Immunoglobulin Gene Rearrangement Studies Research Articles

Non-African Burkitt Lymphoma Presenting With Oral Thrush and an Orbital Mass in a Child

To report an unusual case of orbital involvement with non-African Burkitt lymphoma in a child. A 26-month-old boy developed oral thrush and painless right proptosis with eyelid swelling. Magnetic resonance imaging disclosed a diffuse mass involving the inferior, lateral, and superior right orbit and both maxillary sinuses. Incisional biopsy of the orbital mass was performed. Histopathology disclosed diffuse infiltration of the orbital tissues by a high-grade lymphoid neoplasm that showed positive immunoreactivity for B-cell markers. Immunoglobulin gene rearrangement studies disclosed a characteristic t(8;14) translocation consistent with a small, noncleaved malignant lymphoma of the Burkitt type. Non-African Burkitt lymphoma may manifest with orbital involvement.

Lymphoid-associated antigen expression by acute myeloid leukemia.

The expression of lymphoid-associated antigens (LAA) on blasts in acute myeloid leukemia (AML) and myeloproliferative disorders in myeloid blast crisis (MPD/MBC) has often been used to establish a diagnosis of acute mixed lineage leukemia (AMLL). The purpose of this study was to determine the incidence of LAA expression in AML and MPD/MBC (Ly + AML); to assess lymphoid differentiation at the genomic level in Ly + AML; and to compare features of Ly + AML with AML and MPD/MBC lacking these antigens (Ly-AML). Seventy-four consecutive cases of AML and MPD/MBC were reviewed for blast morphology, TdT reactivity, and cytochemistry results. Blast immunophenotyping was performed by multiparameter flow cytometry. Acute myeloid leukemia was subtyped according to the FAB classification. Acute myeloid leukemia and MPD/MBC cases expressing one or more of the following antigens, CD2, CD3, CD5, CD7, CD19, or CD20, were considered to be Ly + AML. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement studies were performed by Southern blot analysis using probes for JH, Jkappa, and JBI/BII. Sixteen of the 74 cases (22%) were identified as Ly + AML. Of these, the T-cell-associated markers CD7, CD2, and CD5 were expressed on 7(44%), 6(38%), and 4(25%) Ly + AML cases, respectively. The B-cell-associated markers CD19 and CD20 were expressed on two cases (13%) and one (6%) case, respectively. The FAB subtypes were similarly represented among Ly + AML and Ly-AML. Expression of LAA did not correlate with TdT positivity. In nine cases of Ly + AML (7 expressing T-cell-associated antigens and two expressing B-cell-associated antigens), Southern blot analysis revealed no Ig or TCR gene rearrangements. These results suggest that expression of CD2, CD5, and CD7 in otherwise straightforward AML should not be taken as evidence of lymphoid lineage commitment and does not warrant a diagnosis of AMLL.

Establishment of cell lines derived from chronic lymphocytic leukaemic cells by transfection with myc and ras.

Seventy-one samples from 37 cases of chronic lymphocytic leukaemia (CLL) were transfected with human c-myc and/or N-ras. In only three cases did the CLL cells further transform and four cell lines were established following transfection with myc plus ras. Surface marker profiles and immunoglobulin gene rearrangement studies confirm that all cell lines have arisen from the original CLL cells. The cell lines are EBNA positive but negative for EBV latent membrane protein (LMP). Karyotyping of the cell lines is as follows: SA4, 46,XY; SA4-2, 45,XY, 20; RK4, 48,XY,+ 5,+ 12; EHC4, 47,XY, t(14;18)(q32;q21), + 12. Although the majority of CLL cells appear resistant to transformation in vitro with myc and ras with the methods used, it does occur in a small percentage (about 8%). There appeared to be no correlation between the ability to transform in vitro and clinical behaviour.

