MYC translocation in chronic lymphocytic ... - Wiley Online Library

12 downloads 23933 Views 375KB Size Report
May 9, 2008 - M. D. Anderson Cancer Center, Houston, TX. 77030, USA. E-mail: ... of Texas M. D. Anderson Cancer Centre, for cases with t(8;14)(24Ж1;q32), t(2 ...... R.L., Call, T.G., Shanafelt, T.D., Kay, N.E. & Zent, C.S. (2005). Interphase ...
research paper

MYC translocation in chronic lymphocytic leukaemia is associated with increased prolymphocytes and a poor prognosis

Yang O. Huh,1 Katherine I-Chun Lin,1 Francisco Vega,1 Ellen Schlette,1 C. Cameron Yin,1 Michael J. Keating,2 R. Luthra,1 L. Jeffrey Medeiros1 and Lynne V. Abruzzo1 Departments of 1Hematopathology and 2

Leukemia, The University of Texas M.D.

Anderson Cancer Centre, Houston, TX, USA

Received 3 January 2008; accepted for publication 5 February 2008 Correspondence: Yang O. Huh, MD,

Summary Chromosomal translocations that involve MYC, characteristic of Burkitt lymphoma, are rare in chronic lymphocytic leukaemia (CLL). We report the clinical, morphological, immunophenotypic, cytogenetic and molecular genetic features of eight CLL cases with MYC rearrangement. The patients, five men and three women (median age, 71 years) had bone marrow involvement and an absolute peripheral blood lymphocytosis; five had lymphadenopathy; seven had splenomegaly. Prolymphocytes were increased (‡10%) in all cases. Six cases were classified as CLL with increased prolymphocytes (CLL/PL; prolymphocytes 10–55%), and two were classified as CLL in prolymphocytic transformation (CLL/PT; prolymphocytes >55%). All cases co-expressed CD5, CD19, and CD23; five of eight expressed ZAP-70. Of seven cases tested, four had mutated and three had unmutated IGHV genes. Conventional cytogenetic studies demonstrated t(8;14)(q24Æ1;q32) in five cases, t(8;22)(q24Æ1;q11) in two cases, and t(2;8)(p12;q24Æ1) in one case. Seven cases contained additional chromosomal abnormalities. All patients received combination chemotherapy. Two developed Epstein–Barr virus (EBV)-associated diffuse large B-cell lymphomas (DLBCL) that were clonally unrelated to the CLL. At follow-up, two patients are alive, four died of underlying disease, one died of EBV-associated DLBCL, and one died of an unrelated cancer. In summary, MYC rearrangement, which occurs rarely in CLL patients, is associated with increased prolymphocytes, complex cytogenetic abnormalities, and a poor prognosis.

Department of Hematopathology, Box 72, U.T. M. D. Anderson Cancer Center, Houston, TX 77030, USA. E-mail: [email protected]

Keywords: MYC rearrangement, chronic lymphocytic leukaemia, prolymphocytes, transformation, Epstein–Barr virus.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the Western hemisphere. Patients with CLL present with lymphocytosis, bone marrow involvement, and often lymphadenopathy and hepatosplenomegaly. The neoplastic cells have a characteristic immunophenotype; they express B-cell antigens (e.g. CD19), CD5, and CD23, and often also express dim surface immunoglobulin and CD20. The most common cytogenetic abnormalities, best detected by fluorescence in situ hybridization (FISH), are deletions of 13q14, 11q22, 17p13, and trisomy 12 (Dohner et al, 2000). Translocations that involve MYC are very rare in CLL. In a recent study using FISH, no evidence of MYC translocations was identified in 109 cases of CLL (Nelson et al, 2007).

