T-cell-rich B-cell Lymphoma: A Clinicopathologic ...

HEMATOPATHOLOGY Original Article

T-cell-rich B-cell Lymphoma A Clinicopathologic Study of Eight Cases FADY K. BADDOURA, MD, 1 WING C. CHAN, MD, 2 ANEAL S. MASIH, MD, 2 DAN MITCHELL, MT, 2 NORA C.J. SUN, MD, 3 AND DENNIS D. WEISENBURGER, M D 2

histiocytes imparted a lymphoepithelioid appearance in two cases. Although immunoperoxidase stains of frozen tissue were initially suggestive of a peripheral T-cell lymphoma in some cases, paraffin immunoperoxidase stains clearly established the B-cell nature of the large cells, whereas most of the small cells were T lymphocytes. The clonal nature of the large cells was confirmed in seven cases by monotypic immunoglobulin (Ig) light chain restriction or Ig gene rearrangements. EpsteinBarr virus genomic DNA was detected in two of the six cases tested by polymerase chain reaction or Southern blot analysis, but no evidence of a bcl-2 rearrangement was found in any of the five cases examined. These findings indicate that TCRBCL is an uncommon form of NHL with a therapeutic response and overall survival consistent with intermediate grade lymphoma. Paraffin immunoperoxidase stains and occasionally genotypic analysis are required to exclude the diagnosis of PTCL or diffuse lymphocyte predominant Hodgkin's disease. The authors found no morphologic or molecular evidence to support a follicular center cell origin in these cases of TCRBCL. (Key words: T-cell-rich B-cell lymphoma; Non-Hodgkin's lymphoma; Peripheral T-cell lymphoma; T-zone lymphoma; Gene rearrangement; Epstein-Barr virus; bcl-2) Am J Clin Pathol 1995;103:65-75.

Morphological features alone are frequently inadequate for predicting the immunophenotype in diffuse non-Hodgkin's lymphoma, particularly in the mixed cell and large cell types.'" 3 B-cell lymphomas that are likely to be confused with neoplasms of peripheral T-cell origin generally fall into two categories. There are cases with the typical morphological features of peripheral T-cell lymphoma (PTCL), such as those with tumor cells having cerebriform or multilobated nuclei, tumor cells with abundant clear cytoplasm, the presence of a mixed inflammatory cell infiltrate, prominent vasculature, or a high content

of epithelioid histiocytes, but with unequivocal evidence of a B-lineage.4"9 In such cases, the correct diagnosis often is facilitated by the relative abundance of neoplastic B-cells and a paucity of small benign T cells. The second category is B-cell lymphomas in which both the morphologic and immunologic findings suggest a diagnosis of a PTCL because of the large number of small benign T cells that obscure the less abundant neoplastic B cells.610"20 Jaffe and coworkers10 initially described 6 such cases, which included up to 75% benign T cells, and coined the term pseudo-T-cell lymphoma. All of the cases of Jaffe and coworkers10 were preceded by follicular lymphomas and continued to behave in an indolent manner clinically, thus suggesting a beneficial host T-cell response. Ramsay and colleagues12 coined the term T-cell-rich B-cell lymphoma From the 'Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia; ^Department (TCRBCL) in their study of 5 such cases, but included only of Pathology and Microbiology, University of Nebraska Medical cases in which the small T cells exceeded 90% of the entire Center. Omaha, Nebraska; %Departmenl of Pathology, Harbor UCLA nodal population. In the latter study, the tumors appeared to Medical Center, Torrance, California. arise de novo, and the clinical course was rather aggressive. The cases reported in subsequent studies13"20 of TCRBCL have Presented in part at the 80th Annual Meeting of the United Statesbeen rather heterogeneous with regard to the proportion of T Canadian Division of the International Academy of Pathology, Chicells, the relative number and morphology of the large tumor cago, Illinois, March 17-22, 1991. cells, the histologic pattern, and the biology of the disease. Manuscript received June 1, 1993; revision accepted January 10, Therefore, a number of questions still remain, such as (1) What 1994. is a "rich" T-cell content in B-cell lymphoma and what is its Address reprints requests to Dr. Chan: Department of Pathology and clinical relevance? (2) Does TCRBCL represents an early stage Microbiology, University of Nebraska Medical Center, 600 South 42nd of a usual type of diffuse large B-cell lymphoma? (3) Where Street, Omaha, NE 68198-3135.