Lacrimal gland lymphoma: A cytomorphologic and immunophenotypic study

The cytomorphology of lacrimal gland lymphoma has not been specifically described. Herein we present six cases of histologically proven lacrimal gland lymphoma which we analyzed using fine-needle aspiration cytology, cell suspension immunophenotype analysis, and immunoglobulin gene rearrangement studies. Fine-needle aspiration cytology revealed atypical populations of cells comprised of either monomorphic small round lymphocytes with or without plasmacytoid features (4 cases), a mixed population of small and large irregular lymphocytes (1 case), or a population of large irregular lymphocytes (1 case). The initial cytologic diagnosis was malignant lymphoma in all six cases. Cell suspension immunophenotype analysis demonstrated that the lesions were composed predominantly of B-cells that expressed monotypic surface immunoglobulin. Three cases demonstrated an immunoglobulin heavy chain gene rearrangement. The atypical cytologic features and the abnormal immunophenotype were consistently predictive of malignant lymphoma. Given that these lesions are small and biopsy material is often limited, fine-needle aspiration offers the advantage of providing tissue that is ideal for cytologic and cell suspension immunophenotype evaluation, obviating the need to provide surgical biopsy material for this purpose. We conclude that fine-needle aspiration can identify malignant lymphoid lesions of the lacrimal gland and may serve as a valuable adjunct in the assessment of these lesions. Additional study is warranted to determine whether fine-needle aspiration can reliably distinguish between benign and malignant lymphoid proliferations of the lacrimal gland.

Comparison of Cytogenetic Analysis, Southern Analysis, and Polymerase Chain Reaction for the Detection of t(14; 18) in Follicular Lymphoma

This study was undertaken to compare the ability of cytogenetic analysis (CG), Southern analysis (SA) and the polymerase chain reaction (PCR) to detect the t(14; 18) in follicular lymphoma (FL). All methodologies were performed by standard techniques. The probes used for SA included major breakpoint region (mbr) and minor cluster region (mcr) probes. The primers for PCR were identical or similar to those used by other investigators. One hundred fifteen cases of FL were ascertained by morphologic criteria, from which sufficient fresh tissue was available for both CG and molecular analysis. Eleven cases failed by both methods (nonrepresentative sampling). One hundred four cases showed evidence of an abnormal clone by CG and/or immunoglobulin gene rearrangement (IgH) studies. Cytogenetic analysis failed in 2 cases, was positive for t(14; 18) in 91 of the remaining 102 cases (89%) and detected a non-t(14; 18) close in 11 cases. An IgH clonal rearrangement was confirmed in all 104 cases. Southern analysis detected a mbr or mcr rearrangement in 78 of 104 cases (75%). Polymerase chain reaction detected an mbr or mcr rearrangement in 68 of 104 cases (65%). The use of PCR as a clinical test to detect t(14; 18)-positive lymphomas, with single primer sets for the mbr and mcr, will result in a high false-negative rate. The use of additional primers to detect uncommon breakpoints sites will be required to enhance the sensitivity of PCR for detection of t(14; 18) in malignant lymphoma.

Monocytoid B-cell lymphoma arising in extranodal organs

Six cases of monocytoid B-cell lymphoma (MBCL) developing in extranodal sites (thyroid, three; stomach, two; rectum, one) are described. Patients were all women aged 46 to 65 years (median, 53 years). Three patients with thyroid lymphoma presented with an increasing goiter, two with positive serum antithyroid antibodies, and one patient had chronic lymphocytic thyroiditis (CLTH). The histologic type of surgically resected specimens was a diffuse proliferation of atypical lymphoid cells with monocytoid appearance, i.e., abundant pale cytoplasm with distinct cell border and small reniform nucleus. These cells were CD20+, 22+, 24-, 9- showing their B-cell origin. The monoclonal nature of the proliferating cells was confirmed by restricted expression of immunoglobulin (Ig) light chain and/or gene rearrangement study in three cases. Two cases of thyroid lymphoma in which the monoclonality could not be confirmed had histologic appearances characteristic of malignant lymphoma. All three patients with gastric or rectal lymphoma had reactive lymphoid hyperplasia (RLH) near the tumors. These findings showed presence of MBCL in the extranodal sites with invariable coexistence with lymphoid follicles formed by CLTH in thyroid or RLH in stomach and rectum.

