observed in long-term cell cultures and somatic cells of aged men. Its significance in malignant cells is still unknown. A plausible explanation for the Y loss in neoplastic cells might simply be that the genetic information on this chromosome may be useless for malignant clones and, therefore, can be lost during clonal evolution. An interesting finding related to clonal evolution was an i(8q) at the time of the first analysis (Table 1). This anomaly was only present in a subclone, and disappeared at subsequent analyses. Therefore, we conclude that this anomaly, which
222
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Figure 3 Partial karyotypes of chromosome #14 and 14q- indicating a breakpoint in band 14qll due to a small interstitial deletion, clearly shown in the first example, which was interpreted as del(14)(qllq13). A second, more distal interstitial deletion, del(14)(q21q24), brings band q21 and q31 close together forming a new large dark band.
was also described in the case reported by Sparkes [18], is not necessary for the expression of T-CLL and represents a so-called secondary anomaly. Our last chromosome analysis was performed 6 months before the patient died and revealed a chromosomally monoclonal condition. A more consistent finding in T-CLL is structural changes of chromosome 14q. Tandem translocations, t(14;14), in which one chromosome #14 seems to function as a scavenger of the deleted chromatin of its homolog have been described in normal lymphocytes of A-T patients [21] and leukemic cells [16-18]. Therefore, a tandem t{14;14} can hardly be considered specific for T-CLL. Furthermore, this anomaly was absent in our case in benign and leukemic cells. However, it can be hypothesized that a tandem translocation of chromosome #14 might provide a step towards malignancy resulting in a premalignant condition with further steps necessary for the definitive malignant transformation. The observation that tandem translocations of chromosome #14 are not necessarily associated with leukemic cells of A-T patients was also discussed by Wake et al., who described a case of a T-ALL [22]. Based on their findings it was assumed that malignant clones in A-T patients might also originate from chromosomally normal cells and not only those with the tandem t(14;14). Two interstitial deletions of chromosome #14 occurred in our case. First, a small interstitial deletion within region 14ql, which was interpreted as del(14)(qllq13) (Figure 3). The breakpoint 1 4 q l l was previously reported by Zech et al. [23] as nonrandom for T-CLL. These findings gained further significance by the designation of the geue of the ~ chain of the T-cell receptor to band 1 4 q l l [24]. Therefore, we would like to conclude that the expression of the T-cell phenotype of CLL in the present case and those cases reported in the literature (Table 2J is closely associated with breakpoint 14q11. The second interstitial deletion of chromosome #14, classified as del(14)(q21q24) might involve the c-fos oncogene, which was recently mapped to 14q21-31 [25]. Regarding the rcp t(4;20) in our patient, a recent finding assigning the gene for the h u m a n T-cell growth factor [26] on 4q26-28 should be mentioned, although our breakpoint was closer to the centromere. The gene for ctfetoprotein [27], which was mapped to 4q11-12, should be mentioned due to the highly increased et-fetoprotein serum level in our patient. We noted that other authors (Table 2) did not report on increased et-fetoprotein in their patients, therefore, it may be a unique finding in our case although ectopic a-fetoprotein production in lymphoid cells seems not very probable. Another nonrandom cytogenetic feature of T-CLL in A-T seems to be the loss of one chromosome #20, which was observed in all but one case (case 2 in Table 2). The remaining chromosome #20 seems to be involved frequently in rearrangements, as shown in our case and in case 3 in Table 2 [18].
224
R. B e c h e r and C. Dfihrsen
A Ph-like c h r o m o s o m e , as f o u n d in our case, has not b e e n d e s c r i b e d before. It can be a s s u m e d , h o w e v e r , that one of the s m a l l u n i d e n t i f i e d markers s h o w n by Levitt et al. [17] m i g h t r e p r e s e n t a 2 2 q - c h r o m o s o m e . T h i s a s s u m p t i o n s e e m s v e r y probable b e c a u s e the s e c o n d c h r o m o s o m e # 2 2 was m i s s i n g in this karyotype. Supported by the Deutsche Forschungsgemeinschaft (SFB 102, A7). The authors thank Dr. Avery A. Sandberg for his constructive criticisms, Edith Wendehorst and Dagmar Kiihn for excellent technical assistance, and Cordula Massion for preparation of the manuscript.
