James R. Cook, MD, PhD, 1 Sofia Shekhter-Levin, PhD, 2 and Steven H. Swerdlow, MD 3. Abstract

Transcription

1 Hematopathology / ROUTINE CYTOGENETICS FOR SUSPECTED LYMPHOMA Utility of Routine Classical Cytogenetic Studies in the Evaluation of Suspected Lymphomas Results of 279 Consecutive Lymph Node/Extranodal Tissue Biopsies James R. Cook, MD, PhD, 1 Sofia Shekhter-Levin, PhD, 2 and Steven H. Swerdlow, MD 3 Key Words: Cytogenetics; Lymphoma; Lymph node biopsies; DOI: /NAFYB0XW0V3G2JD5 Abstract Classical cytogenetic studies have a critical role in the diagnosis of acute leukemias; however, they are much less widely used in lymphoma diagnosis. To evaluate their utility in this latter setting, G-banded karyotyping was performed on 279 consecutive lymph node or tissue biopsy specimens with suspected lymphoma. Complete cytogenetic studies were successfully obtained in 177 cases (63.4%), including 115 (69.3%) of 166 hematolymphoid neoplasms. Success rates varied with the specific diagnosis (range, 33%-100%). The karyotypes were abnormal in 97 (84.3%) of the hematolymphoid neoplasms. In at least 3 cases of non-hodgkin lymphoma, the findings contributed directly to the specific diagnosis made. In a much larger proportion of cases, characteristic but nonspecific findings were identified. Abnormalities of suggested prognostic importance in follicular lymphoma and in diffuse large B-cell lymphoma were identified in 14 (44%) of 32 cases and 8 (24%) of 33 cases, respectively. Most karyotyped lymphomas will display abnormal findings including many that are not completely specific but support the diagnosis, some that provide additional prognostic information, and, infrequently, some that help establish a diagnosis that might otherwise have been missed. The current World Health Organization (WHO) classification of hematolymphoid neoplasms is based on a multidisciplinary approach that integrates morphologic and immunophenotypic findings with additional ancillary data, often including cytogenetic findings. 1 Classical cytogenetic studies of bone marrow specimens have been used routinely at many institutions for many years, and karyotypic findings are required to classify the acute myeloid leukemias with certainty using the WHO classification. Experience with routine cytogenetic analysis of diagnostic lymph node biopsy specimens or extranodal tissue biopsy specimens for suspected lymphoma has been much more limited, and it is not a widely used diagnostic test. This is, at least in part, due to insufficient data in the literature to indicate how often, and in what setting, routine cytogenetic studies will be successful and will contribute useful diagnostic or prognostic information. There is, however, a large body of literature describing variably specific cytogenetic abnormalities in many types of lymphomas, including some that are reported to be of prognostic significance. 2-6 We reviewed the results of routine classical cytogenetic studies in 279 consecutive diagnostic lymph node and extranodal tissue biopsy specimens submitted for hematopathologic evaluation. In addition to assessing the overall frequency of complete and abnormal studies, the findings were analyzed based on the ultimate diagnosis. Specific attention was given to how often and to what extent the results helped establish the diagnosis, supported the diagnosis, or provided information reported in the literature to be of prognostic importance. Finally, novel translocations were catalogued. 826 Am J Clin Pathol 2004;121: DOI: /NAFYB0XW0V3G2JD5

2 Hematopathology / ORIGINAL ARTICLE Materials and Methods Case Selection This report includes all cases with cytogenetic studies from a 16-month period during which all diagnostic lymph node biopsy specimens and extranodal tissue biopsy specimens received for a complete hematopathologic evaluation were submitted for classical cytogenetic studies as long as there was sufficient material. In some cases in which the initial histologic sections and flow cytometric studies were definitively benign, cytogenetic studies were canceled; such cases were excluded from this study. Diagnoses were made according to WHO criteria 1 based on the histologic findings (available in all cases) together with the results of flow cytometric studies (available in 274 cases [98.2%]), paraffin section immunohistochemical analysis (performed in 205 cases [73.5%]), genotypic studies (performed in 38 cases [13.6%]), and fluorescence in situ hybridization (FISH) studies (performed in 12 cases [4.3%]). In the analysis of follicular lymphoma (FL), grade 1 and grade 2 cases were grouped together (grade 1-2). All reports were reviewed to subjectively evaluate the role of the cytogenetic findings in making the final diagnoses. Cytogenetic Analysis Tissue samples were minced as finely as possible using sterile scissors and forceps. The resulting suspension was cultured in flasks, with caps loosened, in Change B culture media in a carbon dioxide incubator for 24 hours. Ethidium bromide (10 µg/ml concentration) and colcemid (0.1 µg/ml concentration) were added into the flasks before cell harvesting to ensure accumulation of metaphase plates with elongated chromosomes. Cell harvests were performed using standard procedures as previously described. 