Cytogenetic Findings in Mantle Cell Lymphoma Cases With a High Level of Peripheral Blood Involvement Have a Distinct Pattern of Abnormalities

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1 Hematopathology / MANTLE CELL LYMPHOMA Cytogenetic Findings in Mantle Cell Lymphoma Cases With a High Level of Peripheral Blood Involvement Have a Distinct Pattern of Abnormalities Mihaela Onciu, MD, 1* Ellen Schlette, MD, 1 L. Jeffrey Medeiros, MD, 1 Lynne V. Abruzzo, MD, PhD, 1 Michael Keating, MD, 2 and Raymond Lai, MD, PhD 1 Key Words: Mantle cell lymphoma; Leukemia; Lymph node; Conventional cytogenetics Abstract We compared conventional cytogenetic findings in mantle cell lymphomas (MCLs) having an absolute peripheral lymphocytosis of more than 10,000/µL (> /L) at diagnosis ( leukemic ; n = 30) with those in cases having no or minimal lymphocytosis ( nodal ; n = 19). Only cases positive for t(11;14) were included for study. Forty-six cases (94%) had abnormalities in addition to t(11;14). The most frequent abnormalities involved chromosome 13 (26 cases [53%]), followed by chromosomes 1, 3, 7, 8, 9, 10, 12, 15, 17, and 21 (11-18 cases [22%-37%]). There was no difference in the number of aberrations between the 2 groups. Abnormalities of chromosomes 17, 21, and 22 were more frequent, and breakpoints involving 8q24, 9p22-24, and 16q24 were found exclusively in leukemic MCL. Chromosome 17 aberrations involved were structural (breakpoints involving 17p13, 17p11.2, 17q) in leukemic MCL but were only numeric in nodal MCL. Thus, leukemic MCL differs from nodal MCL in their cytogenetic profiles, which may contribute to the clinical presentation. Mantle cell lymphoma (MCL) is a distinct type of non- Hodgkin lymphoma that is characterized by the presence of a balanced chromosomal translocation, the t(11;14)(q13;q32). 1 This abnormality juxtaposes the CCND1 gene (11q13) with the IgH (14q32) gene, resulting in cyclin D1 overexpression. 2-4 It has been shown that MCL often carries karyotypic abnormalities in addition to the t(11;14), as demonstrated by a variety of techniques In most of these studies, del(13)(q14) was identified as the most frequent abnormality, found in 30% to 70% of cases. Other common abnormalities reported involve chromosomes 1, 3, 6, 9, and 17. While it is believed that the t(11;14) is important in upregulating the CCND1 gene, resulting in increased cyclin D1 protein that promotes cell cycle progression, the significance of other chromosomal abnormalities found in MCL has not been examined extensively. Most patients with MCL have nodal-based disease, although low-level peripheral blood involvement is common. In 1 study, up to 77% (27/35) of cases had morphologic evidence of MCL involving the peripheral blood, and most of these cases had a low number of circulating lymphoma cells. 16 Less often, MCL may manifest initially with marked leukemic involvement, with marked absolute lymphocytosis (ie, >10,000/µL [> /L]) in the peripheral blood. 5,17,18 It is possible that leukemic and nodal MCL are biologically different, and the cytogenetic profile of these cases may reflect this difference. However, relatively few cases of leukemic MCL analyzed with conventional cytogenetic methods have been reported. 12,19 Furthermore, there are no studies that have comprehensively correlated cytogenetic abnormalities with the clinical presentation of MCL. We describe the results of conventional cytogenetic studies in 49 well-characterized cases of MCL, 30 that had 886 Am J Clin Pathol 2001;116: American Society of Clinical Pathologists

