Molecular genetic analysis in the diagnosis of lymphoma in fine needle aspiration biopsies. I. Lymphomas versus benign lymphoproliferative disorders

The configurations of immunoglobulin genes, T-cell receptor (TCR) beta chain genes and bcl-2 genes were analyzed by Southern blotting in DNAs derived from 35 fine needle aspiration biopsies from various lymphoproliferative disorders. Only 1 of 16 benign lymphoproliferative disorders showed clonality: the lymph node of a patient with Wiskott-Aldrich immunodeficiency syndrome, in which clonal rearrangement of the TCR beta chain gene was detected. Clonality was demonstrated in all 14 non-Hodgkin's lymphomas (NHLs), 2 of 3 cases of Hodgkin's disease (HD) and 2 cases diagnosed as NHL or angioimmunoblastic lymphadenopathy (AILD). None of the aspirates exhibited rearrangement of the bcl-2 gene. The studies of diagnostically difficult cases proved that molecular genetic analysis of DNA, when appropriately combined with clinical data and light microscopic analysis of the lesions, can be helpful in distinguishing between: (1) a hyperplastic lymph node and NHL or AILD; (2) NHL and well-differentiated lymphocytes; and (3) a hyperplastic lymph node and HD.     

Amplification and novel locations of endogenous mouse mammary tumor virus genomes in mouse T-cell lymphomas.

Endogenous mouse mammary tumor virus genomes are amplified and located in novel cell DNA sequences in many mouse T-cell lymphomas. Transplanted tumors recovered from the same mouse strain and shown to be of independent origin by chromosomal analysis, by the presence of JH immunoglobulin gene rearrangements, or by the integration patterns of exogenous Moloney MuLV genomes frequently showed similar patterns of novel mouse mammary tumor virus-containing cell DNA fragments. This process of amplification and relocation can occur within a limited number of cell generations and in C57BL/6 mice does not lead to the synthesis of mature virus-encoded proteins. In some instances, amplified mouse mammary tumor virus genomes contained novel restriction cleavage sites in the gag-pol region. The restricted time course of occurrence, lack of synthesis of mature virion proteins, and apparent site specificity indicate that this process of retrovirus amplification differs significantly from virus replication after exogenous infection.     

DNA polymerase chain reaction using fine needle aspiration biopsy smears to evaluate non-Hodgkin's lymphoma.

OBJECTIVE: To apply polymerase chain reaction (PCR) analysis to the fine needle aspiration biopsy (FNAB) evaluation of lymphoid proliferations. STUDY DESIGN: We analyzed 37 consecutive archived FNAB malignant lymphoma specimens. Immunophenotypic data from the fine needle aspiration biopsy and excisional biopsy material was available for all specimens. PCR to identify monoclonal rearrangements of the immunoglobulin heavy chain gene, T-cell receptor and translocations involving the bcl-1 and bcl-2 genes was performed. RESULTS: Seventy-eight percent of cases were detected by at least one of these assays. Where DNA analysis was performed on excisional biopsy material, 70% of the cases had identical results; no discordant results for the immunoglobulin heavy chain gene or T-cell receptor were found. In 23% of cases, after review of all available data, a discordant result was thought to be a consequence of a false negative result in DNA analysis of excisional biopsy material. CONCLUSION: These findings indicate that PCR analysis of archived FNAB material, when necessary, provides useful information for diagnosis and staging of malignant non-Hodgkin's lymphomas.     

Fine Needle Aspiration Cytology of High Grade T-Cell Lymphomas In Human T-Lymphotropic Virus Type 1 Carriers

We studied the fine needle aspiration (FNA) cytology of high grade peripheral T-cell lymphomas from eight human T-lymphotropic virus-1 (HTLV-1) positive patients. FNA smears from seven lymphomas showed a distinctive cytologic pattern with a dominance of rounded cells with irregular nuclei and a moderately basophilic cytoplasm. Irregular cells with a pale abundant cytoplasm were present in varying amounts. Some smears contained a few giant cells with cerebriform nuclei. In addition, plasma cells and eosinophils were found. Epithelioid cells were an inconstant finding. On histology these seven lymphomas were assigned to the pleomorphic medium-large cell subtype and all but one were of T-helper phenotype with rearrangements of the T-cell receptor. FNA smears from a lymph node in a patient with a previous histological diagnosis of lymphomatoid papulosis of the gingiva showed a monotonous pattern of large immunoblastic cells with some binucleated variants consistent with a diagnosis of high grade immunoblastic lymphoma, which was confirmed histologically. Our results show that peripheral T-cell lymphomas from HTLV-1 positive patients have cytological patterns which are distinctive enough to allow a conclusive diagnosis of high grade T-cell lymphoma. However, we do not think that the cytology of HTLV-1 positive lymphomas can be differentiated from that of virus-unrelated high grade T-cell lymphomas.     