Persistent polyclonal B lymphocytosis with Epstein-Barr virus antibodies and subsequent malignant pulmonary blastoma.

Persistent polyclonal B lymphocytosis (PPBL), a rare benign lymphoproliferative disorder, occurred in a 25 year old woman. The lymphocytes showed the phenotype cIgM+, FMC7+, CD19+, CD20+, which was similar to that seen in Waldenstrom's macroglobinaemia, but the proliferation was repeatedly shown to be polyclonal both immunologically and by immunoglobulin gene rearrangement studies. Eleven years after presentation the patient developed a malignant pulmonary blastoma, a rare pulmonary tumour of combined epithelial and mesenchymal origin. The failure to develop a lymphoid malignancy over 11 years together with immunological and genetic evidence of polyclonality confirms that PPBL is a benign lymphoproliferative disorder. The subsequent occurrence of a nonhaemopoietic malignancy suggests that benign lymphoid proliferations, like their malignant counterparts, may predispose to the development of neoplasia. A role for Epstein-Barr virus in the pathogenesis of one or both of these rare conditions is suggested by the finding of raised Epstein-Barr viral capsid antigen titres.

Allogeneic Bone Marrow Transplantation in Chronic Lymphocytic Leukemia: 17 Cases. Report of the EBMT.

Allogeneic bone marrow transplantation (BMT) was performed in 17 patients with chronic lymphocytic leukemia (CLL): 15 resistant and 2 untreated forms; 12 males and 5 females with a mean age of 40 years (32-49). The conditioning regimen and graft versus host disease (GVHD) prophylaxis were varied. Successful engraftment was obtained in 15 evaluable cases. Lymphocytosis and clinical symptoms subsided in all but one case. All 15 evaluable patients developed acute GVHD. Among the 17 patients grafted, one early death was observed at the 15th day post-BMT, and one refractory form died 2 months after BMT. Of the remaining 15 patients in complete remission (CR0, 4 died from GVHD, hemorrhage, and graft failure, and 2 relapsed at 7 and 54 months after BMT and died. 9 patients are alive in CR with a mean follow-up of 25.6 months (4-48). Chimerism was complete in 8 patients and partial in the 2 T-depleted cases. In one case, an immunoglobulin gene rearrangement study was performed showing no residual disease. These results suggest that allogeneic BMT might be proposed as an alternative and possibly curative therapy for refractory CLL in young patients when performed earlier in the disease course.

New Agents and Strategies in CLL Treatment.