2 Hematopathology / ORIGINAL ARTICLE marked leukemic involvement (absolute lymphocyte count, >10,000/µL [> /L]) at the time of diagnosis and 19 that manifested as nodal disease and had no or minimal peripheral lymphocytosis. These results demonstrate that the cytogenetic profile of MCL is different in patients with leukemic and nodal tumors. Materials and Methods Study Group The pathology files between 1993 and 2000 at the University of Texas M.D. Anderson Cancer Center, Houston, were searched for lymphoma cases that fulfilled the following criteria: (1) morphologic and immunophenotypic findings compatible with the diagnosis of MCL and (2) availability of the results of the conventional cytogenetic studies that showed the presence of the t(11;14)(q13;q32). Cases of MCL without the t(11;14)(q13;q32) were excluded, so that the diagnosis of MCL is well supported in all cases in this study. For the purpose of this study, we divided these patients into 2 groups, leukemic and nodal, based on the level of absolute lymphocytosis in the peripheral blood. Patients included in the leukemic group were those with marked leukemic involvement, which we arbitrarily defined as an absolute lymphocyte count of more than 10,000/µL (> /L) at the time of initial diagnosis. For the nodal group, all patients had no or minimal peripheral lymphocytosis. Conventional Cytogenetics Conventional G-band karyotype analysis was performed on all cases. Briefly, bone marrow aspirate, peripheral blood, spleen, or lymph node specimens were mixed with Ham F10 medium with 20% fetal calf serum for a total volume of 10 ml and a final concentration of 2 to nucleated cells per milliliter. The culture was incubated overnight at 37 C. Standard harvesting procedures were used: colcemid (0.1 ml K max Colcemid Solution, GIBCO, Grand Island, NY) was added to the culture for 20 minutes, followed by a mol/L concentration of potassium chloride for 30 minutes at 37 C. The fixation procedure consisted of 3 changes of methanol/glacial acetic acid (3:1). A Thermatron drying chamber (Thermatron Industries, Holland, MI) was used for slide preparation. Slides were placed in a 60 C oven overnight, followed by GTG banding. The karyotype reports were written according to the International System for Human Cytogenetic Nomenclature. 20 In the nodal group, specimens used for cytogenetic studies were lymph nodes (13 cases) and bone marrow aspirates (6 cases). In the leukemic group, specimens used for cytogenetic studies were bone marrow aspirates (27 cases), lymph nodes (2 cases), and peripheral blood (1 case). At least 20 metaphases were analyzed in 30 cases, 15 to 19 metaphases were analyzed in 11 cases, and 3 to 15 metaphases were analyzed in 8 cases. Statistical Analysis Comparisons of the frequency of chromosomal abnormalities between the leukemic and nodal groups were assessed using the chi-square test. In samples with multiple clones, only those with 2 or more metaphases were included in the analysis. Results Patient Characteristics The median age of the 49 patients was 65 years (range, years). There were 33 men and 16 women. Nineteen patients had nodal disease at initial assessment. Of this group, 18 of 19 patients had no absolute lymphocytosis in the peripheral blood; 1 patient had mild lymphocytosis (absolute lymphocyte count, 4,800/µL [ /L]). Thirty patients had marked leukemic involvement, with an absolute lymphocyte count ranging from 11,100 to 647,600/µL ( /L; median, 6,600/µL [ /L]) at the time of initial assessment. Three of these patients had no specific diagnosis at the time of referral. The remaining patients had one of the following diagnoses: chronic lymphocytic leukemia, 14; chronic lymphocytic leukemia/prolymphocytic leukemia, 2; prolymphocytic leukemia, 1; splenic lymphoma with villous