Nucleotide sequence analysis of 95 kb near the 3' end of the murine T-cell receptor alpha/delta chain locus: strategy and methodology.

The nucleotide sequence of a region at the 3' terminus of the murine T-cell receptor alpha/delta chain locus is presented. This region, which encodes the constant region genes for alpha and delta chain polypeptides and all 50 joining gene segments for the alpha chain polypeptide, spans 94,647 bp and includes more than 50 noncoding sequence elements important for T-cell receptor gene rearrangement and expression. DNA sequencing of this region included complete analysis of two cosmid clones and five additional restriction fragments using a random subcloning approach with various manual and automated sequencing strategies. The automated sequencing strategies hold considerable promise for future large-scale DNA sequencing efforts.     

Evaluation of T-cell receptor gene rearrangements in patients with recurrent patch/plaque (T2) CTCL (mycosis fungoides)

Cutaneous T-cell lymphoma is typically a clonal neoplasm of epidermotropic CD4+ T-lymphocytes that includes the entity mycosis fungoides (MF). After identification of patients with recurrent MF treated with total skin electron beam therapy (TSEBT) at the Yale University School of Medicine, this study attempted to compare T-cell receptor (TCR) gamma gene rearrangements via polymerase chain reaction (PCR) in both original and recurrent skin biopsies from these patients. Between 1974 and 1996, a total of 95 T2 MF patients were treated with TSEB, and four of these were identified for the study. Slides and tissue samples of both primary and recurrent skin biopsies for each patient were confirmed as being consistent with ME DNA for PCR was isolated from paraffin-embedded tissue samples. Using consensus primers that hybridize with conserved regions of the TCR gene, these regions of the genome were amplified. The PCR products were then analyzed by acrylamide gel electrophoresis. Of the primary and recurrent samples from four patients with a median disease-free interval (DFI) of 1222 days, only two showed evidence of a dominant TCR clone. A number of factors, including lack of sequence homology between the primers and the gene segments, the existence of multiple neoplastic cell lines, DNA degradation in the archival samples, and the presence of reactive as well as malignant lymphocytes, may have prevented the detection of dominant TCR rearranged clones in the samples. Despite the results of this study, TCR analysis via PCR and gel electrophoresis continues to be of utility in the evaluation of patients with MF when used in conjunction with other diagnostic modalities and in cases with nonspecific clinical, histopathological, and immunophenotyping findings.     

Genotypic analysis of DNA isolated from fine needle aspiration biopsies

DNA was isolated from 20 fine needle aspiration (FNA) biopsies from lymphomas, hyperplastic lymph nodes and nonlymphoid malignant tumors. Small aliquots (0.2 microgram to 2.0 micrograms) of DNA from each sample were digested to completion with restriction endonuclease Eco RI and/or Bam HI and electrophoresed in 0.8% agarose minigels. DNA was transferred to a nylon filter after brief treatment in HCl and subsequent denaturation and neutralization. Filters were hybridized to radiolabeled JH, C kappa, TCR beta or bcl-2 probes to determine if these genes were in germline or rearranged configurations in each of the samples. It was possible to demonstrate rearrangement of at least one immunoglobulin gene in each of the samples diagnosed as lymphoma, while all samples derived from hyperplastic lymph nodes and nonlymphoid malignant tumors exhibited a germline pattern for each probe tested. Thus, FNA biopsies can provide suitable and sufficient DNA for genotypic analysis using molecular probes that detect gene rearrangement.     