REFERENCES 1. Daly MB, Swift M (1978): Epidemiological factors related to the malignant neoplasms in ataxia-telangiectasia families. J Chronic Dis 31:624-634. 2. Hecht F, McCaw BK (1977): Chromosome instability syndromes. In: Genetics in Human Cancer. Progress in Cancer Research and Therapy, Vol. 3, JJ Mulvihill, RW Miller, JF Fraumeni, Jr., eds. Raven Press, NY, pp. 105-123. 3. Kohn PH, Whang-Peng J, Levis WR (1982): Chromosomal instability in ataxia telangiectasia. Cancer Genet Cytogenet 6:289-302. 4. Sawitsky A, Bloom D, German J (1966): Chromosomal breakage and acute leukemia in congenital telangiectatic erythema and stunted growth. Ann Intern Med 65:487-495. 5. Gropp A, Flatz G (1967): Chromosome breakage and blastic transformation of lymphocytes in ataxia-telangiectasia. Humangenetik 5:77-79. 6. A1 Saadi A, Palutke M, Krishna Kumar G (1980): Evolution of chromosomal abnormalities in sequential cytogenetic studies of ataxia telangiectasia. Hum Genet 55:23-29. 7. Pfeiffer RA (1970): Chromosomal abnormalities in ataxia telangiectasia (Louis Bar's syndrome). Humangenetik 8:302-306. 8. Shaham M, Becker Y, Lerer I, Voss R (1983): Increased level of bleomycin-induced chromosome breakage in ataxia telangiectasia skin fibroblasts. Cancer Res 43:4244-4247. 9. Barfknecht TR, Little JB (1982): Hypersensitivity of ataxia telangiectasia skin fibroblasts to DNA alkylating agents. Mutat Res 94:369-382. 10. Taylor AMR (1978): Unrepaired DNA strand breaks in irradiated ataxia telangiectasia lymphocytes suggested from cytogenetic observations. Mutat Res 50:407-418. 11. Natarajan AT, Me yers M (1979): Chromosomal radiosensitivity of ataxia telangiectasia cells at different Cell cycle stages. Hum Genet 52:127-132. 12. Littlefield LG, Colyer SP, Joiner EE, DuFrain RJ, Frome E, Cohen MM (1981): Chromosomal radiation sensitivity in ataxia telangiectasia long-term lymphoblastoid cell lines. Cytogenet Cell Genet 31:203-213. 13. Parshad R, Sanford KK, Jones GM, Tarone RE (1985): G2 chromosomal radiosensitivity of ataxia-telangiectasia heterozygotes. Cancer Genet Cytogenet 14:163-168. 14. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier North Holland, New York. 15. McFarlin DE, Strober W, Waldmann TA (1972): Ataxia-telangiectasia. Medicine (Baltimore) 51:281-314. 16. McCaw BK, Hecht F, Harnden DG, Teplitz RL (1975): Somatic rearrangement of chromosome 14 in human lymphocytes. Proc Natl Acad Sci USA 72:2071-2075. 17. Levitt R, Pierre RV, White WL, Siekert RG (1978): Atypical lymphoid leukemia in ataxia telangiectasia. Blood 52:1003-1011. 18. Sparkes RS, Como R, Golde DW (1980): Cytogenetic abnormalities in ataxia telangiectasia with T-cell chronic lymphocytic leukemia. Cancer Genet Cytogenet 1:329-336. 19. Cornforth MN, Bedford JS (1985): On the nature of a defect in cells from individuals with ataxia-telangiectasia. Science 227:1589-1591. 20. Berger R, Bernheim A (1979): Y chromosome loss in leukemias. Cancer Genet Cytogenet 1:1-8.
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21. Hecht F, McCaw BK, Koler RD (1973): Ataxia-telangiectasia--clonal growth of translocation lymphocytes. N Engl J Med 289:286-291. 22. Wake N, Minowada J, Park B, Sandberg AA (1982): Chromosomes and causation of human cancer and leukemia. XLVIII. T-cell acute leukemia in ataxia telangiectasia. Cancer Genet Cytogenet 6:345-357. 23. Zech L, Gahrton G, Hammarstr6m L, Juliusson G, Mellstedt H, Robert KH, Smith CIE (1984): Inversion of chromosome 14 marks human T-cell chronic lymphocytic leukaemia. Nature 308:858-860. 24. Collins MKL, Goodfellow PN, Spurr NK, Solomon E, Tanigawa G, Tonegawa S, Owen MJ (1985): The human T-cell receptor c~-chain gene maps to chromosome 14. Nature 314:273274. 25. Barker OE, Rabin M, Watson M, Breg WR, Ruddle FH, Verma JM (1984): Human cofos oncogene mapped within chromosomal region 14q21--~q31. Proc Natl Acad Sci USA 81:5826-5830. 26. Seigel LJ, Harper ME, Wong-Staal F, Gallo RC, Nash WG, O'Brien SJ (1984): Gene for Tcell growth factor: location on human chromosome 4q and feline chromosome B1. Science 223:175-178. 27. Minghetti PP, Harper ME, Alpert E, Dugaiczyk A (1983): Chromosomal structure and localization of the human ~x-fetoprotein gene. Ann NY Acad Sci 417:1-12.