lymphocytes, 1; MCL, 7; acute lymphoblastic leukemia, 1; and small noncleaved cell lymphoma, 1. Fifteen patients in the leukemic group (50%) received chemotherapy before referral to our institution. In comparison, 7 (37%) patients in the nodal group received chemotherapy before referral to our institution. There was no significant difference in the frequency of previous chemotherapy between the leukemic and nodal groups. Cytogenetic Findings The cytogenetic findings are listed in Table 1. Only 3 cases (6%) carried the t(11;14)(q13;q32) as a sole cytogenetic abnormality: 2 were in the nodal group, and 1 was in the leukemic group. The remaining 46 cases had additional karyotypic abnormalities: 8 cases (16%) had 1 or 2 additional chromosomal changes, 16 cases (33%) had 3 or 4 additional chromosomal changes, and 22 cases (45%) had more than 4 additional chromosomal changes. There were no significant differences in the number of chromosomal abnormalities American Society of Clinical Pathologists Am J Clin Pathol 2001;116:

3 Onciu et al / MANTLE CELL LYMPHOMA Table 1 Analysis of 49 Cases of Mantle Cell Lymphoma by Conventional Cytogenetics Case No. Site Karyotype Leukemic 1 BM* 45,XY,add(10)(p13),t(11;14)(q13;q32), 12,der(13)t(12;13)(q13;q32),add(17)(p13)[9]/44-46,XY,idem,random changes[5] 2 BM* 9/97 : 45,X, X,t(11;14)(q13;q32),add(17)(p13)[13]/45,X, X,idem, 9,+r[4] BM* 6/99 : 48,XX,t(1;11)(p36;q13),del(2)(q31q33),del(3)(q23q25),add(8)(q22), 10,t(11;14)(q13;q32),add(12)(q22),+add(12)(q22)x2, +16,add(17)(p13),+add(17)(p13), 20,+mar[cp20] 3 BM* 46-48,XX,add(8)(q),add(9)(q),t(11;14)(q13;q32),del(13)(q),del(17)(q),+mar[cp20] 4 BM 41,X, Y,der(1)(q32;q21),der(3)t(3;11)(q13;q23),der(3)t(3;21)(q29;q11), 4,der(5)t(5;15)(p15;q11),i(8)(q10),der(11)t(11;14)(q13;q32), del(12)(p13),t(13;17)(q10;q10),der(14)t(3;11)t(11;14)(q13;q32), 15, 21[15]/46,XY[4] 5 BM* 73,XXY,+3,i(8)(q10), 9,+11,t(11;14)(q13;q32)X2;del(12)(q12q21),+13,+14, 15, 17,+20, 21, 22,+3mar[11]/73,XXY,idem,dup(1)(q12q21)[2] 6 BM* 46,XX,t(2;17)(p22;p11),t(11;14)(q13;q32),del(13)(q31)[6]/46,XX[14] 7 BM* 43-46,XX, 11,t(11;14)(q13;q32), 13, 15,der(17;21)(q10;q10), 17,+5 8mar[20] 8 BM 47,XX,add(5)(q35), 8,t(11;14)(q13;q32),i(17)(q10),+2mar[5]/46,XX[16] 9 BM* 46,XX,t(11;14)(q13;q32)[8]/46,XX,idem,+7,+12,+17[5]/46,XX[6] 10 BM* 46,XX,t(11;14)(q13;q32), 15,add(17)(p11.2),+mar[12]/47,XX,idem,+11[8] 11 BM* 44,XY, 3,t(3;16)(q23;q24),add(7)(p22),i(8)(q10),add(9)(p22), 10,t(11;14)(q13;q32),add(13)(q22), 17,add(19)(p13.3),add(21)(p13), 22,+2mar[18]/43-45,XY,idem,random changes[2] 12 BM 42-44, X,Y,add(3)(q29), 5,add(8)(q24),add(9)(q34),add(10)(q26),t(11;14)(q13;q32),add(11)(p15), 13,add(13)(p13), 17, 18, add(22)(q13),del(22)(q11),+2 6mar[cp13]/46,XY[7] 13 BM 7/98 : 46,XY,del(1)(q42),+3,t(11;14)(q13;q32), 15,del(17)(q23),+mar[13]/46,XY[6] LN 8/98 : 46,XY,del(1)(q42),+3,t(11;14)(q13;q32), 15, 17,+mar[13] BM* 7/99 : 46,XY,del(1)(q42),+3,t(11;14)(q13;q32), 13, 15,del(17)(p11.2),+mar[cp3]/46,XY[9] 14 BM* 43,X, X, 4, 9,add(10)(q25),t(11;14)(q13;q32),del(13)(q14q22),del(17)(p11.2), 21,+mar[9]/46,XX[11] 15 BM* 46,XY,+3,add(5)(p15),t(11;14)(q13;q23), 13[2]/46-48,XY,+3,add(5)(p15), 9,t(11;14)(q13;q32), 15, 17, 18,+19,+21,+1 6mar[cp15] 16 BM* 45,XY, 1,del(1)(q25),del(4)(q21),i(6)(p10),del(7)(q22q32), 8,add(9)(p24), 10,t(11;14)(q13;q32),add(13)(p13),+mar[14]/46,XY[24] 17 BM* 5/96 : 46,XY,add(4)(q35),del(7)(q32),t(11;14)(q13;q32),add(16)(q24)[1]/46,XY,idem,i(8)(q10)[10]/46,XY[11] SPLN* 1/98 : 46,XY,del(7)(q32),t(11;14)(q13;q32),add(16)(q24)[2]/46,XY[17] BM* 3/98 : 46,XY,del(7)(q32),t(11;14)(q13;q32),add(16)(q24)[11] 18 BM 11/99 : 40-42,X, Y,+6, 8, 9,t(11;14)(q13;q32), 18, 19, 20, 21, 22[cp19]/46,XY[1] BM* 2/00 : 41-42,X, Y,+6, 