65

Downloaded from http://ajcp.oxfordjournals.org/ by guest on March 20, 2016

Although T-cell-rich B-cell lymphoma (TCRBCL) is a recently recognized form of non-Hodgkin's lymphoma (NHL), limited information regarding its incidence, cellular origin, morphologic spectrum, and biologic behavior is currently available. In this study, the clinicopathologic features of eight patients with TCRBCL are presented. This neoplasm comprised about 1% of all NHLs seen at Emory University Hospital over 2 years. The male-to-female ratio was 1.6, and the mean age at diagnosis was 60 years. At presentation, TCRBCL was nodal in 88% of the patients and widely disseminated in 50% of the patients. A complete remission was seen in three of the five patients treated with combination chemotherapy that was directed at intermediate grade NHL. Three patients received inadequate or incomplete chemotherapy. One of these patients later achieved a complete remission with more intensive therapy. Two of the patients were not evaluable for response to therapy. The actuarial and disease-free survival rates of the group at 5 years were 72% and 21%, respectively. Morphologically, the lymph nodes in seven of eight cases were diffusely obliterated, whereas one had markedly expanded interfollicular zones that lead to an initial diagnosis of T-zone lymphoma. All tumors were characterized by no more than 25% large lymphoid cells, which were scattered in a background of small lymphocytes with round or irregular nuclei. The presence of numerous

66

HEMATOPATHOLOGY Original Article TABLE 1. ANTIBODIES USED IN THE IMMUNOPHENOTYPIC ANALYSIS OF T-CELL-RICH B-CELL LYMPHOMA

Clusters of Differentiation

Antibody

Major Specificity

Source

Tissue Source

Bcell CD19 CD20 CD22 CD74 CDw75

— —

Leu-12 L26 Leu-14 LN2 LN1 Anti-/c Anti-X

BD Dako BD ICN ICN BD, Dako BD, Dako

B cells B cells B cells B cells B cells B cells B cells

F P F P P F, P F, P

OK.T-6 Leu-4 Leu-3 Leu-1 Leu-9 Leu-2 Leu-22 UCHL-1 0F-1

CI BD BD BD BD BD BD Dako TCS

Thymocytes, Langerhans cells T cells T helper/inducer cells T cells T cells T suppressor/cytotoxic cells T cells, granulocytes T cells T cells

F F F F F F P P F

Leu Ml Ki-l/BerH2 CLA Ki-67 HLA-DR

BD Dako Dako Dako BD

RS cells, granulocytes Activated lymphocytes, RS cells Hematopoietic cells Proliferating cells B cells, activated T cells, monocytes

F, P F, P P F F

Tcell

— Miscellaneous CD15 CD30 CD45

— —

CLA = common leukocyte antigen; F = frozen sections; P = paraffin sections; RS = Reed-Stern berg; BD = Becton-Dickinson, Mountain View, CA: CI = Coulter Immunology. Hialeah. FL; Dako = Dakopatts, Santa Barbara. CA; ICN = ICN Biomedicals, Cosa Mesa, CA: TCS = T Cell Sciences. Cambridge. MA.

does TCRBCL fit in the Working Formulation (WF)?21 With this in mind, the cHnicopathologic features of eight cases of TCRBCL are presented, along with a review of the literature, to better understand the morphologic spectrum and biologic behavior of such cases.