7 Metaphase chromosomes were trypsin-giemsa banded, karyotyped, and identified according to the International System of Human Cytogenetics Nomenclature. 8 Cytogenetic studies were classified as complete when clonal abnormalities were identified or when at least 20 metaphases were examined without demonstrable clonal abnormalities. Cases lacking clonal abnormalities but with fewer than 20 metaphases available for examination were considered suboptimal, in accordance with standard cytogenetic practice. Results were analyzed based on the final pathologic diagnoses. In addition, abnormal karyotypes obtained in cases of FL and DLBCL were reviewed, and abnormalities of reported prognostic significance were tabulated. Finally, to evaluate the role of classical cytogenetic analysis as a screening tool for novel abnormalities, we examined successfully obtained karyotypes for the presence of isolated balanced translocations not previously reported in the literature. Statistical Analysis Data were analyzed by using the Student t test or Fisher exact test, as indicated, using GraphPad Prizm software (GraphPad Software, San Diego, CA). Results Yield of Cytogenetic Analysis Of the 279 specimens submitted for classical cytogenetic studies, complete cytogenetic studies were obtained in 177 cases (63.4%) and of those, 105 cases (59.3%) demonstrated karyotypic abnormalities. Among the 166 hematolymphoid neoplasms examined, 115 cases (69.3%) had complete cytogenetic studies, and of those, 97 cases demonstrated abnormalities (84.3%). The remaining cases included 84 cases of benign lymphoid hyperplasia, 11 cases of atypical lymphoid hyperplasia, and 18 cases of other, nonhematolymphoid neoplasms. The yield of analysis varied significantly with the specific diagnosis made Table 1. In addition to the cases with complete cytogenetic studies, 2 suboptimal studies with fewer than 20 metaphases available for examination (1 FL and 1 CD10+ diffuse large B-cell lymphoma [DLBCL]) each demonstrated a nonclonal t(14;18)(q32;q21) that was considered of potential significance. Complete cytogenetic studies were obtained in a significantly higher proportion of cases of hematolymphoid neoplasia than in lymphoid hyperplasias (115/166 [69.3%] vs 45/84 [54%]; P =.02; Fisher exact test). Specific Abnormalities Identified Diffuse Large B-Cell Lymphoma Complete cytogenetic studies were obtained in 33 of the 47 DLBCL cases analyzed Table 2. Of the remaining 14 cases, 10 demonstrated no growth, and 4 were suboptimal with 1 to 15 metaphases (median, 5 metaphases) lacking clonal abnormalities. Overall, the most common abnormalities (>10% of cases) identified in DLBCL were as follows: +mar (12 [36%]); +X and Y (7 [21%] each); +7, +21, t(14;18)(q32;q21), and del(6q) (5 [15%] each); and +8, +11, 15, t(8;14)(q24;q32), del(11q), and add(1p) (4 [12%] each). To determine whether cases of DLBCL with a germinal center phenotype displayed different karyotypic abnormalities from other cases of DLBCL, we compared the findings in CD10+ and CD10 DLBCL. CD10 cases were more likely than CD10+ cases to yield a complete cytogenetic study, although this difference did not reach statistical significance (22/27 [81%] vs 11/20 [55%]; P =.06; Fisher exact test). The presence of either t(14;18) or trisomy 7 was associated significantly with the expression of CD10 (P =.03; Am J Clin Pathol 2004;121: DOI: /NAFYB0XW0V3G2JD5 827

3 Cook et al / ROUTINE CYTOGENETICS FOR SUSPECTED LYMPHOMA Table 1 Yield of Classical Cytogenetic Analysis by Diagnosis * Diagnosis No. of Cases Complete Abnormal Benign lymphoid hyperplasia (54) 1 (2) Atypical lymphoid proliferations 11 8 (73) 0 (0) B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma 16 7 (44) 7 (100) Lymphoplasmacytic lymphoma 1 1 (100) 0 (0) Splenic marginal zone lymphoma 4 4 (100) 2 (50) Plasma cell neoplasm 2 0 (0) Extranodal marginal zone B-cell lymphoma (MALT lymphoma) 10 8 (80) 7 (88) Nodal marginal zone B-cell lymphoma 5 4 (80) 4 (100) Follicular lymphoma (89) 31 (97) Grade (87) 19 (95) Grade 3A (92) 11 (100) Grade 3B 1 1 (100) 1 (100) Mantle cell lymphoma 3 3 (100) 3 (100) Diffuse large B-cell lymphoma (70) 31 (94) CD (55) 10 (91) CD (81) 21 (95) B-cell lymphoma, not otherwise specified 3 1 (33) 1 (100) Atypical Burkitt lymphoma 3 2 (67) 2 (100) Lymphoblastic lymphoma, precursor T-cell type 2 1 (50) 1 (100) Peripheral T-cell lymphoma 7 6 (86) 5 (83) Angioimmunoblastic T-cell type 3 3 (100) 2 (67) Peripheral T-cell, unspecified 2 2 (100) 2 (100) Anaplastic large cell type, ALK 2 1 (50) 1 (100) Hodgkin lymphoma, nodular lymphocyte predominant 3 2 (67) 0 (0) Hodgkin lymphoma, classical (45) 3 (30) PTLD, monomorphic 1 1 (100) 0 (0) Acute myeloid leukemia 1 0 (0) Other tumors, nonhematolymphoid 18 9 (50) 7 (78) ALK, anaplastic lymphoma kinase; MALT, mucosa-associated lymphoid tissue; PTLD, posttransplant lymphoproliferative disorder. * Data are given as number (percentage). Includes cases diagnosed as angioimmunoblastic T-cell lymphoma (AITL) or AITL-like T-cell lymphoma. Fisher exact test). The other abnormalities showed no significant difference between CD10+ and CD10 DLBCL. The 1 case of t(14;18)(q32;q21) in a CD10 DLBCL was shown by further FISH analysis to represent an IGH/MALT1 rather than an IGH/bcl-2 translocation, as previously described. 