Indolent Small Intestinal CD4+ T-cell Lymphoma Is a Distinct Entity with Unique Biologic and Clinical Features

Enteropathy-associated T-cell lymphomas (EATL) are rare and generally aggressive types of peripheral T-cell lymphomas. Rare cases of primary, small intestinal CD4+ T-cell lymphomas with indolent behavior have been described, but are not well characterized. We describe morphologic, phenotypic, genomic and clinical features of 3 cases of indolent primary small intestinal CD4+ T-cell lymphomas. All patients presented with diarrhea and weight loss and were diagnosed with celiac disease refractory to a gluten free diet at referring institutions. Small intestinal biopsies showed crypt hyperplasia, villous atrophy and a dense lamina propria infiltrate of small-sized CD4+ T-cells often with CD7 downregulation or loss. Gastric and colonic involvement was also detected (n = 2 each). Persistent, clonal TCRβ gene rearrangement products were detected at multiple sites. SNP array analysis showed relative genomic stability, early in disease course, and non-recurrent genetic abnormalities, but complex changes were seen at disease transformation (n = 1). Two patients are alive with persistent disease (4.6 and 2.5 years post-diagnosis), despite immunomodulatory therapy; one died due to bowel perforation related to large cell transformation 11 years post-diagnosis. Unique pathobiologic features warrant designation of indolent small intestinal CD4+ T-cell lymphoma as a distinct entity, greater awareness of which would avoid misdiagnosis as EATL or an inflammatory disorder, especially celiac disease.     

MicroRNA Profiling of Primary Cutaneous Large B-Cell Lymphomas

Aberrant expression of microRNAs is widely accepted to be pathogenetically involved in nodal diffuse large B-cell lymphomas (DLBCLs). However, the microRNAs profiles of primary cutaneous large B-cell lymphomas (PCLBCLs) are not yet described. Its two main subtypes, i.e., primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) and primary cutaneous follicle center lymphoma (PCFCL) are characterized by an activated B-cell (ABC)-genotype and a germinal center B-cell (GCB)-genotype, respectively. We performed high-throughput sequencing analysis on frozen tumor biopsies from 19 cases of PCFCL and PCLBCL-LT to establish microRNA profiles. Cluster analysis of the complete microRNome could not distinguish between the two subtypes, but 16 single microRNAs were found to be differentially expressed. Single microRNA RT-qPCR was conducted on formalin-fixed paraffin-embedded tumor biopsies of 20 additional cases, confirming higher expression of miR-9-5p, miR-31-5p, miR-129-2-3p and miR-214-3p in PCFCL as compared to PCLBCL-LT. MicroRNAs previously described to be higher expressed in ABC-type as compared to GCB-type nodal DLBCL were not differentially expressed between PCFCL and PCLBCL-LT. In conclusion, PCFCL and PCLBCL-LT differ in their microRNA profiles. In contrast to their gene expression profile, they only show slight resemblance with the microRNA profiles found in GCB- and ABC-type nodal DLBCL.     

Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas.

We have determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myc genes contained missense mutations. This strongly supports the notion that the c-myc proto-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.     

Molecular genetic analysis in the diagnosis of lymphoma in fine needle aspiration biopsies. II. Lymphomas versus nonlymphoid malignant tumors

The configurations of immunoglobulin genes and T-cell receptor beta chain genes were analyzed by Southern blotting in DNA derived from nonlymphoid malignant tumors and lymphomas. Gene rearrangements were not detected in any of the 35 cases of nonlymphoid malignant tumors. On the contrary, they were shown in all 14 cases of non-Hodgkin's lymphomas, 2 of 3 cases of Hodgkin's disease and 2 cases diagnosed as non-Hodgkin's lymphoma or angioimmunoblastic lymphadenopathy. The differentiation by light microscopy between lymphoma and nonlymphoid malignant tumors was a diagnostic problem in five cases; the molecular genetic analysis of DNA was contributory in all five diagnostically difficult aspirates. By gene rearrangement studies, the diagnosis of lymphoma was confirmed in two cases and nonlymphoid malignant tumors were accurately indicated in aspirates diagnosed finally as rhabdomyosarcoma (one case) and carcinoma (two cases).     

An unusual case report of indolent T-cell lymphoproliferative disorder with aberrant CD20 expression involving the gastrointestinal tract and bone marrow

Background

Indolent T-cell proliferative disorder of the GIT is a rare and provisional entity in the revised WHO 2016 classification. The patients usually have prolonged survival with persistent disease even without any treatment.