8, 9,t(11;14)(q13;q32), 18, 19, 20, 21, 22[cp12]/46,XY[8] 19 PB 41-44,XY,del(1)(q13), 5, 7, 13,der(14)t(11;14)(q13;q32),+1 5mar[18]/46,XY[2] 20 LN 46,XY,t(11;14)(q13;32)[7] 21 BM 46,XY,t(11;14)(q13;q32)[12]/48,XY,idem,t(6;9)(q22;p24),+7,+12[8] 22 BM 42-44,XY, 3, 8,del(11)(q14),t(11;14)(q13;q32),add(12)(p13), 13,del(13)(q22q32), 21,+1 2mar[11]/46,XY[38] 23 BM 46,XY,t(11;14)(q13;q32),add(22)(q13)[8]/46,XY[12] 24 BM 47,XY,t(11;14)(q23;q32),+12[3]/46,XY[16] 25 BM 45-48,XY,+3, 7,del(10)(q24),t(11;14)(q13;q32),+21,+mar[cp4]/46,XY[16] 26 BM 46,XX,add(9)(p24),t(11;14)(q13;q32),del(13)(q21), 15,add(16)(q24),+1 3mar[26] 27 LN 3/97 : 71-74,XXY,+Y,del(1)(p34), 2,del(6)(q21),+del(6)(q21),+10, 11, 13, 14, 15, 17,+18,+20,+21,+22,+4 6mar[20] PB 3/97 : 73-79,XXY,del(1)(p13p31), 2,del(3)(p24),del(6)(q21),+del(6)(q21), 8, 14, 15,+6 10mar[3]/41-46,XY,random changes[6] BM* 7/97 : 42-45,XY,del(1)(p13p31), 2,del(3)(p24),add(5)(q35),del(6)(q21),add(9)(p24),t(11:14)(q13;q32), 13, 14, 15, 18,+r,+1 3mar[11] 28 BM* 46,XY,t(11;14)(q13;q32),add(13)(q34)[17]/44-46,XY,idem[3] 29 BM 45,X, X,del(1)(p13p22), 2,der(8)t(2;8)(q13;q24),del(9)(p12),t(11;14)(q13;q32),t(12;18)(q13;q23), 13,add(15)(p15),add(21)(p13), +2mar[9]/46,XY[11] 30 BM* 7/97 : 46,XY,del(7)(q22),add(8)(q24),t(11;14)(q13;q32)[4] BM* 1/98 : 46,XY,del(8)(q22)[2]/46,XY[18] Nodal 31 LN 45,XY,t(11;14)(q13;q32), 13[9]/46,XY[4] 32 LN 44-46,XY,del(1)(p11p22),del(6)(q21),add(11)(q23),t(11;14)(q13;q32),+mar[cp13]/46,XY[6] 33 LN* 43-44,X, X, 1,add(1)(p36.3),t(11;14)(q13;q32), 14, 17,+1 3mar[cp12]/46,XX[7] 34 LN 46,XY,t(11;14)(q13;q32)[18]/47,XY,idem,+5[1]/46,XY,idem,del(1)(p36),+5,t(7;11)(q36;q13)[1] 35 LN 45,XX,t(1;6)(p22;p21), 6, 10,t(11;14)(q13;q32), 12,add(13)(p12),der(14)add(14)(p11)t(11;14)(q13;q32),+18,del(20)(q13), +mar[18]/46,xx[2] 36 LN* 43-46,XY,del(6)(q14),del(9)(q13), 9,t(11;14)(q13;q32), 15,+2 3mar[cp6]/46,XY,random changes[2]/46,xy[7] 37 LN* 56-58,XY,+X,+4,+5,t(11;14)(q13;q32),+19,+1 4mar[2]/79,XXY,+8,+11,t(11;14)(q13;q32),+12,+13, 17,+18, 20,+21,+6mar[1] 38 LN* 43-47,XX,t(1;2)(p31.1;p23),+3,del(6)(q21), 10,t(11;14)(q13;q32),+1 2mar[24]/46,XX[1] 39 LN* 89-95,XXXX,add(1)(p11)x2, 3,+5,del(7)(q31)x2, 8,+9, 10,del(11)(p11)x2,t(11;14)(q13;q32)x2, 13, 17x2,+r,+4 6mar[9]/46,XX[8] 40 BM 46,XY,t(11;14)(q13;q32)[1]/45,X, Y[4]/46,XY[25] 41 BM* 4/97 : 46,XY,add(3)(q29),t(11;14)(q13;q32)[2]/46,XY[10] LN* 5/97 : 46,XY,add(3)(q29),t(11;14)(q13;q32)[7] 42 BM* 47,XY,t(1;6)(p22;q25), 5,+7, 8, 9, 9, 10,t(11;14)(q13;q32),+12, 13, 13,add(20)(p13),+6mar[5]/47,XY,idem,add(4)(q35), +6mar[10]/48,XY,idem,add(4)(q35),+18,+6mar[5] 43 BM 40-43,X, Y, 5,t(11;14)(q13;q32), 12,add(13)(p11), 15, 16, 18, 19, 20[5]/46,XY[14] 44 BM 46,X, X,+2,+3,del(7)(q22), 9,t(11;14)(q13;q32), 13, 14,+1 3mar[cp12]/46,XX[8] 45 LN 46,XY,t(11;14)(q13;q32), 13,+mar[5]/43-46,XY,idem,add(4)(p16),+mar[3]/46,XY[3] 46 LN 46,XY,t(11;14)(q13;q32)[5]/46,XY[1] 47 LN 90-91,t(11;14)(q13;q32)x2[3]/46,XX[9] 48 BM 45,X, Y,del(1)(p22),del(6)(q15),t(11;14)(q13;q32)[3]/46,XY,del(1)(p22),del(6)(q15),t(11;14)(q13;q32)[18] 49 LN 47,XX,der(1)(p22p32)del(1)(q21q25), 2, 4,t(11;14)(q13;q32),+4 5mar[2]/46,XX[1] BM, bone marrow; LN, lymph node; PB, peripheral blood; SPLN, spleen. * Following chemotherapy. Date (mo/y) when sample was obtained for patients with more than 1 sample. 888 Am J Clin Pathol 2001;116: American Society of Clinical Pathologists