Deregulation of the MYC oncogene, located on chromosome 8 at band q24Æ1, has been shown to play a decisive role in lymphomagenesis. Rearrangement of MYC with the immunoglobulin heavy chain locus on chromosome 14 at band q32, or less commonly, with the kappa (2p12) or lambda (22q11Æ2) light chain loci, is a consistent genetic abnormality in Burkitt lymphoma (Diebold et al, 2001). However, MYC rearrangement is not unique to Burkitt lymphoma. MYC translocations are found in 5–15% of cases of diffuse large B-cell lymphoma (DLBCL), and the frequency of MYC rearrangement is much higher in cases of DLBCL with a high proliferation rate (Lin & Medeiros, 2007). MYC rearrangement also has been reported infrequently in other types of lymphoma, particularly in

First published online 9 May 2008 ª 2008 The Authors doi:10.1111/j.1365-2141.2008.07152.x Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 142, 36–44

MYC Rearrangement in Chronic Lymphocytic Leukaemia neoplasms that have undergone transformation to more aggressive disease, for example, blastoid variant of mantle cell lymphoma (Hao et al, 2002) and transformation of follicular lymphoma to DLBCL (Lossos et al, 2002). Occasional cases of B-cell malignancies that harbor both the t(14;18)(q32;q21Æ3) and MYC rearrangement have been reported (Karsan et al, 1993; Kanungo et al, 2006). MYC rearrangement also has been reported in rare cases of B-cell prolymphocytic leukaemia (PLL) with more than 55% prolymphocytes (Brennscheidt et al, 1994; Lens et al, 1999; Merchant et al, 2003; Kuriakose et al, 2004). We describe the clinical, morphological, immunophenotypic, cytogenetic and molecular genetic findings in eight cases of CLL with Burkitt type translocations.

Materials and methods Case selection We searched the files of the Clinical Cytogenetics Laboratory, in the Department of Hematopathology at The University of Texas M. D. Anderson Cancer Centre, for cases with t(8;14)(24Æ1;q32), t(2;8)(p12;q24Æ1), or t(8;22)(q24Æ1;q11Æ2) in patients with a diagnosis of CLL. We identified eight cases out of 3405 CLL patients (0Æ2%) (untreated and previously treated) newly seen at our institution between January 1997 and August 2007. One patient (case 7) has been reported previously (Merchant et al, 2003).

Morphological examination For all cases we reviewed peripheral blood smears, and bone marrow aspirate smears, clot sections, touch imprints and core biopsy specimens. Peripheral blood smears were stained with May–Grunwald–Giemsa stain and a manual 100-cell white blood cell (WBC) differential count was performed. Bone marrow aspirate clot sections and decalcified core biopsy specimens were routinely processed and stained with haematoxylin and eosin (H&E). In bone marrow biopsy specimens, cellularity was assessed and the pattern of lymphocytic infiltration was classified as nodular, interstitial, diffuse, or a combination of these patterns. Differential 500 cell counts were performed on either bone marrow touch imprints or bone marrow aspirate smears stained with Wright-Giemsa. Particular attention was paid to the cytology of the lymphocytes with respect to atypical morphological features, including indented or clefted nuclei, plasmacytoid features, and the presence of prolymphocytes, large cells, or immature-appearing cells. We classified the disease as CLL with increased prolymphocytes (CLL/PL) when the percentage of prolymphocytes was 10–55%, as suggested in the World Health Organization classification (Muller-Hermelink et al, 2001). We classified cases as CLL in prolymphocytic transformation (CLL/PT) when prolymphocytes exceeded 55% of lymphoid cells in the blood and/or bone marrow

aspirate smear. Two patients underwent biopsy of their gastrointestinal tract.

Immunophenotypic analysis A bone marrow aspirate specimen from each patient was assessed by three- or four-colour flow cytometry, as described previously (Huh et al, 2001). The lymphocyte population was gated using right angle light scatter and CD45 expression. The panel of monoclonal antibodies used included reagents specific for: CD5, CD10, CD11c, CD19, CD20, CD22, CD23, CD38, CD45, CD79b, FMC7 and immunoglobulin light chains. Ten thousand events were acquired to a FACSCalibur (Becton Dickinson, San Jose, CA, USA) and the data were analyzed using CellQuest software (Becton Dickinson). For each case the CLL score was calculated according to the system of Matutes et al (1994), subsequently modified by Moreau et al (1997). The intensity of CD20 expression was graded according to the mean channel fluorescence (MCF) as weak (MCF 1000) (Huh et al, 2001). Immunohistochemical stains were performed using routinely fixed and processed paraffin-embedded tissue sections of bone marrow core biopsy and/or aspirate clot specimens, as described previously (Admirand et al, 2004). The panel used to evaluate the bone marrow specimens included monoclonal antibodies specific for: cyclin D1 (SP4, 1:40; Labvision/ Neomarkers, Fremont, CA, USA), CD20 (L26, 1:700; DAKO, Carpinteria, CA, USA), Ki-67 (MIB-1, 1:100; DAKO), BCL2, and ZAP70 (1:100; Upstate Cell Signaling Systems, Lake Placid, NY, USA). Additional antibodies used to evaluate the gastrointestinal tract biopsy specimens included CD3 (SK7, 1:100; DAKO), CD5 (4C7, 1:20; Labvision/Neomarkers), CD10 (56 C6, 1:70; Novocastra Vision Biosystem, Norwell, MA, USA), CD15 (MMA, 1:40; BD Biosciences, San Jose, CA, USA), CD30 (Ber-H2; 1:20, Signet Covance, Berkeley, CA, USA), and BCL-6 (PIF6, 1:40; Novocastra Vision Biosystem). In situ hybridization analysis for Epstein–Barr virus (EBV) RNA was performed on formalin fixed, paraffin-embedded tissue sections using a probe for EBV-encoded RNA (EBER) and a hybridization kit according to the manufacturer’s (DAKO) instructions with appropriate positive and negative controls.