9 Follicular Lymphoma Complete cytogenetic studies were obtained in 32 of 36 cases of FL Table 3. Of the remaining 4 cases, 3 demonstrated no growth, and 1 contained 4 metaphases with only a single cell containing a t(14;18)(q32;q21). The mean ± SD number of abnormalities identified per abnormal karyotype was 6.1 ± 0.85 and was higher in grade 3 than in the grade 1-2 FL group (9.4 ± 1.6 vs 3.9 ± 0.6; P <.001; Student t test). The most common abnormalities identified (>10% of cases) were as follows: t(14;18)(q32;q21) (27 [84%]); +mar (8 [25%]); +7 and +12 (7 [22%] each); +X (6 [19%]); and +5, +8, +21, del(5q), and del(10q) (4 [13%] each). Abnormalities with breakpoints at 1p36.1 or 1q21, previously reported as frequent secondary changes in FL, 10 were identified in 4 (13%) and 3 (9%) of the cases, respectively. The finding of t(14;18) was more common in the grade 1-2 group (18/20 [90%]) than in grade 3 FL (9/12 [75%]), although this difference was not statistically significant (P =.27; Fisher exact test). The t(3;14)(q27;q32), presumably involving the bcl-6 gene, was identified in 2 grade 3 FLs and in none of the grade 1-2 FLs. Marginal Zone Lymphoma Of the 19 cases of marginal zone lymphoma (MZL) analyzed, 16 yielded complete cytogenetic studies, including 4 of the nodal type, 4 of the splenic type, and 8 of the extranodal mucosa-associated lymphoid tissue (MALT) type Table 4. The 3 remaining MZLs demonstrated no growth (1 nodal and 1 MALT) or yielded a suboptimal study (1 MALT with only 9 metaphases available). The mean ± SD number of abnormalities per abnormal karyotype was lower in MALT lymphomas (1.7 ± 1.1) than in splenic MZL (4.5 ± 3.5) and nodal MZL (4.0 ± 1.6), although this difference did not reach statistical significance (P =.15 and P =.11, respectively; Student t test). The only recurring abnormalities within this category were: +X (1 splenic and 1 nodal MZL), +3 (1 nodal MZL and 2 extranodal MALT cases), and +18 (2 nodal MZLs and 2 extranodal MALT cases). The t(1;14)(p22;q32), believed to be specific for MALT lymphoma, 3,11,12 was identified in 1 case of pulmonary MALT lymphoma. Two cases of splenic MZL showed partial or complete loss of chromosome Am J Clin Pathol 2004;121: DOI: /NAFYB0XW0V3G2JD5

4 Hematopathology / ORIGINAL ARTICLE Table 2 Diffuse Large B-cell Lymphomas With Complete Cytogenetic Studies Case No. CD ,X, Y,inv(3)(p11.2q27),add(14)(q32)[cp3] ,XX,dup(1)(q42q25),dup(2)(p21p23),t(9;12)(p13;p13),del(13)(q14q22),+21[8]/47,XX,+9[2]/46,XX[11] ,X, Y,t(9;22)(p13;p11.2),+mar[14]/46,X, Y,idem,del(12)(p11.2p12.2)[9] ,XX,+X,add(1)(p36.3),inv(1)(p13q25)del(1)(q25)x1-2,+del(1)(p13p36.1)x1-2,del(2)(q31q35), 3 or inv(3)(q21q26)add(3)(p12),+4, 5,del(6)(q15),dup(7)(q31q34),t(8;14)(q24;q32), 10,del(11)(q23),+12x1-2,add(12)(p12),add(19)(p13.3),del(20)(q11.2q13.3), +1-3mar[cp+1-11]/46,+1-XX[8] ,XX,t(2;11)(q21;p11.2)[12],der(4;10)(p10;q10)[12],t(6;19)(p21.3;p13.3)[12],i(6)(p10)[12],+6[12],t(8;14)(q24;q32)[12],+8[3], t(9;12)(q12;q13)[12],add(14)(p11.2)[12],del(16)(q22q24)[11][cp12]/46,xx[8] ,XY,add(8)(q24)[2]/46,XY[5] ,XXYY,i(1)(q10), 2,del(3)(p11.2p21),inv(4)(p12q21), 4,add(6)(q23)x2,add(8)(q24),add(9)(p22)x2, 11, 11, 13,t(13;14)(p10;q10), 15, 16,add(16)(p12),i(17)(q10),add(17)(q23)x12,der(19)t(11;19)(q13;q13.3),add(20)(p13), 21, 22, 22,add(22)(p13), +mar[cp17]/46,xy[3] ,XX,t(6;10)(p23;p13)[4]/46,XX[6] ,XXY, Y,del(1)(p13)x2,del(3)(q21),add(4)(q21), 14,add(14)(q32),add(15)(q26),+16,add(17)(p13)x2[cp7]/46,XY[3] ,XY,t(5;17)(p15.3;q12)[12]/46,XY[6] ,t(X;2)(p22.1;q31), X[15]/46,XX[5] ,X, Y,t(1;2)(p13;p23)[4]/45,X, Y[15] ,XXXX,+X,add(8)(p11.2)x2[3],del(11)(q13)x2[2],add(11)(q23)x3[1],add(13)(q34)x2,add(14)(q32)x2,+19,+1-4mar[3][cp3]/46,XY[17] ,XX[19] ,XX, 8,der(8)t(6;8)(p11.2;p23),der(11)t(8;11;15)(q13;q13;q24),der(15)t(8;11;15)(q13;q13;q24),der(20)t(12;20)(q13;q13.3), +r[13]/46,xx[7] ,XY,+X,del(1)(p34.3),del(7)(q22q32),+8,add(10)(q26),add(12)(q24)[cp18]/46,XX[2] ,XX,+X,del(6)(q13q27),der(6)t(6;17)(p21.3;q11.2),+idic(9)(q12),dup(11)(q13q13), 17,+21[20],+mar[7][cp20] ,X,+X,+X, Y,add(5)(q35),+9,add(12)(q24), 12,+mar1,+mar2[cp11]/46,XY[9] ,XY,+X,t(8;14)(q24;q32),del(11)(q13q23)[7]/46,XY,t(8;14)(q24;q32),del(11)(q13q23),t(11;17)(q13;q21)[8]/46,XY[6] ,XY,t(3;12)(q23;p13)[2] ,X, Y,+3,del(6)(q13q25)x2,+der(6)t(1;6)(q12;p21.3),+7,+7,+12,der(14)t(14;?)(q32;?),+20[cp11]/46,XY[5] ,XX,+3,del(6)(q15q21),+11,t(14;18)(q32;q21),der(14;21)(p10;q10), 15, 15,der(22)t(1;22)(q21;p11.2),+mar[20] CD ,XY,+mar[7]/46,XY[11] ,X, X,del(2)(p23),add(2)(p25)[12-16],add(4)(p15.2),+4[59],add(8)(p23),+12,dup(13)(q12q34),add(13)(q32),add(13)(q32), t(14;18)(q32;q21), 15, 17,hsr(21)(q22), 22,+1-2mar[14-16][cp16]/46,XX[4] ,X, Y,del(1)(q42),add(2)(q37), 6,+7,del(9)(p13),add(13)(q34)x2[16],add(15)(p11.2)[4],i(18)(q10)[16],+add(18)(p11.3), i(21)(q10),+21,der(21;22)(p10;q10)[3-9],+22[14],+mar[9][cp16]/46,xy[4] ,X, X[11]/46,XX[9] ,XX[5],t(X;15)(p22.1;q15)[8],add(1)(p36)[8],del(4)(q12)[8],+7[12],t(8;14)(q24;q32)[14],t(14;18)(q32;q21)[14],add(15)(q15)[5], i(17)(q10)[14],add(17)(q25)[9],+18[9],del(22)(q11.2)[3], 22[3][cp14]/46,XX[6] ,XX[4],X[2],add(2)(q33)[7],add(2)(q33)[6], 3,+7[3],+8[2], 9, 9,+11[3],del(11)(q23)[5],del(11)(q23), 14,t(14;18)(q32;q21)[3], t(14;18)(q32;q21)[1],+17,+17[2],+18[4],+18[3],der(18)t(14;18)(q32;q21)[1],+20[2],+1-10mar[7][cp7] ,XX,add(6)(q27)x2,add(7)(q36),+7,+8,t(8;22)(q24;q11.2),del(9)(p22),+11, 14,t(14;18)(q32;q21), 15,+21,+21,add(21)(q22), add(22)(q13),+2-6mar[cp3]/46,xx[2] ,XX[25] ,XX,+X,add(1)(p36.3),+3,t(7;7)(p22;q32),+11,ins(12;?)