To yield an accurate chromosomal picture, it is crucial that cytogenetic investigations be performed on the tissue representative of tumor and not on the adjacent nonmalignant (stromal) tissue and that appropriate culture methods be utilized for the growth of tumor cells. When sufficient tissue is available, selected tumor material should be divided for morphologic, immunologic, and cytogenetic studies. For best results and to retain viability, the lymphoid tissue should be transported in sterile media at room temperature to the cytogenetics laboratory as quickly as possible. The tissue should never be fixed or frozen. Any necrotic tissue should be removed before disaggregation. The tissue is mechanically disaggregated, which can be followed by enzymatic disaggregation if deemed necessary. The minced tissue is divided between the appropriate cultures containing RPMI 1640 (Irvine Scientific, Santa Ana, CA, USA) with 20% fetal bovine serum (FBS), HEPES, glutamine, penicillin, streptomycin, and heparin. Tumor cells are spontaneously dividing; hence the cultures do not require mitogens to achieve metaphase preparations. Depending on the availability of the tissue material for cytogenetic preparations, the unstimulated cultures are set up in the following preference: direct-overnight with 10 μL of a low concentration (10 μg/mL) of Colcemid (Irvine Scientific, Santa Ana, CA, USA) overnight, 24-hour, and 48-hour cultures and incubated at 37°C.

Approximately 40 minutes before the initiation of harvest, the cells are treated with Colcemid, 0.2 μg/mL. After hypotonic treatment (0.4% KCl solution for 20 minutes at 37°C), the cells are prefixed by adding 2 mL of freshly prepared chilled 3:1 methanol–glacial acetic acid fixative and gently inverting the tubes. This is followed by three additional fixation cycles and air-dried slide preparation. The slides are generally “aged” in a hot oven (100°C) for 30 minutes or in a 60°C oven overnight. The slides are then G-banded with a trypsin pretreatment. When available, at least 20 metaphases are analyzed. Karyotypes of Giemsa-banded metaphase chromosomes are typically described according to the most recent version of the International System of Cytogenetic Nomenclature (ISCN 2009).²¹ Abnormal clones are defined as either two or more cells with the same structural abnormality or gain of the same chromosome, or the presence of three or more cells with loss of the same chromosome. However, if an abnormal karyotype contains a known NHL-associated translocation in only one cell, it is included in the nomenclature. All normal cells are also included in the nomenclature.²²–²⁴

Depending on the availability and requirement for differential diagnosis, FISH procedures are performed either on cell suspensions prepared from cryopreserved cell pellets or on 4- to 5-μm unstained paraffin embedded tumor tissue sections. FISH probes to investigate the majority of the specific NHL-related translocations or gene disruptions are commercially available. Many laboratories generate homebrew FISH probes for detection of common abnormalities to make the testing more cost effective. The protocol for FISH studies has been outlined in another article by Karen D. Tsuchiya elsewhere in this issue. Our laboratory protocol has been described in detail previously.²²–²⁴

A rare disease in Eastern countries and the most common leukemia of adults in Western nations, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) accounts for about 7% of NHL. The term SLL is used for the nodal form with the tissue morphology and immunophenotype of CLL. Of all hematologic malignancies, CLL/SLL shows the highest genetic predisposition.²⁵ Cytogenetic abnormalities have been detected in nearly 80% of CLL/SLL, specifically by FISH techniques.²⁶–²⁸ The malignant CLL cells have a poor response to conventional mitogens; thus previous studies identified the cytogenetic abnormalities at a different frequency than those observed with specific FISH probes.^27,29–³¹ Interphase studies demonstrated that del(13q) is the most common abnormality detectable by FISH.³¹ This is followed by trisomy 12 and deletions of 11q22–q23, 17p13, and 6q21.²⁷ The use of mitogenic agents such as CD40L and immunostimulatory CpG-oligonucleotide DSP30 and IL2, has improved the percentage of metaphase chromosome abnormalities in CLL cases. CLL/SLL primarily depicts genomic imbalances; more chromosomal abnormalities and rare reciprocal translocations are observed with an overall karyotypic evaluation than focused FISH studies.²⁸ The expression of tyrosine kinase ZAP-70 is associated with IGHV unmutated CLL genotype.¹⁷ The distribution of genomic abnormalities varies depending on the mutational status of VH.²⁵ Trisomy 12, del(13q), del(11q), and del(17p) are frequently observed independent of each other. However, combinations of these abnormalities appearing together are not uncommon. The del(11q) and del(17p) are recurrently noted as secondary changes. Although rare, trisomies of 3, 8, and 18 and del(6q) have been observed, recurrently.³²