ABSTRACT: We report chromosomal studies of a 27-year-old male patient with ataxia telangiectasia who developed a chronic T-cell lymphocytic leukemia. The leukemic cells grew spontaneously although a better yield of metaphases could be obtained after PHA stimulation. Chromosome analysis revealed a hypodiploid leukemic clone (44 chromosomes) and a single remaining normal metaphase. An isochromosome 8q was detected in a subclone at an early analysis, which was lost during clonal evolution. At the time of the last analysis, 6 months before the patient died, the diploid metaphases disappeared completely. The karyotype of the final chromosome study showed a monoclonal condition with the following abnormalities: 44,X,- Y , 4 q - , 6 p - , 1 4 q - , 1 9 p + ,20q + , - 2 0 , 2 2 q - . Loss of the Y chromosome was limited to the leukemic cells. Comparing our data with the chromosome abnormalities reported in the literature, breakpoints at band 14q11-12 (location of the gene for the alpha chain of the Tcell receptor), loss of a normal chromosome #20, as well as structural abnormalities of the remaining chromosome 20p seem to be nonrandom in T-CLL arising in patients with ataxia telangiectasia. A Philadelphia-like marker that seems to be a peculiar feature of the case described here, however, resembles a small marker chromosome qualified as unidentifiable in a similar case of T-CLL reported in the literature. INTRODUCTION According to a generally accepted hypothesis, cancer may originate from specific changes of the genome which express features typically related to neoplastic cell growth i n c l u d i n g metastatic spread. Therefore, syndromes presenting with chromosome instability and a high risk for malignancies apparently provide a good opportunity to study chromosome changes before and after neoplastic transformation. The occurrence of a higher than normal rate of various neoplasms in the condition of ataxia telangiectasia (A-T) has been well established [1]. Chromosomal instability has been shown to be a typical feature of A-T [2]. The instability is reflected by an elevated level of s p o n t a n e o u s chromosome breakage in peripheral lymphocytes and fibroblasts from some but not all affected i n d i v i d u a l s [3-7]. In addition, A-T cells have shown to be more sensitive to DNA damaging agents like bleomycin [8], various alkylating drugs [9], and x-rays [10-13]. This hypersensitivity has been interpreted as a result of deficient DNA repair. Clinically, A-T is an autosomal recessive
From the Innere Universit~itsklinik(Tumorforschung)West GermanTumor Center (R. B.), and the Medizinische Universit~tsklinikEssen, HaematologischeAbteilung(C. D.), Essen, Germany. Address requests for reprints to Dr. B. Becher, Inhere Universitdtstdinik [Tumofforschung), Westdeutsches Tumorzentrum, Hufelandgtrasse 55, D-4300 Essen 1, F.R.G. Received February 3, 1986; accepted March 28, 1986.
217 © 1987 ElsevierSciencePublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet26:217-225 (1987) 0165-4608/87/$03.50
218
R. Becher and C. Dfihrsen disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasias, and various degrees of expression of endocrine and immunologic disorders. Here we describe chromosomal abnormalities of a patient with A-T who developed a chronic T-cell lymphoproliferative disorder and discuss the significance of these changes for the classification of the disease.