(q13;?),+13,+21[10]/51,idem,der(3)t(1;3)(q21;q27)[4] 88 46,XY,trp(11)(q13q23),dup(21)(q21q22)?hsr(21)(q22)[20] ,XXXX,add(1)(p36.3)x2,add(3)(p11.2),del(3)(p11.2),del(6)(q23)[cp4]/46,XX[8] Other B-Cell Lymphomas Complete cytogenetic studies were obtained in 15 of 27 cases of other B-cell lymphomas Table 5. Two cases of atypical Burkitt lymphoma each demonstrated relatively simple karyotypes including t(8;14)(q24;q32). A third case of atypical Burkitt lymphoma demonstrated no growth. Each of 7 successfully studied cases of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) was cytogenetically abnormal. The only recurring abnormality identified in the CLL/SLL cases was trisomy 12 (4 cases). The remaining 9 cases of CLL/SLL demonstrated no growth (3 cases) or were suboptimal (6 cases with 1 to 11 metaphases; median, 8 metaphases). Each of 3 cases of mantle cell lymphoma was karyotypically abnormal with findings that included t(11;14)(q13;q32). One B-cell lymphoma that could not be further classified demonstrated a complex karyotype with numerous nonspecific abnormalities. Two additional cases of unclassifiable B-cell lymphoma yielded suboptimal studies with 6 and 8 metaphases available. T-Cell Lymphomas Complete cytogenetic studies were obtained in 7 of 9 T- cell lymphomas submitted for analysis Table 6. The remaining cases, an anaplastic lymphoma kinase negative anaplastic large cell lymphoma (ALCL) and a γδ T-cell lymphoblastic lymphoma, demonstrated no growth. Of the cases with complete studies, 1 was diagnosed definitively as Am J Clin Pathol 2004;121: DOI: /NAFYB0XW0V3G2JD5 829

5 Cook et al / ROUTINE CYTOGENETICS FOR SUSPECTED LYMPHOMA Table 3 Follicular Lymphomas With Complete Cytogenetic Studies Case No./ Grade Grade ,XY[20] ,XY,t(14;18)(q32;q21),+18[5] ,XX,del(9)(p22)[6]/46,XX[13] ,XY,t(14;18)(q32;q21),+21[2]/47,idem,del(1)(p32p36.1)[2]/50-51,idem,+X,+del(1)(p32p36.1),+12,+21[cp12]/46,XY[4] ,XY,add(1)(p36),t(3;6)(p10;q10),del(4)(q21q25),del(10)(q24),del(13)(q14q22),t(14;18)(q32;q21)[8]/46,XY[11] ,X, Y,+X,+12,t(14;18)(q32;q21),add(14)(q32)[10]/46,XY[8] ,XX,i(6)(p10),+7,inv(12)(p11.2q13),+12,t(14;18)(q32;q21),add(21)(p11.2),+mar[16]/46,XX[4] ,X, X,t(14;18)(q32;q21)[2]/45,X, X,dup(1)(q21;q23),t(14;18)(q32;q21)[12]/90,XX, X, X,dup(1)(q21q23)x2, t(14;18)(q32;q21)x2[2]/46,xx[4] ,XY,+X,t(14;18)(q32;q21)[2]/47,XY,+8,t(14;18)(q32;q21)[5]/46,XY[11] ,XY,del(6)(q15q25)[9],del(6)(q15q25)[6],add(7)(p22)[6], 12[8],t(14;18)(q32;q21)[9],+mar1[7],+mar2[8],+mar3[2][cp9]/46,XY[11] ,XY,t(14;18)(q32;q21)[19] ,XX,t(14;18)(q32;q21)[16]/46,XX[3] ,XY,t(1;10)(q42;q24),del(2)(p23),t(14;18)(q32;q21),+der(18)t(14;18)(q32;q21)[3]/46-48,XY,t(1;10)(q42;q24), 2, 12, t(14;18)(q32;q21),+der(18)t(14;18)(q32;q21),+der(?)t(?;12)(?;11.2)x1-2,+mar[cp15]/46,xy[2] ,XY,del(5)(q22q31),add(9)(p24),t(14;18)(q32;q21),+der(18)t(14;18)(q32;q21)[14]/46,XY[6] ,X, X,t(14;18;22)(q32;q21;q11.2)[3]/44-48,X, X,+5[7],i(6)(p10),+6,+7,t(14;18;22)(q32;q21;q11.2),add(22)(q13)[cp14]/46,XX[3] ,XY,t(14;18)(q32;q21)[7]/46,XY[3] ,X,add(X)(q21.3),add(2)(q34), 6,t(14;18)(q32;q21),add(17)(p11),+mar1,+mar2[11]/46,XX[8] ,XY,add(1)(p36.3),t(14;18)(q32;q21)[3]/46,XY[3] ,XY, 13,der(14)t(14;18)(q32;q21)del(14)(q13q22),der(18)t(14;18)(q32;q21),+r[7]/46,XY[8] 5675A 46,XY,t(14;18)(q32;q21)[2]/46,XY[4] Grade 3A 63 49,XX,+X,+X,add(1)(q42)x2,t(3;14)(q27;q32),+21[6]/50,idem,+7[4]/46,XX[9] ,XY,+Y,der(1;6)(p10;q10),+8,+20,+21[cp15]/46,XY[4] ,XY,+X,dup(1)(q21q44),+7,t(14;18)(q32;q21)[13]/46,XY[7] ,XX,t(6;8)(q23;p23),t(14;18)(q32;q21)[16]/46,XY[4] ,XX,add(4)(p16),del(5)(q12q33),+5,t(14;18)(q32;q21),add(15)(p11.2),+16[7]/49-50,XX,idem,+2[6],del(2)(q11.2q31)[3], +12[4][cp7]/46,XX[6] ,XX,del(3)(q12q21),t(14;18)(q32;q21)[6]/48,idem,+7,+9,del(10)(q24q26) ,X, X,der(2;12)(p10;q10),del(4)(q21q25),del(5)(q22q35),der(6;17)(p10;q10),dup(7)(q32q36)x2,+7,del(9)(q12q22), 9,del(10)(q24q26),+10,t(14;18)(q32;q21),+17,+18,+18,add(19)(q13.3),del(22)(q12),+mar[cp21] ,XXYY,t(1;19)(q23;p13.3)x2[14],del(3)(q21q25)x2[14],add(4)(q35)[4], 7[6],inv(9)(p11q13)x2[15], del(10)(q24)x2[14],t(14;18)(q32;q21)x2[15], 14[3],del(15)(q24)[10],del(15)(q24)[4], 17[4],der(19)t(1;19)(q23;q13.3)[3], 20[4],+mar1[4],+mar2[4][cp16]/46,XY[3] ,XY,+del(5)(q11.2q33),del(6)(q21),+del(6)(q21),+del(7)(q22q36), 13,t(14;18)(q32;q21),+16,+17,+18,add(19)(q13), +20,+21[cp12]/46,XY[6] 8977A 68-77,XY,+X,+Y,inv(1)(p22p36.1)x1-2,del(1)(p22p36.1),+del(1)(q11.2),add(2)(q21),+3,+5,+7,+8,+11,+12,t(14;18)(q32;q21)x2, 9901A add(17)(p11.2),add(18)(q22),+22,+mar[cp5] 86-91,XXYY,add(2)(q31),add(4)(p16)x2,+5,add(6)(q21)x2,del(8)(p21)x2, 8, 8,t(9;14;18)(p24;q32;q21)x2,+9,+9, 10,del(12)(q22)x2,+12,+12,add(13)(p11.2)x2, 14, 14, 15, 15, 16,+19,+mar1,+mar2,+mar3[cp16]/46,XY[4] Grade 3B ,XY,dup(1)(q21q12),t(3;14)(q27;q32),del(6)(q21q25)[19],+8[2],+12,add(12)(q24.3),del(13)(q12q14)[19][cp19]/46,XY[1] angioimmunoblastic T-cell lymphoma (AITL), while 2 others displayed AITL-like features. The karyotypes in these cases were less complex than those obtained for the 2 cases of peripheral T-cell lymphoma, unspecified (PTCLU), or the 1 case of anaplastic lymphoma kinase negative ALCL. The only