Fludarabine Monophosphate (FLU) is an adenosine analogue with a high-level of anti-leukemic activity in patients (pts) with CLL. FLU (25-30 mg/m(2)/day × 5 every 4 weeks) was given to 78 previously treated pts with CLL. The overall response rate was 58% (CR 11%, nodular CR 26%, PR 21%). Subsequently, prednisone (P, 30 mg/m(2)/day × 5) was added to the FLU regimen and administered to 169 pts. The response rate, up to this time, was similar to the FLU regimen 52% (CR 12%, nodular CR 28%, PR 12%). The major toxicity was febrile episodes which occurred in one course in 5 for both groups of pts. The median survival was also similar in both groups (FLU 74 weeks and FLU + P 103 weeks). Thus, no advantage was noted with the addition of P to FLU. FLU and FLU + P have been administered to 36 pts and 62 pts respectively who have had no prior treatment. The response rate for FLU was 83% (CR 39%, nodular CR 36%, PR 8%) and FLU + P was 82% (CR 29%, nodular CR 34%, PR 19%). Several of the FLU + P group developed persistent thrombocytopenia despite a good anti-leukemic response. The rates of decrease of the circulating and marrow lymphocyte counts were similar for the FLU and FLU + P groups. No survival difference has been noted to date in these groups. Time to development of progressive disease in the previously treated pts FLU and FLU + P is 104 weeks and is similar for the CR and nodular CR pts 118 weeks vs. 97 weeks and for the PR pts 89 weeks. All pts appear eventually to recur. Insufficient data is available to evaluate the time to progression of the previously treated pts. Two parameter flow-cytometry for CD19 and CD5, Kappa/Lamda ratio and immunoglobulin gene rearrangement studies are able to detect residual bone marrow disease in a majority of pts with CR and nodular CR (BM lymphocytes <30%). As relapse appears to be inevitable in the previously treated group who respond, post-remission therapy will include autologous bone marrow transplantation for pts <60 years of age and recombinant alpha-Interferon for pts >60 years of age. Post-remission therapy for the previously untreated responders will not be given to allow an estimate of the unmaintained time to progression. Bone marrow will be stored in first remission for future use, if the pts recur. Combination therapy studies utilizing FLU combined with Ara-C or Doxorubicin are being conducted.

Cellular and Molecular Characterization of Two Cases of Castleman's Disease, Plasma Cell Variant.

We report the cellular and molecular characterization of two cases of Castleman's disease, plasma cell variant, that differed in their clinical presentation and course. Patient 1 had Castleman's disease in association with Kaposis's sarcoma unrelated to human immunodeficiency virus (HIV) infection and died while he was receiving an aggressive chemotherapeutic regimen for Kaposi's sarcoma. Patient 2 had an isolated retroperitoneal lymphoid mass with an adjacent enlarged limph nodes and his symptoms disappeared completely following the surgical removal of both. Pathologic and immunohistochemical analyses in both cases, revealed that there was a massive infiltration of polyclonal plasma cells in the interfollicular areas of the lymph nodes. Immunoglobulin gene rearrangement studies confirmed the polyclonal nature of B-lineage cells in the involved lymph nodes. Southern blot experiments failed to demonstrate the presence of EBV genome copies in the same lymph nodes. These paradigmatic cases lend further support to the notion that Castleman's disease is an extremely heterogeneous entity.

EPSTEIN-BARR VIRUS—DETERMINED CLONALITY IN POSTTRANSPLANT LYMPHOPROLIFERATIVE DISEASE

Analysis of the genomic termini of Epstein-Barr virus can provide valuable insight into the cofactor role of EBV in the development of B cell lymphomas and lymphoproliferative disease. We report EBV genomic findings in pathologic specimens from 10 patients who developed lymphoma or lymphoproliferative disease after renal or bone marrow transplantation. Endonuclease restriction patterns of EBV genomic termini are highly variable in size in both the episomal and linear configuration. This variability in fragment size permits direct assessment of tissue clonality in EBV-infected material. Hybridization with terminus-specific probes also reveals configuration of viral genome (circular and latent vs. linear and replicative). Nine of 10 patients had tumors with mono- or biclonal episomal markers, and 4 of 10 had evidence of linear or replicative virus. Analyses of virally determined markers were compared to immunoglobulin gene rearrangement studies, histologic immunophenotyping, cytogenetics, and clinical outcome. These 10 cases represent a spectrum of lymphoproliferative disorders ranging from benign polyclonal to malignant monoclonal disease. The molecular data lend credence to two important aspects of viral pathogenesis: (1) the finding of a homogeneous episomal population in the monoclonal tumors suggests that EBV infection is an early event in tumorigenesis that occurs before clonal expansion; and (2) therapeutic efficacy of acyclovir has been shown only in presence of polyclonal disease but may impact on intermediate stages where linear replicative virus can be found. Finally, the various assessments of tumor clonality were compared, and although heterogeneity was seen among patients and among diagnostic methods, analyses at the molecular level using virus and immunoglobulin gene specific probes were concurrent and provide the more sensitive means for detection of clonality.