Conventional cytogenetic analysis and fluorescence in situ hybridization Conventional cytogenetic analysis was performed on G-banded metaphase cells prepared from bone marrow aspirate specimens in all cases using standard techniques. Twenty metaphases were analyzed and the results were reported using the International System for Human Cytogenetic Nomenclature. FISH for MYC rearrangement was performed on cultured bone marrow cultures in three cases (cases 1, 3, and 8) using either an IGH/MYC dual-colour, dual-fusion translocation probe

ª 2008 The Authors Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 142, 36–44

37

Y. O. Huh et al nated as unmutated if there were fewer than 2% mutations (>98% homology to germline sequences), or as mutated if there were 2% or more mutations (£98% homology to germline sequences) compared to the germ-line sequences (Fais et al, 1998).

(case 1) (Vysis Inc., Downers Grove, IL, USA) or a dual-colour break-apart probe for MYC (cases 3 and 8) (Cancer Genetics, Inc., River Vale, NJ, USA). In two cases where material was available for study (cases 1 and 8), FISH analyses for common cytogenetic abnormalities associated with CLL were performed on interphase cells from the corresponding bone marrow cultures using a panel of probes designed to detect deletion 13q14Æ3, deletion 13q34, trisomy 12, deletion of TP53 at 17p13, and deletion of ATM at 11q22Æ3 (Vysis). FISH for MYC rearrangement was performed on formalinfixed paraffin-embedded (FFPE) tissue sections obtained from a bone marrow core biopsy (case 7) using a dual colour, dual fusion probe, as described previously (Merchant et al, 2003). In cases 1 and 3, FISH for MYC rearrangement was also performed on FFPE tissue sections of the gastrointestinal tract biopsy specimens using a dual-colour break-apart probe for MYC (Cancer Genetics, Inc.).

Results Clinical findings The clinical and laboratory data are summarized in Table I. There were five men and three women, with a median age of 71 years (range, 49–76 years). All had CLL for a variable period of time ranging from 3 months to 10 years before MYC rearrangement was detected. Two patients (cases 1 and 3) were previously untreated; six patients had been previously treated with a variety of chemotherapeutic agents (Table I). All patients except one (case 8) had anaemia and an absolute lymphocytosis in the peripheral blood. The median haemoglobin was 97 g/l (range 89–137). The lymphocyte counts ranged from 2 to 339 · 109/l, with a median lymphocyte count of 73 · 109/l. The median platelet count was 134 · 109/l (range 72–176 · 109/l). Five patients had lymphadenopathy, and seven had splenomegaly. Because of progressive disease, all patients received treatment with combination chemotherapy following detection of MYC rearrangement as listed in Table I. Two patients are alive, one without evidence of disease for the follow-up period of 50 months (case 3). One patient (case 1) developed an EBVpositive DLBCL that involved the oesophagus, stomach, duodenum, and left lung. It arose 9 months after MYC

Sequence analysis of the IGHV genes In seven of eight cases (cases 1–5, 7, 8), sequence analysis of the immunoglobulin heavy chain variable region (IGHV) genes was performed using DNA extracted from formalinfixed, paraffin-embedded tissue sections obtained from bone marrow clot sections or gastrointestinal tract biopsy specimens, as described previously (Yin et al, 2007). In case 1, sequence analysis was performed on RNA extracted from peripheral blood mononuclear cells. In order to determine the level of somatic mutation, patients’ sequences were aligned to the germline sequences in the V-BASE 2 database (Retter et al, 2005). The IGHV mutation status was desigTable I. Clinical features at the time of MYC rearrangement detection. Duration of CLL* (months)