CASE HISTORY
The patient, a 27-year-old male was first admitted to the medical department of the University of Essen, in July 1983. Evaluation of his past history revealed a moderate but generalized muscular weakness during the first year of his life. Then he developed a slowly progressive incoordination of gait and a dysarthric speech leading to the diagnosis of cerebellar ataxia. At the age of 6 years he was confined to a wheelchair. In 1966 he was operated for an acute appendicitis with perforation. At the age of 20 years, first ocular and then cutaneous telangiectasias developed. He suffered from herpes zoster of the trigeminal nerve in 1981. On admission he presented with an increased peripheral leukocyte count (52,200/V3). Physical examination showed a slightly underweight patient (55 kg, height 165 cm). Besides oculocutaneous telangiectasias his skin was covered with depigmented vitiligo-like areas of variable size. In addition, he had a mild bilateral gynecomastia. The right testicle was not palpable and the size of the left one in the lower normal range. There were no lymph nodes palpable and there was no hepatosplenomegaly. Neurologic examination showed severe cerebellar ataxia, ocular dysmetria, nystagmus, dysarthric speech, intentional tremor, dysdiadochokinesis, and inability to stand or move without help; tendon reflexes were absent. He had a sock-like hypanesthesia with reduction of the vibration sense. Laboratory studies showed a decrease of IgA (<42 mg/dl) and igE (<5 mU/ml) and an increase of IgM (702 mg/dl). Alpha-l-fetoprotein was markedly increased (359 ng/ml). The serum testosterone level was in the lower normal range (491 ng/ dl). Raised basal serum concentration of LH (13.3 mU/ml) and FS-H (19.4 mU/ml) with further increase after stimulation with LH-RH (45.3 mU/ml) and 28.9 mU/ml, respectively) were compatible with primary hypogonadism. The hemoglobin was 13.7 g/dl, the leukocyte count 52,500/V~1 with 73% atypical, medium sized lymphocytes with irregular, partly indented nuclei. The bone marrow showed an infiltration with lymphocytes of the same type. The platelet count was normal (436,000/ ~1). Immunologic studies indicated a chronic T-cell leukemia (CLL). Eighty-nine percent of the mononuclear peripheral blood cells (PBMC) rosetted with sheep erythrocytes. About 98% of them reacted strongly with pan-T and suppressor T-cell reagents. In approximately 60% of the cells a weak reaction was found with anti-helper T-cell antibodies. 95% of PBMC were positive for Leu-2a, 55% for Leu-3a. Whereas all ceils staining for Leu-3a simultaneously reacted with Leu-2a, about one-third of the cells only exhibited positively for Leu-2a. A panel of monoclonal antibodies failed to demonstrate an increased expression of antigens associated with thymocytes, B-lymphocytes, or monocytes. The leukemic lymphocytes responded normally to stimulation with concanavalin A and slightly weaker to PHA and pokeweed mitogen (PWM). After stable disease for 8 months, the leukocyte count increased up to 230,000/p,1 and the patient was subsequently treated with chemotherapy consisting of chlorambucil and prednisone, which proved to be ineffective. The intensification of chemotherapy by cyclophosphamide (100-150 mg/day) induced a reduction of leukocyte count but was followed by a severe hemorrhagic cystitis. After discontinuation of cyclophosphamide, leukocyte counts increased again, exceeding 80,000/p.1. A certain reduction was accomplished by intermittent leuko-
T-CLL in A-T
2 19
pheresis performed at intervals of 15-30 days and led to i m p r o v e m e n t of the patient's general condition. The patient finally died from recurrent severe urinary bleeding on June 2, 1985; autopsy was refused by his relatives. Evaluation of the family history revealed no other cases of A-T, however, there was a high frequency of various other neoplasias in the paternal line of the patient's family.