Detection of B- and T-Cells in Paraffin-Embedded Tissue Sections: Diagnostic Utility of Commercially Obtained 4KB5 and UCHL-1

Although many recent studies have begun exploring the diagnostic utility of anti-B- and anti-T-cell antibodies that work on paraffin-embedded tissue sections, the most optimal panel to use remains uncertain. In addition, many of the published reports have used antibodies obtained before their commercial formulation and distribution. For these reasons, B5-fixed paraffin-embedded tissue samples from 174 reactive or neoplastic lymphoid and hematopoietic proliferations were immunostained with the commercially obtained antibodies 4KB5, UCHL-1, and, in selected cases, L26. In reactive nodes, 4KB5 stained B-cell areas and UCHL-1 T-cell areas. Seventy-nine percent of the B-cell neoplasms were 4KB5 positive, and only 2% were UCHL-1 positive. Two cases of myeloma were 4KB5 and UCHL-1 negative. The 4KB5-negative B-cell lymphomas (ML-B) were all L-26 positive. UCHL-1 stained 78% of the T-cell lymphomas (ML-T), and 4KB5 stained 14%. In the five 4KB5-positive putative T-cell lymphomas, immunoglobulin and T-cell receptor gene rearrangement studies were performed. In Hodgkin's disease (HD), Reed-Sternberg (RS) cells were UCHL-1 negative in 75% of cases and occasionally positive in 25%. Definite 4KB5 positivity of RS cells was identified in both cases of lymphocyte predominant HD and in one case of nodular sclerosing HD with monomorphic large cell areas. With the exception of 1 of 15 acute nonlymphocytic leukemias that was UCHL-1 positive, all acute leukemias were 4KB5 and UCHL-1 negative. In summary, commercially obtained 4KB5 and UCHL-1 form a useful but not absolutely specific or sensitive paraffin section immunoperoxidase panel for the categorization of B- and T-cell lymphoid neoplasms. Addition of L26 appears to add to the sensitivity and specificity of the panel. Definite immunophenotypic distinction of HD from non-Hodgkin's lymphomas, particularly of T-cell type, often was not possible.

Detection of monoclonal B lymphocytes in bone marrow and peripheral blood of multiple myeloma patients by immunoglobulin gene rearrangement studies.

To investigate whether B lymphocytes are involved in the malignant cell clone of multiple myeloma (MM), we performed immunoglobulin gene rearrangement analysis of mononuclear cells and separated B lymphocytes, isolated from bone marrow and peripheral blood of MM patients. The B lymphocytes were separated by immunomagnetic beads, coated with an HLA class II specific antibody. Southern blot analysis with a JH probe revealed in the bone marrow of three out of seven patients identical immunoglobulin gene rearrangements in the B lymphocytes when compared to the plasma cells. Out of 10 patients, two patients with a high tumour burden were found to have monoclonal B lymphocytes in the peripheral blood. These results suggest that B lymphocytes in the bone marrow are part of the myeloma clone and that they can circulate in the peripheral blood. Although previous studies indicated that the ratio of K to lambda bearing lymphocytes in the peripheral blood can provide evidence for B cell monoclonality, we did not find a correlation between the results of K/lambda analysis and immunoglobulin gene rearrangement.