Lymphocytes (·109/l)

Therapy after MYC detection

Outcome (survival after MYC detection)

FCR, pegfilgrastim, Cidofovir, R F, C

Alive, CR, EBV + DLBCL (23 months) Died, EBV + DLBCL (48 months) Alive, CR (40 months) Died with disease (3 months) Died with disease (15 months) Died with disease (3 months) Died, oropharyngeal SCCA, CLL in CR (40 months) Died with disease (1 month)

Platelets (·109/l)

Spleen

LN

Previous therapy

81

150

Yes

Yes

None

91

10

111

Yes

No

24 6

122 129

7 73

176 174

Yes Yes

No Yes

Chlorambucil, prednisone None F, C

F

108

94

339

72

Yes

Yes

F, R, Campath

Compound 506

67

M

102

89

99

117

No

Yes

F

F, C

7

71

M

12

137

23

134

Yes

No

F, C

Hyper-CVXD, R

8

66

M

48

130

2

113

Yes

Yes

FCR, Campath

Ofatumumab, OFAR

Case

Age

Sex

Hb (g/l)

1

74

M

3

97

2

74

F

48

3 4

72 49

F M

5

76

6

CHOP-R Hyper-CVAD

LN, lymph node; CR, complete remission; DLBCL, diffuse large B-cell lymphoma; F, fludarabine; C, cyclophosphamide; R, Rituximab; CHOP, cyclophosphamide, hydroxydoxorubicin, vincristine, prednisone; Hyper-CVAD, cyclophosphamide, vincristine, cytarabine, doxorubicin; hyperCVXD, fractionated cyclophosphamide, vincristine, liposomal daunorubicin, dexamethasone; SCCA, squamous cell carcinoma; OFAR, oxaliplatin, fludarabine, alemtuzumab, rituxan. *Duration of CLL before MYC was detected.

38

ª 2008 The Authors Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 142, 36–44

MYC Rearrangement in Chronic Lymphocytic Leukaemia abundant pale cytoplasm. However, in sections obtained from the jejunum, many of the large cells had multilobated nuclei with multiple eosinophilic nucleoli. Mitotic figures were easily found and there were areas of geographic necrosis. In case 2, fragments of focally ulcerated colonic mucosa were infiltrated by DLBCL, also associated with areas of necrosis. Neither case contained a low-grade component.

rearrangement was identified in the bone marrow. The patient was treated with segmental resection of jejunum followed by chemotherapy with rituximab and cidofovir. This patient is alive after 23 months without evidence of CLL or DLBCL. Six patients died with disease, with survival ranging from 1 to 40 months after the MYC translocation was identified. Four died of the underlying disease. One patient (case 2) died from complications of an EBV-positive DLBCL in the rectum that arose 3 years after the identification of MYC rearrangement in the bone marrow, and one died of an unrelated cancer.

Results of immunophenotypic and in situ hybridization analyses Immunophenotypic analysis performed by flow cytometry on bone marrow aspirates in all cases demonstrated that the neoplastic cells expressed CD5, CD19, and CD23. Surface immunoglobulin expression was dim in four cases (cases 1, 4, 5, and 8), but bright in the other four cases (cases 2, 3, 6, and 7). Expression of CD22 or CD79b was negative (cases 1, 3, 4) or dim (case 8) in four cases tested. CD20 expression was strong in three cases (cases 1, 3, and 4) and moderate in one case (case 8). FMC-7 was negative in seven cases (cases 1, 3–8) and positive in one (case 2). Based on these results, the CLL score was 4 or 5 in seven cases (cases 1, 3–8). The one case that was positive for FMC-7 (case 2) had a CLL score of 3. Of the five cases evaluated for CD38 expression, three were positive (cases 1, 3, and 4) and two were negative (cases 5 and 8). The results of immunohistochemical stains performed on formalin-fixed, paraffin-embedded sections of bone marrow core biopsy specimens are summarized in Table II. Staining with antibody to Ki-67 in seven cases revealed