no more than 25% large B cells and at least 75% small T cells. However, a lesser percentage of T cells (65%) was accepted in one case with a high number of histiocytes. Lymphomas exhibiting neoplastic follicles at initial diagnosis were excluded from this study. Survival data were interpreted by life-table analysis.22

MATERIALS AND METHODS Histology and Patient Population The cases retrieved from the diagnostic pathology files of Emory University Hospital (cases 1, 2, 4, and 8) were collected over a 2-year period from January 1989 to January 1991, and constituted about 1% of all newly diagnosed cases of nonHodgkin's lymphoma (NHL). Four additional cases were found in thefilesof the University of Nebraska Medical Center (cases 3, 6, and 7) and Harbor UCLA Medical Center (case 5). Lymph node biopsies were available from all 8 cases, and additional diagnostic material, including biopsies of skin (1 of 8), bone marrow (2 of 8), and liver (1 of 8), cytospin preparations of pleural fluid (1 of 8), and nodal fine-needle aspirate smears (1 of 8), also was reviewed. The percentages of small and large lymphoid cells in reference to all lymphoid cells and histiocytes were obtained in each case by light microscopic analysis of 10 random high-power fields in hematoxylin-and-eosin stained and immunostained (CD20 and CD43) paraffin sections. The numerical criteria used for inclusion of a case in this study were

Immunohistochemistry

All nodal specimens but two (cases 1 and 5) were received in the fresh state. Tissue sections cut from 10% buffered formalinor B5-fixed paraffin blocks were available for morphologic analysis and immunophenotypic studies in each case. Serial 5 /rnithick sections from the paraffin and snap-frozen tissue blocks were mounted on poly-D-lysine coated slides and immunostained using a battery of monoclonal antibodies to B cells, T cells, CD 15, CD30, HLA-Dr, and Ki-67, as well as polyclonal antibodies to immunoglobulin K and X light chains using an avidin-biotin-complex immunoperoxidase technique23 (Table 1). Molecular Studies Frozen Tissue. DNA was extracted from six fresh specimens (cases 2-4, 6-8) according to a standard procedure.24 Ten ng from each sample were digested with Bam HI, Eco RI or Hind III enzymes (Bethesda Research Laboratory, Gaithersburg,

A.J.C.P.-January 1995


CDla CD3 CD4 CD5 CD7 CD8 CD43 CD45RO

BADDOURA ET AL.

67

T-cell-rich B-cell Lymphoma TABLE 2. CLINICAL FEATURES OF EIGHT PATIENTS WITH T-CELL-RICH B-CELL LYMPHOMA Case No.

Age (years) ISex

Presentation

Stage

Initial Diagnosis

Initial Therapy

Follow-up

Painful right inguinal LN; hepatosplenomegaly; positive liver scan and BM biopsy Solitary skin lesion on left forearm; history of treated lymphoblastic lymphoma with leukemic phase 6 yr before presentation Fever and night sweats, left cervical LN and Waldeyer's ring mass

1VA

Diffuse mixed small and large B-cell lymphoma (TCRBCL)

MACOP-B

IA

Cutaneous lymphoma (not otherwise classified)

Chlorambucil, prednisone

I1B

CAPBOP

CR; relapse in axillary, mediastinal, and peripancreatic nodes at 7 yr; Rx with DHAP; alive with disease at 8 yr

62/M

Weight loss and fatigue; generalized LN and pulmonary nodules

IVB

CHOP

CR; alive with NED at 23 mo

64/M

IVB

COPP

One Rx cycle given due to poor drug tolerance; lost to follow-up at 4 mo

IVB

Diffuse large B-cell lymphoma

COPP

Died at 8 wk during second Rx cycle

70/M

Weight loss, nausea, periumbilical pain, and hypercalcemia; supraclavicular and axillary LN; hepatosplenomegaly Fever, malaise, nausea, and vomiting; generalized LN; splenomegaly; positive BM biopsy and peripheral blood smear Generalized LN

B-immunoblastic lymphoma with a high content of epithelioid histiocytes Diffuse mixed small and large B-cell lymphoma (TCRBCL) T-immunoblastic lymphoma