Trisomy 12, frequently associated with atypical morphology,³² is found in nearly one third of CLL with abnormal karyotypes and in about 15% of all CLL as detected by FISH,²⁹ with the minimum common gain being 12q13. Other additional recurrent aberrations among the trisomy 12 cases include trisomies of 18 and 19,³³ t(14;18)(q24;q32) IGH-BCL2 fusion,³⁴ t(14;19)(q32;q13) leading to IGH–BCL3 fusion,³⁵ and del(14)(q24q32) with one breakpoint in IGH locus.³⁶ Variant translocations including the 2p12 and 22q13 (IGK and IGL, respectively) are also observed. After initial FISH studies,²⁹ abnormalities of 13/13q were accepted as the most frequent alteration in CLL/SLL. These are observed in 50% of CLL by FISH and in about 20% of abnormal karyotypes. The critical deletion region involves 13q14 and excludes the RB1 gene.^37,38 Monoallelic as well as biallelic losses of 13q14, and monosomy/nullisomy of 13, have been observed in CLL/SLL. Deletions of 11q, involving the minimum deletion region 11q22–q23 which included the ATM gene, are observed mostly by interphase FISH studies.^27,30,39 Structural abnormalities of 17p including deletions of TP53 are also seen in CLL/SLL and are associated with an unfavorable prognosis.^28,40 Most cases containing translocation t(11;14)(q13;q32) linking the IGH–CCND1 genes may now be classified as mantle cell lymphoma (MCL); however, this may also rarely be noted CLL/SLL.

Progression of CLL/SLL to B-PLL is rare; however, an acute transformation to a diffuse large cell lymphoma, known as Ritcher’s syndrome, takes place in some cases and a very low percentage develop HL. Complex karyotypes, deletions of TP53 and ATM, and involvement of 8q24 (MYC region) and 9p21 (CDKN2A) are indicators of transformation,⁴¹ while isolated 13q14.3 deletions are associated with a favorable prognosis.²⁷

A neoplasm of B prolymphocytes with more than 55% of lymphocytes being prolymphocytes, B-cell prolymphocytic leukemia (B-PLL) is an extremely rare disease, comprising approximately 1% of lymphocytic leukemias.⁴² Chromosome abnormalities that are common in CLL/SLL are also characteristic of B-PLL.^26,28,40 Complex karyotypes with nearly 50% cases containing the deletion of 17p including the TP53 gene have been demonstrated in B-PLL. In addition, deletions of 13q14 are also noted by FISH analysis. Trisomy 12 is rare in B-PLL.⁴³ The initial finding of nearly 20% cases of B-PLL containing t(11;14)(q13;q32)⁴⁴ is now refined, and these cases are more likely considered to be leukemic variants of mantle cell lymphoma.^43,45

Constituting about 2% of the lymphoid neoplasms, splenic B-cell marginal zone lymphoma (SMBZL) is seen mostly among patients older than 50 years of age.⁴⁶ SMBZL is composed of small lymphocytes that surround and replace the splenic white pulp germinal center, efface the follicle mantle, and merge with a peripheral (marginal) zone of larger cells. Lymphoma cells may be found in the peripheral blood as villous lymphocytes (splenic lymphoma with circulating villous lymphocytes [SLVLs]).⁴⁷ Deletion of 7q is the most frequent abnormality in SMBZL and is observed in nearly 40% of the cases; the critical region is 7q31–32. Gain of 3q and numerous other abnormalities have also been described.⁴⁸–⁵⁰ In addition, dysregulation of CDK6 gene as a result of a t(2;7)(p12;q21) has been demonstrated in some cases.^51,52 The t(11;18), common in extranodal marginal zone lymphoma of mucosa associated lymphoid tissue (MALT), is absent in SMBZL. The presence of a 7q deletion may be associated with an unfavorable outcome.^47,53