Case presentation

The 46 years old male patient has been followed up for more than 6 years without chemotherapy. Repeated gastrointestinal biopsies showed expansion of the lamina propria extending to the submucosa by small to medium sized lymphocytes with minimal cytologic atypia. The lymphoid cells were positive for CD3, CD43, TIA-1, CD2, CD7 and the B-cell marker CD20; but negative for CD4, CD8, PAX5, CD56, cyclinD1, granzyme (GraB) and Epstein Barr virus-encoded RNA (EBER). Ki-67(MIB1) index was less than 10%. Molecular tests demonstrated a clonal rearrangement for T-cell receptor γ (TCR γ) gene but immunoglobulin chain (IgH, IgK, IgL) gene remained germline. Recognition of possible aberrant CD20 expression in indolent T-cell LPD is important to avoid potential diagnostic pitfall and improper treatment.

Conclusions

We present an unusual case of indolent T-cell lymphoproliferative disorder with aberrant CD20 expression, Recognition of this unusual immunophenotype of indolent T-cell LPD of GI helps to eschew misdiagnosis of B-cell and other high grade lymphomas and inappropriate aggressive treatment.

     

An Epstein-Barr Virus (EBV) mutant with enhanced BZLF1 expression causes lymphomas with abortive lytic EBV infection in a humanized mouse model

Immunosuppressed patients are at risk for developing Epstein-Barr Virus (EBV)-positive lymphomas that express the major EBV oncoprotein, LMP1. Although increasing evidence suggests that a small number of lytically infected cells may promote EBV-positive lymphomas, the impact of enhanced lytic gene expression on the ability of EBV to induce lymphomas is unclear. Here we have used immune-deficient mice, engrafted with human fetal hematopoietic stem cells and thymus and liver tissue, to compare lymphoma formation following infection with wild-type (WT) EBV versus infection with a "superlytic" (SL) mutant with enhanced BZLF1 (Z) expression. The same proportions (2/6) of the WT and SL virus-infected animals developed B-cell lymphomas by day 60 postinfection; the remainder of the animals had persistent tumor-free viral latency. In contrast, all WT and SL virus-infected animals treated with the OKT3 anti-CD3 antibody (which inhibits T-cell function) developed lymphomas by day 29. Lymphomas in OKT3-treated animals (in contrast to lymphomas in the untreated animals) contained many LMP1-expressing cells. The SL virus-infected lymphomas in both OKT3-treated and untreated animals contained many more Z-expressing cells (up to 30%) than the WT virus-infected lymphomas, but did not express late viral proteins and thus had an abortive lytic form of EBV infection. LMP1 and BMRF1 (an early lytic viral protein) were never coexpressed in the same cell, suggesting that LMP1 expression is incompatible with lytic viral reactivation. These results show that the SL mutant induces an "abortive" lytic infection in humanized mice that is compatible with continued cell growth and at least partially resistant to T-cell killing.     

Cytomorphology and immunocytochemistry of fine needle aspirates from blastic non-Hodgkin's lymphomas

Fine needle aspirates from 54 consecutive patients with primary or recurrent blastic (high-grade malignant) non-Hodgkin's lymphomas (NHLs) were analyzed by cytomorphology and immunocytochemistry. The cytologic diagnoses induced follicular center-cell-derived (centroblastic or anaplastic centrocytic) lymphoma (31 cases), immunoblastic lymphoma (11 cases), lymphoblastic lymphoma (9 cases) and histiocytic lymphoma (3 cases). Immunocytochemistry showed a B-cell phenotype of the neoplastic lymphocytes in all lymphoblastic lymphomas, 29 follicle center-cell lymphomas and 4 immunoblastic lymphomas. Four of the immunoblastic lymphomas were of T-cell origin while one case was not evaluable due to necrosis. A histiocytic origin was confirmed in two of the three cases that had a cytologic diagnosis of histiocytic lymphoma; the third case was shown by immunocytochemistry to be a true Ki-1-positive large cell lymphoma. Histologic and immunohistochemical analysis were performed on surgical biopsies from 18 patients. The results were in agreement with those on the fine needle aspiration (FNA) material in 14 cases. Three lymphomas could be phenotyped on aspirated material while marker studies on excised material were inconclusive. One lymph node aspirate contained mostly necrotic cells, which were unsatisfactory for adequate immunocytochemistry. However, sections from a removed tonsil from the same patient could be used for conclusive histology and phenotyping. In conclusion, the high diagnostic accuracy of combined cytomorphologic and immunocytochemical assessment of FNA samples validates the use of the technique in the diagnostic work-up of blastic (high-grade malignant) NHLs. In fact, the diagnostic accuracy seems so high that the technique can safely be used in the final diagnosis of blastic NHLs.     