IIIA

CAPBOP

81/F

Left axillary LN

IA

B-immunoblastic lymphoma, plasmacytoid type Diffuse mixed small and large T-cell lymphoma (T-zone lymphoma)

PR; switched to CHOP and CEPP; alive with progressive disease at 30 mo CR (adjuvant localized radiotherapy 1 mo prior to CR); relapse in thoracic and abdominal nodes with left pleural effusion and rib fracture at 12 mo; Rx with 1 cycle of chlorambucil and prednisone; died of disease at 13 mo

38/M

42/F

59/M

CHOP

PR = partial response; CR •= complete response; NED = no evidence of disease; LN - lymphadenopathy; BMT = bone marrow transplant; Rx = therapy; + CHOP therapy; MACOP-B MACOP-B = = methotrexate methotrexate + + doxorubicin doxorubicin + + cyclophosphamide cyclophosphamide + + vincristine vincristine + + prednisone prednisone + bleomycin; bleomycin; CHOP L: = cyclophosphamide + doxorubicin doxorubicin + + vincristine vincristine + + prednisone; prednisone; CAPBOP CAPBOP = = cyclophosphamide cyclophospha~:j~ +' Jdoxorubicin ~'~ +' procarbazine *~-'-- + ' Llbleomylide + cin + vincristine + prednisone; COPP = cyclophosphamide + vincristine + procarbazine + prednisone; DHAP = decadron + high-dose cytosine arabinoside iide + c/s-platinum; CEPP = Cytoxan + etoposide + prednisone + procarbazine.

MD) and size-fractionated by electrophoresis in 0.7% agarose gel.25 The DNA was then transferred to a nylon filter in 20 X SSC (1 X SCC = 150 mM NaCl, 15 mM NaCitrate, pH 7.4). Hybridization was performed in 50% formamide at 42 °C for 24 to 48 hours using DNA probes labelled with 32P by the random primer method. 26 The filters were then washed, and followed by autoradiography at —70 °C. The nylon filters were reused by stripping the labelled probe from the filter with 0.5% NaOH and 1.0% sodium dodecyl sulfate at 50 °C, placed back on radiographic film to confirm the absence of radioactive bands, and then rehybridized with another 32P-labelled probe. The gene probes used included an Ig heavy chain gene joining

region (JH) probe, 27 a K gene joining region (J.) probe, 28 and a X gene constant region (Cx) probe 29 (which were provided by Dr. Philip Leder), as well as a T-cell receptor /3-chain constant region (Q,) probe (Oncogene Science, Gaithersburg, MD). The presence or absence of Epstein-Barr virus (EBV) genome in samples from cases 3, 6, and 7 was determined by Southern blot analysis after Bam HI digestion, as described above, using a 32P labeled probe for EBV (1.9 kb Xho 1 fragment, which was provided by Dr. Mary Raab-Traub). 30 Paraffin Tissue. DNA was extracted from deparaffinized tissue sections in all cases, but one (case 3) using the method of Wright and Manos. 31 Extracts from cases 1 and 5 consisted of

Vol. 103-No. I


66/F

PR; Rx with Ara-C and cw-platinum; NED at 9 mo; relapse in liver at 23 mo; Rx with CHOP; NED at 29 mo; autologous BMT at 32 mo; alive with NED at 43 mo CR; relapse in cervical node at 9 mo; treated with VP16, melphalan, and cyclophosphamide; NED at 14 mo; autologous BMT at 15 mo; relapse in right inguinal node at 40 mo; alive with disease at 42 mo

68

HEMATOPATHOLOGY Original Article 100

L

80-

>

> DC

3

60

O

40J

5 < BO

O

20

20

-1—

—t—

40

60

-1

80

100

MONTHS

RESULTS Clinical

FIG. 1. (Top) a, Actuarial and b, disease-free survival curves in T-cellrich B-cell lymphoma. FIG. 2. (Middle) Architectural effacement of the node by a T-zonal infiltrate (case 9) (hematoxylin and eosin, X40). FIG. 3. (Bottom) Scattered large transformed lymphocytes in a predominantly small cell infiltrate (case 5) (hematoxylin and eosin, X600).