CHROMOSOME STUDIES Cytogenetic studies were performed from u n s t i m u l a t e d bone marrow and PHA stimulated lymphocytes cultured in RPMI 1640 medium. Standard G- and C-banding methods were utilized for chromosome analyses, e.g., short-term trypsin treatment for G-banding and alkaline treatment for C-banding [14]. Dates and results of our chromosome studies are summarized in Table 1. Abnormal cells were observed in u n s t i m u l a t e d cells after i n c u b a t i o n overnight for approximately 16 hours and after PHA stimulation during a culture time of 72 hours. The quality, as well as the number, of metaphases clearly improved after PHA stimulation. Besides the leukemic cells, normal cells (46,XY) were detected on two occasions (during the first and second study) which, in contrast to the leukemic ones, still contained the Y chromosome. The leukemic cells had a hypodiploid modal n u m b e r of 44 chromosomes. A representative G-banded karyotype is shown in Figure 1. None of the examined metaphases contained a t(14;14) marker. The following numerical and structural abnormalities were found in all metaphases: - Y , 4 q - , 6 p - , 1 4 q - , 19p +, 2 0 q + , - 2 0 , 2 2 q - . The structural changes were interpreted in the following way: rcp t(4;20)(q13;q12), interstitial del(14)(q21q24). Region 14ql of the deleted chromosome #14 appeared atypically condensed due to a small interstitial deletion of part of region ql, designed as del(14)(qllq13). The 19p + marker is composed of a translocation of 6p on the p arm of chromosome #19. The additional light stained chromatin between 6p and 19q could most probably constitute the deleted part of the q arm of chromosome #22, resulting in a t(6;19;22)(p11;p13;q13) as shown in Figure 2. C-banding studies revealed that the 19p+ marker contained only one centromere. In addition, no i n v o l v e m e n t of the Y chromosome in the complex rearrangements could be detected by C-banding. One G-banded metaphase was missing the 2 2 q - marker, which was interpreted as a random loss because no second identical
Table 1 Date
Chromosomal findings in u n s t i m u l a t e d and PHA stimulated cultures Total number of metaphases Mode a
5/7/84
8b
7/2/84
14c
12/14/84
35c
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Karyotypes
The morphology of metaphases allowed no banding (1) 46,XY , (3) 44,X, - Y4q- ,6p - ,14q - ,19p + ,20q +, - 20,22q(1) 44,X, - Y4q- ,6p - ,i(8q),14q- ,19p + ,20q +, - 20,22q(1) 42,X,-Y4q- , - 5,6p- ,i(8q),- 13,- 14,19p + ,20q+ ,22q43(2) (5) 44,X, - Y 4 q - ,6p - ,14q- ,19p + ,20q+ , - 20,22q44(32) (1) 43,X, - Y4q - ,6p - ,14q - ,19p + ,20q +, - 20, - 21,22q 45(1) (1) 43,X, - Y4q- ,6p - ,14q - , - 15,19p + ,2Oq-, - 20,22q-
aNumber of cells in parentheses. bUnstimulatedcells. CPHAstimulation.
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Figure 2 Illustration of the complex rearrangement t(6;19;22) demonstrating the breakpoints involved. cell was found. At the time of the second study a subclone with an i(8q) and corresponding loss of one normal chromosome #8 was observed which, however, disappeared in subsequent analyses. DISCUSSION Most patients with a diagnosis of A-T are characterized by poor prognosis and short survival [15]. Our patient, as well as the other cases with A-T and T-CLL reached a relatively high age, ranging from 25 to 47 years, as shown in Table 2 [16-18]. This observation may support the hypothesis that a certain age might be a prerequisite for the manifestation of the disease, due to long generation times of CLL cells. Cytogenetic changes of malignant clones of CLL in A-T patients are of specific interest because "normal cells" of these patients are cancer prone, possibly due to a defect related to DNA repair [19]. Our chromosomal findings, compared with those reported in the literature, are summarized in Table 2. The leukemic clones of the published cases including ours were all hypodiploid with modal numbers of 41--44 chromosomes. The patient presented here, to our knowledge is the first male individual with this condition. Therefore, the behavior of the Y chromosome could be studied in benign and malignant cells. From various types of leukemias it is well known [20] that loss of the Y chromosome may occur during clonal evolution. This phenomenon was also observed in long-term cell cultures and somatic cells of aged men. Its significance in malignant cells is still unknown. A plausible explanation for the Y loss in neoplastic cells might simply be that the genetic information on this chromosome may be useless for malignant clones and, therefore, can be lost during clonal evolution. An interesting finding related to clonal evolution was an i(8q) at the time of the first analysis (Table 1). This anomaly was only present in a subclone, and disappeared at subsequent analyses. Therefore, we conclude that this anomaly, which
222
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Figure 3 Partial karyotypes of chromosome #14 and 14q- indicating a breakpoint in band 14qll due to a small interstitial deletion, clearly shown in the first example, which was interpreted as del(14)(qllq13). A second, more distal interstitial deletion, del(14)(q21q24), brings band q21 and q31 close together forming a new large dark band.