Identification of two murine loci homologous to the v-cbl oncogene.

The virally transduced oncogene v-cbl transforms fibroblasts in vitro and induces early B-cell-lineage lymphomas in vivo. A series of probes derived from a molecular clone of v-cbl were used to map related sequences in the mouse genome. Analyses of Chinese hamster x mouse somatic-cell hybrids showed that two related genes, cbl-1 and cbl-2, were located on chromosomes 6 and 9, respectively. Restriction enzyme studies of DNA from hybrid cells containing either chromosome 6 or 9 suggested that cbl-1 resembles v-cbl and may be a processed gene, whereas cbl-2 has a complex genomic structure. Analyses of Mus domesticus/M. spretus interspecific backcross mice showed that Cbl-1 maps between the immunoglobulin kappa light chain and T-cell receptor beta chain loci and that Cbl-2 is tightly linked to Thy-1.     

Ig V region gene expression in small lymphocytic lymphoma with little or no somatic hypermutation

Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V kappa gene) rearrangement in small lymphocytic non-Hodgkin's lymphomas that express Ig bearing a major kappa-L chain associated cross-reactive Id, designated 17.109. Previously, we identified the 17.109-cross-reactive Id in chronic lymphocytic leukemia as a serologic marker for expression of a highly conserved V kappa gene, designated Humkv325. Using sense-strand oligonucleotides specific for the 5'-end of this V kappa gene and antisense oligonucleotide specific for a J kappa region consensus sequence, we could amplify specifically Humkv325 when juxtaposed with J kappa through Ig gene rearrangement. This allowed us to amplify rearranged V kappa genes from DNA isolated from minute amounts of lymphoma biopsy material for molecular analyses. Our studies demonstrate that 17.109-reactive SL NHL, with or without associated CLL, rearrange, and presumably express, Humkv325 without substantial somatic diversification. Our data suggest that malignant B cells in SL NHL, in contrast to NHL of follicular center cell origin, may express immunoglobulin variable region genes with little or no somatic hypermutation.     

Detection of Epstein-Barr virus-associated antigens and DNA in salivary gland biopsies from patients with Sjogren's syndrome

Sjogren's syndrome (SS) is an autoimmune disorder characterized by lymphocytic infiltration of salivary and lacrimal glands. To determine whether Epstein-Barr virus (EBV) might play a role in the pathogenesis of this disorder, we used monoclonal antibodies and DNA probes to detect evidence of viral gene products and genomes in these patients' tissue biopsies and saliva. Cytoplasmic staining of epithelial cells (i.e., ductal and/or acinar cells) with monoclonal antibody against the EBV-encoded early antigen (EA-D) was noted in 8/14 salivary gland biopsies from SS patients. This antibody did not react with normal salivary glands or salivary gland tumors, nor with other tissues from SS patients. The reactive antigen in SS biopsies had a m.w. of 52,000 on the basis of immunoblotting experiments, similar to the EA-D antigen found in lymphoblastoid cells lytically infected with EBV. EBV DNA was detected in parotid biopsies from two SS patients in amounts ranging from 0.1 to 3 pg per 20 micrograms of cellular DNA. Southern blotting was used to demonstrate the reactivity with Bam V, Eco D, and Bam M probes. Parotid saliva samples from 8/20 SS patients contained EBV DNA detectable by slot blot hybridization. EBV DNA was not detected in saliva of age-matched controls, rheumatoid arthritis patients lacking sicca symptoms, or patients with benign parotid tumors. The presence of EBV in salivary gland and saliva samples was associated with clinically more severe SS, as manifested by extraglandular symptoms such as vasculitis, occurrence of "pseudolymphoma", and marked abnormalities in immunoglobulin levels. These results demonstrate an elevated content of EBV in salivary glands of SS patients, and suggest that this virus may play a role in pathogenesis.