Features

The clinical features of the eight cases are summarized in Table 2. There were five men and three women, and the age at diagnosis ranged from 38 to 81 years (median, 63 years). The involved sites at initial presentation were primarily nodal in seven patients and extranodal in one patient. Four patients had widespread disease with tumor involving visceral organs at diagnosis (stage IV), including two with bone marrow involvement. Although all eight patients received some form of combination chemotherapy, five patients (cases 1,3,4, 7, 8) received adequate doses of a regimen having curative potential, and three of these achieved a complete remission. A continuous complete remission also was obtained in case 2 after reinduction with chemotherapy followed by an autologous bone marrow transplantation (BMT). Case 5 received only one treatment cycle because of poor tolerance to chemotherapy, and was lost to follow up 4 months after diagnosis. The tumor in case 6 responded well to therapy; however, this patient was in very poor physical condition and died 8 weeks after diagnosis during the second course of chemotherapy. Four of the remain-

A.J.C.P.-January 1995


DC O.

degraded nucleic acids that were unsuitable for further analysis. PCR amplification for the detection of the EBV EBNA-2 gene was then performed on cases 2, 4, 6-8. One fig of DNA was mixed with 0.1 nM of each dNTP in 1.0 mM MgCl 2 , 1.0 unit of Taq DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT), as well as 1.6 and 1.4 /*M of the 5' and 3' primers, respectively, in a total volume of 25 fd. Amplification was achieved after 40 cycles in an air thermocycler (Idaho Technology, Idaho City, ID) using the following conditions: 95 °C for 10 seconds, 55 °C for 10 seconds, and 72 °C for 15 seconds. The polymerase chain reaction (PCR) primers were complementary to the EBV EBNA-2 gene sequence,32 and the specificity of the amplification product was confirmed by Southern hybridization using an internal oligonucleotide probe. 32 The detection of bcl-2 translocation was performed on the previously mentioned six cases using the primer sets described by Gribben and colleagues.33 An air thermocycler (Idaho Technology) was used and the reagents were modified as follows: DNA 1.5 /ig/reaction, primer concentration 1.0 fiM, dNTP 200 fiM, Taq 1.0 unit, MgCl2 2.25 mM, KC1 20 mM, Tris HC1 buffer 50 mM at pH 8.5, and 500 Mg/mL of bovine serum albumin in a total volume of 25 fiL. The reaction parameters were modified as follows: initial denaturation for 10 seconds at 94 °C, annealing for 10 seconds at 55 °C for the major breakpoint region (MBR) primers, and 58 °C for the minor cluster region (MCR) primers, and extension for 20 seconds at 72 °C. After 25 cycles, 2.5 /iL of the amplified product were removed and reamplified for 30 cycles using nested primers. The conditions were similar to the first amplification except that the annealing temperatures for the nested major and minor break point primers were 60 °C and 66 °C, respectively. Ten /tL of the product were electrophoresed in a 2.0% agarose gel (Nu Sieve FMC, Rockland, ME) and transferred to a nylon membrane. The membrane was probed with internal oligonucleotides end labelled with y 32P dATP (Life Technologies, Gaithersburg, MD). Positive controls for rearrangements at the MBR and MCR consisted of DNA from the cell line RL-7 and DNA from a patient shown previously to have a rearrangement at the MCR by Southern blot analysis, respectively.

90

85

Diffuse

Diffuse

Diffuse

Diffuse

Diffuse

Diffuse

Diffuse

T-zonal

1

2

3

4

5

6

7

8

Irregular

Round 10.0 10

9

ND Equivocal

5

ND

ND ND

8

NA

NA

7

18