recurring abnormalities within this category were: +5 (1 AITL-like case and 1 ALCL), del(1p) (1 ALCL and 1 PTCLU), and del(3q) (1 AITL-like case and 1 PTCLU). Hodgkin Lymphoma Twelve cases of Hodgkin lymphoma yielded complete cytogenetic studies, with 9 demonstrating an apparently normal chromosome complement Table 7. An additional 13 cases demonstrated no growth (7 cases) or were suboptimal studies (6 cases with 1-19 metaphases; median, 12 metaphases). Abnormalities were noted in 3 cases of classical Hodgkin lymphoma and in none of the cases of nodular lymphocyte predominant Hodgkin lymphoma analyzed. The only recurring abnormality noted was trisomy 5, which was present in 2 cases. Reactive Hyperplasia and Nonhematopoietic Neoplasms Among the 84 cases of reactive hyperplasia, 21 cases demonstrated no growth and another 18 cases were suboptimal studies with 1 to 19 metaphases obtained (median, 13 metaphases). Of the 45 cases of benign lymphoid hyperplasia for which complete cytogenetic studies could be obtained, 44 cases demonstrated only normal findings. In the 830 Am J Clin Pathol 2004;121: DOI: /NAFYB0XW0V3G2JD5

6 Hematopathology / ORIGINAL ARTICLE Table 4 Marginal Zone Lymphomas With Complete Cytogenetic Studies Case No./Tumor Type and Location Mucosa-associated lymphoid tissue 3214, lung 49,XY,t(1;14)(p22;q32),+3,+12,+18[11]/46,XY[7] 786, orbit 47,XY,del(3)(p12p21.1),+3[6]/46,XY[13] 794, orbit 48,XX,+18,+mar[cp9] 1001, orbit 46,XY[20] 4045, orbit 46,XX,del(11)(?q21)[7]/46,XX[12] 7768, orbit 47,XX,inv(9)(p11q13),+10[16]/46,XX,inv(9)(p11q13)[4] 7769, parotid 46,XY,i(18)(q10)[8]/46,XY[10] 8023, parotid 46,XX,add(11)(p15)[cp2] Nodal ,X,del(X)(q22q26),add(1)(p36.3),dup(6)(p23p25)[11-13],dup(10)(p13p12)[3],del(16)(q13q22),+18, der(19)t(13;19)(p13.3;q14.3),+21[13-15][cp15]/46,xx[4] 1357* 46,X,t(X;5)(q26;q15)[16]/46,XX[3] ,XY,der(1)t(1;1)(p36.1;q21),der(8)t(3;8)(q11.2;p11.2)[17]/46,XY[3] ,XX,+3,del(11)(q23),del(13)(q12q14),+18[8]/47,XX,+X[2]/46,XX[11] Splenic ,XX+X,del(6)(q12q23), 7,add(8)(p23), 17,add(20)(q13.3)[cp8]/81-89,XXXX,+X,del(6)(q12q23)x2, 7, 7,add(8)(p23)x2, i(13)(q10), 17, 17,add(20)(q13.3)x2[cp3]/46,XX[13] ,XX[20] ,XY[20] ,XX,del(7)(q22q34)[13]/46,XX[7] * As described in detail elsewhere, 14 the following revised karyotype was assigned following additional fluorescence in situ hybridization studies: 46,X,t(X;5)(q28;q22). Table 5 Other B-Cell Lymphomas With Complete Cytogenetic Studies Case No./ Diagnosis Atypical Burkitt ,XY,t(8;14)(q24;q32)[10] ,XY,t(8;14)(q24;q32),t(9;22)(p24;q11.2)[7]/46,XY[11] CLL/SLL ,XX,+12,add(14)(q32)[4]/47,idem,add(3)(p25)[5]/46,XX[6] ,XY,+12[11]/46,XY[9] ,XY,inv(1)(p13q44),del(6)(q23q25)[3] ,XY,i(17)(q10)[3]/45,XY,i(17)(q10),add(9)(q34),dic(19;21)(p13.3;p11.2)[cp12]/46,XY[5] ,XX,+12[3]/46,XX[17] ,XY,+12[17]/47,XY,add(11)(q25),+12[cp2] ,XY, 14,+r[4]/46,XY,t(7;10)(q22;q24),del(13)(q12q14),del(14)(q22q31)[2]/46,XY[13] LPL ,XY[20] Mantle cell ,XY,add(10)(q24),t(11;14)(q13;q32)[5]/46,XY[5] ,XX,add(3)(q27),t(9;18)(p13;p12),t(11;14)(q13;q32)[7]/46,XX[13] ,XY,add(1)(p36.3),add(1)(p22),del(2)(q33),add(2)(q37), 5,add(5)(q37), 7,t(11;14)(q13;q32),+17,+20,+1-2mar[cp9]/46,XY[7] PTLD, monomorphic, DLBCL type ,XY[18] B-cell lymphoma, NOS ,XY,+X[3]/50,XY,+X,+X,trp(1)(q21q32)[16],add(1)(p36.3)[11],+2[16],add(2)(p23),+der(3;4)(p10;q10)[11], add(6)(p25)[16],+12[5],der(14;15)(p10;q10)t(14;?)(q32;?)[16],+18[11],i(21)(q10),add(22)(p11.2)[16],+mar[16][cp16] CLL/SLL, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma; DLBCL, diffuse large B-cell lymphoma; LPL, lymphoplasmacytic lymphoma; NOS, not otherwise specified; PTLD, posttransplant lymphoproliferative disorder. remaining case, a tonsillectomy specimen, the histopathologic and flow cytometric analyses demonstrated lymphoid hyperplasia. Genotypic studies did not demonstrate evidence of a clonal B-cell or T-cell population by polymerase chain reaction or Southern blot studies. Cytogenetic analysis, however, displayed the following karyotype: 46,XY,t(12;13)(q13;q32)[4]/46,XY[13]. The patient was otherwise well, and the significance of this cytogenetic abnormality identified remains unclear. Complete cytogenetic studies also were obtained for 7 cases of metastatic carcinoma, 1 poorly differentiated malignant neoplasm of uncertain lineage, and 1 benign thymoma. Apparently Am J Clin Pathol 2004;121: DOI: /NAFYB0XW0V3G2JD5 831

7 Cook et al / ROUTINE CYTOGENETICS FOR SUSPECTED LYMPHOMA Table 6 T-Cell Lymphomas With Complete Cytogenetic Studies Case No./ Diagnosis T-cell lymphoma Angioimmunoblastic ,XY[16] Angioimmunoblastic-like 86 47,XY,+5[4]/46,XY[16] ,XX,del(3)(q12q21)[7]/46,XX[13] Peripheral, unspecified ,Y, X,del(1)(p13p22),inv(2)(p11.2p13),del(3)(q11.2q25),+3,der(4)add(4)(p16)add(4)(q25),del(5)(q13q22), del(6)(q21q25),del(8)(q21.1q22),dup(12)(q13q24.1),add(13)(q34),add(16)(p13),+mar[18]/46,xy[5] ,XY,+5,add(6)(p23),inv(11)(q14.2q23),+19,add(19)(p13.3),add(21)(q22),+22[14]/50,idem,+add(21)(q22)[2]/46,XY[1] Anaplastic large cell lymphoma, ALK ,X,t(X;6)(p11.2;p23),del(1)(p22p36),del(7)(q22q34),i(17)(q10), 22,+1-4mar[cp5]/89-93,idemx2[2]/46,XX[14] Precursor T lymphoblastic lymphoma ,XX,add(7)(p11.2)[12]/46,XX[6] ALK, anaplastic lymphoma kinase. Table 7 Hodgkin Lymphomas With Complete Cytogenetic Studies Case No/Type Classical ,XX[20] ,XY[20] ,XY[20] ,XY[19] ,XY,del(13)(q12q14)[5]/46,XY[14] ,XX,+5,+9,+10,+15,+18,+19,+21,+22[3]/46,XX[17] ,XY[19] ,X, X,+i(2)(q10),add(4)(q35),+5,+i(5)(p10), der(7)t(1;7)(q21;q34),+i(9)(p10),del(10)(p12.2), add(13)(p13),+add(19)(p13.3), 21,add(22)(p13), +2r[cp5]/46,XX[16] ,XY[20] ,XX[20] Nodular lymphocyte predominant ,XY[20] ,XY[18] Table 8 Abnormalities of Suggested Prognostic Significance in Follicular Lymphoma * Cytogenetic Abnormality Grade 1-2 (n = 20) Grade 3 (n = 12) All (n = 32) 6 breaks 3 (15) 8 (67) 11 (34) 1p21~22 0 (0) 1 (8) 1 (3) 6q23~27 1 (5) 4 (33) 5 (16) 17p 1 (5) 2 (17) 3 (10) t(14;18) + c-myc 0 (0) 0 (0) 0 (0) Any of the above 5 (25) 9 (75) 14 (44) * Data are given as number (percentage). P =.006; grade 1-2 vs grade 3. P =.01; grade 1-2 vs grade 3. normal karyotypes were found in 1 case of carcinoma and in the thymoma, while the remaining cases each demonstrated complex karyotypes with numerous numeric and structural abnormalities (data not shown). Impact on Diagnosis Made In 3 cases, the final diagnosis was based explicitly, in part, on the cytogenetic abnormalities identified (2.6% of hematolymphoid neoplasms successfully karyotyped). Two of these cases were B-cell lymphomas that displayed high-grade features but greater morphologic pleomorphism than classic Burkitt lymphomas. Following identification of t(8;14)(q24;q32) in each case, either as an isolated finding or as part of a simple karyotype, a diagnosis of atypical Burkitt lymphoma was made. In the third case, a diagnosis of splenic MZL was made based in part on the demonstration of del(7)(q22q34), an abnormality associated with up to 40% of splenic MZLs. 13 In the remaining cases with abnormal karyotypes (81.7% of hematolymphoid neoplasms karyotyped), the findings were considered to provide variably significant additional, although nonspecific, support for the final diagnosis issued. For example, t(14;18)(q32;q21) was identified in 84% of cases of FL (27/32), strongly supporting that diagnosis. In no case did the results of cytogenetic analysis alter a diagnosis that had been issued before completion of such studies. Cytogenetic studies also did not assist in further clarifying the 11 cases of atypical lymphoid proliferations or further classifying the 3 cases of B-cell lymphoma, not otherwise specified, in this series. Prognostic Information in Abnormal s As shown in Table 8, 44% (14/32) of cases of FL demonstrated at least 1 of the following abnormalities that have been reported to be of prognostic significance: 6 or more chromosome breaks, 1p21~22 abnormalities, 6q23~27 abnormalities, 17p abnormalities, or the presence of both t(14;18)(q32;q21) and a rearrangement of c-myc at 8q24. 3,4 These abnormalities were more likely to be present in grade 3 cases than in grade 1-2 cases (P =.01; Fisher exact test). 832 Am J Clin Pathol 2004;121: DOI: /NAFYB0XW0V3G2JD5

8 Hematopathology / ORIGINAL ARTICLE Table 9 Abnormalities of Suggested Prognostic Significance in 33 Diffuse Large B-Cell Lymphomas Cytogenetic Abnormality In DLBCL, 24% of cases (8/33) demonstrated one or more of the following abnormalities reported to be of prognostic significance: abnormalities of chromosome 1 at band 1q21~q23, del(6)(q21~25), trisomy 6, or the presence of both t(14;18)(q32;q21) and a c-myc rearrangement at 8q24 3,5,6 Table 9. Comparison of CD10+ and CD10 cases showed no significant difference in the incidence of these abnormalities (data not shown). Identification of Novel Abnormalities Three cases of non-hodgkin lymphoma (NHL) contained previously unreported balanced translocations as isolated karyotypic abnormalities (2.6% of cases of hematolymphoid neoplasms with complete cytogenetic studies). One of these cases, a nodal MZL with t(x;5)(q28;q22), has been described in detail elsewhere. 14 In addition, 1 hepatic DLBCL contained a t(6;10)(p23;p13), and analysis of 1 splenic DLBCL demonstrated a t(5;17)(p15.3;q12). Discussion No. (%) of Cases der(1)(q21~23) 2 (6) del(6)(q21~25) 4 (12) +6 1 (3) t(14;18) + c-myc 2 (6) Any of the above 8 (24) Although the cytogenetic findings in malignant lymphoma have been studied extensively for many years, 2,3 there is little information in the literature concerning the utility of the routine use of classical cytogenetic studies in the evaluation of lymph node or other solid tissue biopsy specimens for suspected lymphoma. This report demonstrates that in the course of routine practice, complete cytogenetic studies can be obtained for the majority of such specimens. The rate of successful karyotyping was higher in cases of hematolymphoid neoplasia than in benign lymphoid hyperplasia. There was also considerable variation in the rate of complete analysis depending on the type of lymphoma present. For example, 89% of FL cases (32/36) yielded complete cytogenetic studies compared with only 44% and 45% of cases of CLL/SLL (7/16) and classical Hodgkin lymphoma (10/22), respectively. These findings are similar to those in previous studies of these specific entities, 2,3 although some investigators have reported higher rates of successful analysis in classical Hodgkin lymphoma. 