4 Hematopathology / ORIGINAL ARTICLE Table 2 Frequent (>20% of Cases) Chromosomal Abnormalities in 49 Cases of Mantle Cell Lymphoma * Overall Chromosome Incidence Trisomy Monosomy Specific Additions Specific Deletions Translocations 1 17 (35) 0 0 1p31p36 (1) 1p31p36 (2) 1p31p36 (2) 1p22 (4) 1p22 (2) 3 13 (27) 6 3 3q25q29 (2) 3q25q29 (1) 3q25q29 (1) 7 11 (22) 3 2 7q22 (3) 7q32 (2) 8 16 (33) 0 6 8q24 (3) 8q24 (1) i8q10 (4) 9 15 (31) 1 7 9p22-24 (3) 9p12-13 (2) 9p22-24 (1) 9q34 (2) (24) p11-13 (1) 10q24-26 (2) 10q24-26 (1) (24) p13 (1) 12p13 (1) 12q12-13 (1) 12q12-13 (2) 12q22-24 (1) 12q22-24 (1) (53) q14 (1) 13q14 (1) 13q31q34 (2) 13q31q34 (2) 13q31q34 (1) (22) q15 (1) (37) p13 (2) 17p11.2 (1) 17p11.2 (2) 17p11.2 (1) 17q (1) 17q (1) 17q (2) (22) p13 (2) 21q10-11 (2) * Data for incidence are given as number (percentage). Specific additions, deletions, or translocation of the hot spots on specific chromosomes. Numbers in parentheses are the number of additions, deletions, or translocations. between the leukemic and nodal groups. Both numeric and recurrent structural abnormalities were identified in both groups. Only 5 cases showed near-triploid or near-tetraploid karyotypes. Two cases were leukemic, and 3 cases were nodal. None of these 5 cases had a blastoid appearance. The chromosomes that were most frequently abnormal were chromosome 13 in 26 cases (53%), chromosome 17 in 18 cases (37%), chromosome 8 in 16 cases (33%), and chromosome 1 in 17 cases (35%). Chromosomes 3, 7, 9, 10, 12, 15, and 21 were involved in 11 to 18 cases (22%-37%). The changes seen in these chromosomes are summarized in Table 2. The frequencies of various cytogenetic abnormalities differed in leukemic and nodal MCL, as summarized in Table 3. Abnormalities involving chromosomes 17, 21, and 22 were significantly associated with leukemic manifestation. Chromosome 17 abnormalities were identified in 16 Table 3 Differences in Chromosomal Abnormalities Between the Leukemic and Nodal Cases of Mantle Cell Lymphoma * Chromosome Leukemic (n = 30) Nodal (n = 19) P Del 8q24 4 (13) 0 (0).1 9p (13) 0 (0).1 16q24 3 (10) 0 (0) (53) 2 (11) < (37) 0 (0) < (20) 0 (0) <.05 * Data are given as number (percentage). (53%) of 30 leukemic MCLs, compared with 2 (11%) of 19 nodal MCLs (P <.05). In leukemic MCL, abnormalities of chromosome 17 involved structural aberrations in 10 (62%) of 16 cases, including recurrent breakpoints involving 17p13 and 17p11.2. In contrast, both nodal MCLs had monosomy 17. Chromosome 21 abnormalities were observed in 11 (37%) of 30 leukemic MCLs compared with none of the nodal MCLs (P <.05). Abnormalities of chromosome 22 were present exclusively in 6 leukemic MCLs and were numeric in 5 (P <.05). There were 3 other notable differences between the leukemic and nodal groups. Aberrations involving 8q24 in 4 cases (13%), 9p22-24 in 4 cases (13%), and 16q24 in 3 cases (10%) were found exclusively in leukemic MCL. Although these differences were not statistically significant, this is probably due to the relatively low frequency of these abnormalities. We compared the frequency of chromosomal changes in patients who had or had not received previous treatment, and chromosome 17 and Y abnormalities correlated with therapy. Fourteen (64%) of 22 cases of treated MCL had chromosome 17 abnormalities compared with 5 (19%) of 27 cases of untreated MCL (P <.05). No cases of treated MCL had chromosome Y abnormalities, compared with 5 (19%) of 27 cases of untreated MCL (P <.05). There was no significant difference in the distribution of specific breakpoints involving chromosome 17 between the 2 groups. None of the 7 cases with follow-up cytogenetics data (cases 2, 13, 17, 18, 27, 30, and 41) developed abnormalities of American Society of Clinical Pathologists Am J Clin Pathol 2001;116:

5 Onciu et al / MANTLE CELL LYMPHOMA chromosome 17 after chemotherapy administered at our institution. No other chromosomal abnormalities correlated with a history of chemotherapy. Of the 49 cases in this study, only 3 cases (6%) had high-grade morphologic features. Owing to their small number, no further statistical analysis was performed. Discussion There are relatively few studies reported in the literature that have extensively assessed cytogenetic abnormalities in MCL. 7,13,21,22 These studies have been performed using a variety of techniques, including conventional cytogenetics, fluorescence in situ hybridization, and comparative genomic hybridization; thus, it is difficult to discern a comprehensive view of these findings. Most of these studies also are limited by a lack of clinical information, and, thus, the relative frequencies of each chromosomal abnormality may be biased, depending on the stage or clinical manifestation of disease. Also, some MCL cases reported in previous studies were diagnosed morphologically, with no documentation of cyclin D1 overexpression or the presence of the t(11;14)(q13;q32). We hypothesized that leukemic and nodal MCLs are biologically different, and the cytogenetic profile of these cases may reflect this difference. Thus, we comprehensively examined chromosomal abnormalities in 49 well-characterized cases of MCL as determined by conventional cytogenetics. Thirty patients had marked leukemic involvement at the time of initial diagnosis, which we arbitrarily defined as an absolute lymphocyte count of more than 10,000/µL (> /L), and 19 patients had nodal disease. Each case of MCL carried the t(11;14) and had compatible morphologic and immunophenotypic features. Forty-six cases (94%) in this series had karyotypic abnormalities in addition to the t(11;14), in agreement with the results of 1 previous study of MCL using conventional cytogenetics. 13 The frequent presence of these karyotypic abnormalities suggests their importance in the pathogenesis of MCL, consistent with findings in previous studies of transgenic mice that have shown that cyclin D1 overexpression, by itself, is insufficient for tumorigenesis. 23,24 As indicated in Table 2, karyotypic abnormalities often are close to or overlap with the loci of known or putative oncogenes or tumor suppressor genes, such as the p16 INK4a at 9p22-24, p53 at 17p13, erbb-2 at 17p11.2, cyclin D2 at 12q12-13, and ING1 at 13q34. Some of these genes have been shown previously to be important in MCL, including p16 and p53, both of which are more likely to be inactivated in highgrade MCL cases that are associated with a worse prognosis. 21,25-27 Other genes, such as cyclin D2, ING1, and erbb-2, have not been examined in MCL. ING1 is a putative tumor suppressor gene. 28 We identified a significant correlation between specific chromosomal changes and clinical presentation. As shown in Table 3, abnormalities involving chromosomes 17, 21, and 22 were more common in leukemic than in nodal MCL. In addition, abnormalities involving 8q24, 9p22-24, and 16q24, although present at low frequency, were found exclusively in leukemic MCL. Another interesting finding is that the types of chromosome 17 abnormalities were different between these 2 groups. Only monosomy 17 was observed in nodal MCL. In contrast, leukemic MCL cases had breakpoints involving chromosome 17, including 17p13 and 17q, the loci harboring the p53 and the erb-b2 genes, respectively. These findings suggest that both genes may be involved in the pathogenesis of leukemic MCL. The biologic significance of these karyotypic differences between the nodal and leukemic groups needs to be studied further. Nevertheless, the differences may reflect genetic abnormalities that are associated with disease progression. Thus, nodal MCL cases acquire additional cytogenetic abnormalities as they progress to the leukemic phase of the