The Clinical Spectrum, Pathology, and Clonal Analysis of Epstein-Barr Virus–Associated Lymphoproliferative Disorders in Heart–Lung Transplant Recipients

This study presents the clinical and laboratory observations on posttransplant lymphoproliferative disorders (PTLDs) occurring in 5 of 53 heart-lung transplantation recipients. Cervical lymph nodes, tonsils, lungs, and gastrointestinal tract were the common sites of involvement by PTLDs. The histopathologic findings showed a spectrum of lymphoid and immunoblastic proliferation ranging from diffuse hyperplasia to malignant lymphoma, immunoblastic or large cell type. All cases were associated with a primary Epstein-Barr virus infection, and viral DNA was demonstrated within the lesional tissue in three cases. Immunohistochemical and immunoglobulin gene rearrangement studies revealed a B-cell proliferation that was monoclonal in three cases and polyclonal in two cases. Compared with PTLDs arising in other organ transplant recipients, this series is remarkable for a high incidence of PTLDs (9.4%), a short interval to tumor diagnosis (2.2 months, mean), involvement of the primary allograft in three cases (60%), and the frequent development of bronchiolitis obliterans. Possible reasons for this distinct clinicopathologic profile are discussed.

Pseudoleukemia in Down’s Syndrome: Analysis of Immunophenotype and Gene Rearrangement

This report demonstrates a case of transient abnormal myelopoiesis (TAM) evolving in a patient with Down's syndrome. A diagnosis was established after the patient's blast cell count decreased considerably three weeks after the initial leukemic phase. The blast population in the authors' case expressed Leu-9 (CD7), 6D1, and TdT+. Cytochemistries showed some of the blast population to be peroxidase positive and Sudan black positive. Platelet peroxidase by electron microscopic examination showed some positive blasts. Therefore, surface markers and cytochemical studies in this case suggested an abnormal proliferation involving a pluripotential stem cell capable of expressing myeloid and lymphoid characteristics. Cytogenetics was performed at birth and showed 47,XY,+21/48,XY,+21,+mar, confirming the diagnosis of Down's syndrome. The origin of the chromosomal fragment was uncertain. It was of interest that during the remission phase of his pseudoleukemia there was a concomitant decrease in the extra chromosomal fragment. Immunoglobulin and T-cell antigen receptor gene rearrangement studies showed only germline patterns, indicating that the lymphoid cells in the blast population were not clonally expanded. Therefore, immunoglobulin and T-cell antigen receptor rearrangement analysis and immunophenotyping are extremely valuable techniques in distinguishing between TAM and acute lymphoblastic leukemia in patients with Down's syndrome.

Two patients Illustrating lymphoma transition and response to therapy in Sjögren's syndrome

Two patients with Sjögren's syndrome (SS) who subsequently developed malignant B-cell lymphomas are reported in detail. The first patient had both benign- and malignant-appearing lymphoid infiltrates on the same submandibular gland specimen and was successfully treated with combined chemotherapy and irradiation. The second patient developed cutaneous lymphoid infiltrates difficult to diagnose by light microscopy but containing a monoclonal IgM-Kappa population revealed by immunoperoxidase staining and immunoglobulin gene rearrangement studies. Her lesions resolved rapidly and completely on cyclophosphamide, recurred rapidly when this drug was discontinued, and resolved again on a second course of cyclophosphamie which is currently maintained at 50 mg daily. Both patients are doing well without recurrence two and three years after initial treatment. This clinical experience is presented to emphasize: (1) the clinical use of molecular biologic techniques to define the earliest appearance of malignant transformation in Sjögren's syndrome, and (2) the successful outcome that can be achieved with prompt institution of appropriate treatment. The phenomenon of lymphoma development in SS is discussed with regard to immunoregulatory abnormalities predisposing to malignancy in the setting of autoimmune disease.

Phenotypic, cytogenetic and molecular characterization of a new B-chronic lymphocytic leukaemia (B-CLL) cell line

A lymphoid cell line was established from a patient with B-cell chronic lymphocytic leukaemia (B-CLL) by infecting blood lymphocytes with Epstein-Barr virus (EBV). Immunoglobulin gene rearrangement studies and the presence of a chromosome marker (isochromosome 17q) provided the formal proof that the line has originated from the neoplastic B cells. The morphology and phenotype indicate that the EBV-induced cell line has reached a plasma cell-like stage of differentiation.