was also described in the case reported by Sparkes [18], is not necessary for the expression of T-CLL and represents a so-called secondary anomaly. Our last chromosome analysis was performed 6 months before the patient died and revealed a chromosomally monoclonal condition. A more consistent finding in T-CLL is structural changes of chromosome 14q. Tandem translocations, t(14;14), in which one chromosome #14 seems to function as a scavenger of the deleted chromatin of its homolog have been described in normal lymphocytes of A-T patients [21] and leukemic cells [16-18]. Therefore, a tandem t{14;14} can hardly be considered specific for T-CLL. Furthermore, this anomaly was absent in our case in benign and leukemic cells. However, it can be hypothesized that a tandem translocation of chromosome #14 might provide a step towards malignancy resulting in a premalignant condition with further steps necessary for the definitive malignant transformation. The observation that tandem translocations of chromosome #14 are not necessarily associated with leukemic cells of A-T patients was also discussed by Wake et al., who described a case of a T-ALL [22]. Based on their findings it was assumed that malignant clones in A-T patients might also originate from chromosomally normal cells and not only those with the tandem t(14;14). Two interstitial deletions of chromosome #14 occurred in our case. First, a small interstitial deletion within region 14ql, which was interpreted as del(14)(qllq13) (Figure 3). The breakpoint 1 4 q l l was previously reported by Zech et al. [23] as nonrandom for T-CLL. These findings gained further significance by the designation of the geue of the ~ chain of the T-cell receptor to band 1 4 q l l [24]. Therefore, we would like to conclude that the expression of the T-cell phenotype of CLL in the present case and those cases reported in the literature (Table 2J is closely associated with breakpoint 14q11. The second interstitial deletion of chromosome #14, classified as del(14)(q21q24) might involve the c-fos oncogene, which was recently mapped to 14q21-31 [25]. Regarding the rcp t(4;20) in our patient, a recent finding assigning the gene for the h u m a n T-cell growth factor [26] on 4q26-28 should be mentioned, although our breakpoint was closer to the centromere. The gene for ctfetoprotein [27], which was mapped to 4q11-12, should be mentioned due to the highly increased et-fetoprotein serum level in our patient. We noted that other authors (Table 2) did not report on increased et-fetoprotein in their patients, therefore, it may be a unique finding in our case although ectopic a-fetoprotein production in lymphoid cells seems not very probable. Another nonrandom cytogenetic feature of T-CLL in A-T seems to be the loss of one chromosome #20, which was observed in all but one case (case 2 in Table 2). The remaining chromosome #20 seems to be involved frequently in rearrangements, as shown in our case and in case 3 in Table 2 [18].
224
R. B e c h e r and C. Dfihrsen
A Ph-like c h r o m o s o m e , as f o u n d in our case, has not b e e n d e s c r i b e d before. It can be a s s u m e d , h o w e v e r , that one of the s m a l l u n i d e n t i f i e d markers s h o w n by Levitt et al. [17] m i g h t r e p r e s e n t a 2 2 q - c h r o m o s o m e . T h i s a s s u m p t i o n s e e m s v e r y probable b e c a u s e the s e c o n d c h r o m o s o m e # 2 2 was m i s s i n g in this karyotype. Supported by the Deutsche Forschungsgemeinschaft (SFB 102, A7). The authors thank Dr. Avery A. Sandberg for his constructive criticisms, Edith Wendehorst and Dagmar Kiihn for excellent technical assistance, and Cordula Massion for preparation of the manuscript.
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21. Hecht F, McCaw BK, Koler RD (1973): Ataxia-telangiectasia--clonal growth of translocation lymphocytes. N Engl J Med 289:286-291. 22. Wake N, Minowada J, Park B, Sandberg AA (1982): Chromosomes and causation of human cancer and leukemia. XLVIII. T-cell acute leukemia in ataxia telangiectasia. Cancer Genet Cytogenet 6:345-357. 23. Zech L, Gahrton G, Hammarstr6m L, Juliusson G, Mellstedt H, Robert KH, Smith CIE (1984): Inversion of chromosome 14 marks human T-cell chronic lymphocytic leukaemia. Nature 308:858-860. 24. Collins MKL, Goodfellow PN, Spurr NK, Solomon E, Tanigawa G, Tonegawa S, Owen MJ (1985): The human T-cell receptor c~-chain gene maps to chromosome 14. Nature 314:273274. 25. Barker OE, Rabin M, Watson M, Breg WR, Ruddle FH, Verma JM (1984): Human cofos oncogene mapped within chromosomal region 14q21--~q31. Proc Natl Acad Sci USA 81:5826-5830. 26. Seigel LJ, Harper ME, Wong-Staal F, Gallo RC, Nash WG, O'Brien SJ (1984): Gene for Tcell growth factor: location on human chromosome 4q and feline chromosome B1. Science 223:175-178. 27. Minghetti PP, Harper ME, Alpert E, Dugaiczyk A (1983): Chromosomal structure and localization of the human ~x-fetoprotein gene. Ann NY Acad Sci 417:1-12.