disease. In support of this concept, there are more similarities than differences in the complexity and overall pattern of the karyotypic abnormalities between the nodal and leukemic groups. We also considered the possibility that karyotypic differences between the nodal and leukemic cases may be attributed to the use of specimens from different anatomic sites, as most of the specimens in the leukemic group were from the bone marrow, whereas most of the specimens in the nodal group were from the lymph nodes. However, we consider this possibility unlikely, because karyotypic results from multiple specimens of different anatomic sites were available in 4 cases (Table 1, cases 13, 17, 27, 41), and the results were consistent from site to site. We included only unequivocal cases of MCL that have t(11;14) detectable by conventional cytogenetics. This selection criterion is rigorous and may have excluded a proportion of MCL cases that did not grow well in culture and/or did not have the t(11;14) detectable by conventional cytogenetics. Nevertheless, this selection criterion carries an important advantage the diagnosis of MCL in our study cases was well supported. In addition, our study is relatively reproducible owing to this simple but objective selection criterion. Excluding the t(11;14), chromosome 13 abnormalities have been reported most frequently in MCL. Of these, del(13)(q14) has been reported commonly. 8,29 This deletion has been found in cases of leukemic follicular lymphoma 30 and some aggressive cases of multiple myeloma. 31 In our series, only 2 cases had this abnormality. In the present study, we observed that monosomy 13 was most common and that 13q31-34 was the most frequently involved breakpoint. 890 Am J Clin Pathol 2001;116: American Society of Clinical Pathologists

6 Hematopathology / ORIGINAL ARTICLE 13q31-34 is known to harbor a candidate tumor suppressor gene, ING1, which is frequently rearranged in head and neck squamous cell carcinoma. 32 We did not observe any significant difference in the overall frequency of chromosomal aberrations in MCL between patients who had or had not received previous chemotherapy. Nevertheless, we found that abnormalities of chromosome 17 were more frequent in the tumors of previously treated patients, and it is possible that chromosome 17 abnormalities are therapy induced. However, we believe that this possibility is unlikely. Seven patients had sequential cytogenetic studies, before and after chemotherapy, and in no case were chromosome 17 abnormalities found in MCL after treatment. Thus, we believe that the chromosome 17 abnormalities detected in this study are most likely intrinsic to MCL. The association between loss of chromosome Y and the untreated group is difficult to explain. Loss of chromosome Y is known to occur in the bone marrow of elderly patients with or without evidence of malignancy, most likely as an age-related nondisjunctional phenomenon. 33,34 The occurrence of chromosome Y loss in hematologic disease, including B-cell lymphoma, is related directly to patient age, does not seem to correlate with disease progression or prognosis, and may be a secondary event. 34,35 We confirm the observations of others that most cases of MCL have nonrandom chromosomal abnormalities in addition to the t(11:14)(q13;q32). Our findings also support the concept that aberrations of these specific chromosomal regions are important in the pathogenesis of these neoplasms. The differences between the cytogenetic profiles of leukemic and nodal MCL suggest that these karyotypic abnormalities contribute to the clinical and biologic behavior of MCL. 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