An indolent and rare disease of small mature B lymphoid cells with oval nuclei and abundant cytoplasm with “hairy” projections, hairy cell leukemia (HCL) constitutes 2% of lymphoid leukemias, occurring at a median age of 50 years and exhibiting a strong male predominance (5:1 male to female ratio).⁵⁴ Structural rearrangements of 14q including translocations involving 14q32 and deletions of 14q22−24 have been described.⁵⁵ Further, abnormalities of chromosome 5 including gains in 5q13–31 have been observed.^56,57

Lymphoplasmacytic lymphoma (LPL) involves small B lymphocytes, plasmacytoid lymphocytes, and plasma cells, usually involving bone marrow and sometimes lymph nodes and spleen. Walderstom macroglobulinemia (WM) is found in a large subset and is usually defined as LPL with BM involvement. These LPLs are characterized by excessive proliferation of an immunoglobulin M (IGM)-producing clone of malignant plasmacytoid cells.⁵ The distinction between marginal zone lymphoma (MZL) and LPL is sometimes difficult. These cases may be described as small lymphocytic lymphoma with plasmacytic differentiation.⁵⁸ No specific cytogenetic abnormality is currently defined as a hallmark of LPL. Previously these lymphomas were associated with a characteristic translocation t(9;14)((p13;q32) involving the PAX5/IGH genes⁵⁹; however, the translocation is rarely observed in LPL and is also observed in other B-cell neoplasms.^60,61 Although deletion of 6q is nonspecific for LPL, it is most frequently noted in LPL/WM (6q21 deletions by FISH).⁶² Cases with a deletion of 6q have been associated with an adverse prognosis.

Composed of morphologically heterogeneous small B cells including marginal zone (centrocyte-like) cells, small lymphocytes, scattered immunoblasts, and centroblast-like cells, MALT lymphoma is an extranodal lymphoma that comprises 7% to 8% of all B-cell lymphomas.⁶³ The differential diagnosis of MALT lymphoma includes the reactive inflammatory processes including Helicobactor pylori gastritis that typically precede the lymphoma and other small B-cell lymphomas (follicular lymphoma, mantle cell lymphoma, small lymphocytic lymphoma). The t(11;18)(q21;q21) is the most frequent translocation that occurs in MALT lymphoma. The API2 (BRIC3) gene on 11q21 and the MALT1 gene on chromosome 18q21 are disrupted and recombined to form a API2–MALT1 fusion gene on the derivative chromosome 11.⁶⁴–⁶⁶ Most often this appears as a sole abnormality.⁶⁷–⁶⁹ Approximately 15% of MALT lymphomas reveal the t(11;18) when molecular or FISH techniques are utilized; the majority of these are the gastric and pulmonal MALT cases.^70,71 The t(11;18) appears to be specific for MALT lymphomas and has not been observed in nodal and splenic marginal zone B-cell lymphomas.^64,65,72 Three other recurrent and mutually exclusive translocations are observed in MALT lymphoma. The t(14;18)(q32;q21), which deregulates the MALT1 gene through IGH juxtaposition,^73,74 is observed in nearly 11% of MALT lymphomas, more frequently in ocular adnexel and liver lymphomas.^70,71 This translocation can be distinguished from the cytogenetically identical t(14;18)(q32;q21) involving the IGH and BCL2 by using FISH techniques. The t(3;14)(p14.1;q32) resulting in the transcriptional deregulation of FOXP1 on 3p14.1⁷² was identified in 10% of MALT lymphomas of thyroid, ocular adnexae/orbit, and skin. The t(1;14)(p22;q32), which juxtaposes the BCL10 gene with the IGH locus,^75,76 is seen in 2% of MALT lymphomas.⁷¹ A variant t(1;2)(p22;p12) involving the IGK locus has also been described.⁷⁷

Trisomies of 3 and 18 are seen in nearly 30% of all MALT lymphomas, including those that are translocation negative. Trisomies are more commonly seen in intestinal, salivary gland, and orbital adnexal lymphomas.⁷⁰ Partial gains of 3q (3q27), 9q (9q34), and 18q are common in MALT.^78,79 Deletions of 6q have also been observed in aCGH studies.⁸⁰ MYC amplifications, del(9p), and del(17p) are seen in progressive disease.⁶⁹–⁷¹ The t(1;14) and partial or complete trisomy 18 predict adverse clinical behavior.⁸¹ FISH studies on paraffin-embedded tissues help in the differential diagnosis of marginal zone and MALT lymphomas.