15,16 This series also demonstrates that in routine clinical practice, more than 80% of karyotyped malignant lymphomas will demonstrate abnormal findings, with 90% to 100% abnormal karyotypes obtained for most types of lymphoma. The lowest incidence of cytogenetic abnormalities was found in classical Hodgkin lymphoma (3/10 [30%]), consistent with the results of previous studies. 15 It is very difficult to precisely quantify the contribution of abnormal cytogenetic findings to the final diagnosis in a specific case, partially because the value of such studies will vary depending on the nature of other ancillary studies performed and the validity of the initial histopathologic evaluation. For example, in a case of FL with flow cytometric studies demonstrating CD10 expression by monoclonal B cells or a bcl-2 translocation identified by polymerase chain reaction studies, the demonstration of t(14;18)(q32;q21) by classic cytogenetic studies provides little additional diagnostic information. In the absence of such other ancillary studies or if a mistaken diagnosis of follicular hyperplasia is made, the finding of t(14;18) by classical cytogenetic studies would be of far greater value. Within the present series, performed at a tertiary medical center with ready access to a wide array of immunohistochemical, flow cytometric, and genotypic studies, classical cytogenetic analysis contributed variably important, usually noncritical additional information in many cases. Classical cytogenetic studies provided critical information in 2.6% of the successfully analyzed neoplasms. Two high-grade B-cell lymphomas were classified as atypical Burkitt lymphomas based on the presence of t(8;14)(q24;q32) as part of a simple karyotype. Because this translocation involving c-myc and IGH may be seen as a secondary abnormality in other forms of lymphoma, such as with the t(14;18)(q32;q21), knowledge of any additional karyotypic abnormalities is useful in reaching a final interpretation. The cytogenetic studies provide information, therefore, that would not be obtained by using more focused genotypic or FISH studies looking for a c-myc rearrangement alone. One splenic lymphoma was diagnosed as splenic MZL in part owing to the presence of an isolated del(7q), an abnormality reported in up to 40% of cases of splenic MZL but in fewer than 10% of other B-cell lymphomas. 13 Although FISH could have been used to identify this abnormality, such a directed investigation would not have revealed other diagnostically significant karyotypic abnormalities such as those associated with a mantle cell lymphoma, FL, or another splenic MZL mimic. 17 In a much larger proportion of cases, the karyotypic abnormalities that were identified, although not specific, provided variably significant additional support for the diagnosis made. The abnormalities documented among the specific forms of lymphoma in this unselected series were Am J Clin Pathol 2004;121: DOI: /NAFYB0XW0V3G2JD5 833

9 Cook et al / ROUTINE CYTOGENETICS FOR SUSPECTED LYMPHOMA generally similar to those reported in the literature. For example, t(14;18)(q32;q21) was identified in 84% of cases of FL (27/32) and in 15% of cases of DLBCL (5/33), and each of the cases of mantle cell lymphoma studies demonstrated the t(11;14)(q13;q32). 2,3 The three most common secondary abnormalities found in the FL cases in this series also have been reported by others in a similar proportion of cases: +7 (21%-23%), +12 (20%-30%), and +X (18%-23%). 10,18 Abnormalities with less specific but well-recognized diagnostic associations also were found, such as trisomy 12 in CLL and trisomy 3 in some MALT lymphomas. 12,19,20 Some cytogenetic abnormalities with well-described diagnostic associations, such as t(11;18)(q21;q21) with extranodal MALT lymphoma 12,21 or isochromosome 7q with hepatosplenic T-cell lymphoma, 22 were not identified in this unselected series, likely related to sample size and the overall frequency of these abnormalities. In general, the demonstration of any abnormal karyotype provides the pathologist with independent confirmation of the presence of a neoplasm; however, cases in which this is the only nonhistologic supportive evidence are relatively few. Even this observation must be interpreted with some caution, because a clonal population was identified in 1 specimen, even though no other evidence of a neoplasm could be identified. The cytogenetic studies led to a more extensive workup that could have led to the discovery of a subtle neoplasm. Other investigators have reported that cases of apparent benign lymphoid hyperplasia with clonal cytogenetic abnormalities are at very high risk for the subsequent development of an overt lymphoma, although the same authors also diagnosed a subsequent lymphoma in 7 of 17 cases in which the initial biopsies revealed benign hyperplasia, with normal or incomplete cytogenetic studies. 23 In addition, while it is reassuring not to have found cytogenetic abnormalities in any of the atypical lymphoid proliferations we examined, given that some overt neoplasms display normal karyotypes, this observation also must be interpreted cautiously. The finding of an abnormal karyotype in only 1 of 84 cases of reactive hyperplasia suggests that classical cytogenetic studies are a poor screening tool and unnecessary in the workup of cases in which the histologic and immunophenotypic findings indicate lymphoid hyperplasia. Cases also can be cultured and then held pending a decision about whether cytogenetic studies are appropriate. Classical cytogenetic studies also have a role in providing prognostic information in a moderate number of cases, at least in the more common and extensively studied NHL. Abnormalities of 1p21~22, 6q23~27, or 17p, or 6 or more chromosome breaks have been reported to be adverse prognostic indicators in FL and were identified in almost half of the FL cases in this study. These adverse prognostic indicators were more frequently identified in the grade 3 FL. In addition, there are other histologic correlations with certain abnormalities, such as trisomy 7 that has been associated with the higher grade FL 10 and transformation to DLBCL. 24 In our series, grade 3 FL cases also demonstrated a greater number of abnormalities, a greater number of chromosomal breaks, and a lower incidence of the t(14;18)(q32;q21) than did FLs in the grade 1-2 group. It should be recognized, however, that simply finding other abnormalities in addition to the t(14;18)(q32;q21) does not imply clinical or histologic disease progression. Very few FLs have the latter translocation as an isolated finding (13% in the present series; 3% in Horsman et al 10 ), although they are all grade 1-2. Similarly, abnormalities of 1q21~23, del(6)(q21~25), +6, or t(14;18) with concurrent c-myc rearrangements have been reported to be adverse prognostic indicators in DLBCL. These abnormalities were present in 24% of the DLBCLs (8/33) reported herein. Karyotypic abnormalities that might influence prognosis or have treatment implications also have been described in many other forms of NHL, 2,3 but the number of cases in many of the specific categories of NHL limited further analysis in the present series. In addition to identifying known prognostic factors, cytogenetic studies also can further elucidate the heterogeneity of DLBCL. Both the t(14;18)(q32;q21) and trisomy 7 were associated significantly with CD10+ DLBCL, a phenotype associated with DLBCL of a germinal center type gene profile. 25 In fact, the only t(14;18) identified in a CD10 DLBCL in the present series was later shown to represent an IGH/MALT1 translocation rather than the expected IGH/bcl-2 translocation. 9 Both trisomy 7 and t(14;18) are common abnormalities in FL, and their significant association with CD10+ DLBCL likely reflects transformation of an underlying FL in these cases. 3,4,26 As noted in preceding text, trisomy 7 has been shown to be a frequent abnormality in cases of FL that transform to DLBCL. 24 Another advantage of classical cytogenetic analysis, which is not possible using more directed FISH or genotypic studies, is the ability to demonstrate unexpected novel cytogenetic abnormalities. Of the 115 hematopoietic malignant neoplasms karyotyped in the present series, 3 cases (2.6%) contained novel balanced translocations. These findings are of great interest because they can lead to the discovery of previously uncharacterized oncogenes involved in lymphomagenesis. Our study demonstrated that complete classical cytogenetic studies can be obtained in a large proportion of diagnostic lymph node and extranodal tissue biopsy specimens in routine practice. Although the proportion of these unselected cases in which critical or novel information is obtained is small, in many cases the results provide variably specific support for the diagnosis made using a moderately extensive multiparameter approach to lymphoma diagnosis. 834 Am J Clin Pathol 2004;121: DOI: /NAFYB0XW0V3G2JD5

10 Hematopathology / ORIGINAL ARTICLE As illustrated, cytogenetic studies can provide prognostic information in a significant minority of cases, although further studies are required to assess the most cost-effective manner to identify these abnormalities and to determine how many are truly independent prognostic indicators. In addition, the presence of at least 1 t(14;18)(q32;q21) in a DLBCL that represents a MALT1 and not a bcl-2 translocation highlights 1 potential limitation of this technique, which morphologically identifies chromosomal regions rather than specific genes. While the ultimate relative value of classical cytogenetic studies in the evaluation of suspected lymphoma remains to be determined, the value of such data likely will continue to increase with growing experience and larger studies. From the 1 Department of Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH; 2 Department of Human Genetics, University of Pittsburgh, Pittsburgh Cytogenetics Laboratory at the University of Pittsburgh Medical Center, Magee-Women s Hospital, and University of Pittsburgh Cancer Institute; and 3 Department of Pathology, Division of Hematopathology, University of Pittsburgh School of Medicine, Pittsburgh, PA. Address reprint requests to Dr Swerdlow: Dept of Pathology, Division of Hematopathology Room C606-PUH, University of Pittsburgh School of Medicine, 200 Lothrop St, Pittsburgh, PA Acknowledgment: We are grateful to the expert technologists of the Pittsburgh Cytogenetics Laboratory for their thorough cytogenetic analysis and karyotyping. References 1. Jaffe E, Harris N, Stein H, et al. Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press; World Health Organization Classification of Tumours. 2. Donner LR. 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