Peripheral T-cell lymphoma with Reed-Sternberg-like cells of B-cell phenotype and genotype associated with Epstein-Barr virus infection.

We report three cases of nodal peripheral T-cell lymphoma (PTCL) with Reed-Sternberg-like (RS-like) cells of B-cell pheno- and/or genotype. Histologic analysis in all cases revealed diffuse nodal effacement by atypical lymphoid cells of variable size. Two of the three cases had features of angioimmunoblastic T-cell lymphoma (AILT). Large mononuclear and binucleated cells with prominent eosinophilic nucleoli and abundant cytoplasm resembling classic RS cells and mononuclear variants were scattered throughout all biopsies. The lymphoma cells in the three cases were of T-cell lineage (CD3+, CD43+, and CD45RO+). The RS-like cells from all cases were CD30 and CD15 positive. In contrast to the neoplastic T cells, the RS-like cells lacked all T-cell markers and in two cases were positive for CD20. Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) and EBER 1 (2/2) were detected in the RS-like cells in all cases. The neoplastic T cells were negative for EBV. Polymerase chain reaction (PCR) analysis demonstrated clonal rearrangements of the T-cell receptor gamma chain gene in the three cases. PCR analysis of microdissected RS-like cells for immunoglobulin heavy chain gene rearrangements in cases 1 and 3 showed an oligoclonal pattern. The presence of RS-like cells in PTCL represents a diagnostic pitfall, because in one case this observation led to a misdiagnosis of Hodgkin's disease (HD). The oligoclonal expansion of EBV-infected cells may be related to underlying immunodeficiency associated with T-cell lymphomas and AILT in particular. This phenomenon may provide the basis for some cases of Hodgkin's disease after T-cell lymphomas and suggests that they are clonally unrelated neoplasms. The expression of LMP1 appears to be crucial for the immunophenotype and probably for the morphology of the RS and RS-like cells appearing in diverse lymphoid malignancies, including HD, chronic lymphocytic leukemia, and PTCL.

Cutaneous lymphomatoid granulomatosis: correlation of clinical and biologic features.

Lymphomatoid granulomatosis (LYG) is a rare angiocentric and angiodestructive Epstein-Barr virus-associated B-cell lymphoproliferative disorder (EBV-BLPD), varying widely from an indolent process to an aggressive large cell lymphoma. The skin is the extrapulmonary organ most commonly involved in LYG. We studied 32 skin lesions from 20 patients with known pulmonary LYG, using immunohistochemistry, in situ hybridization for EBV, and polymerase chain reaction for the presence of antigen receptor gene rearrangements (IgH and TCR) to better define both the clinicopathologic spectrum and pathogenesis of the cutaneous lesions. We describe two distinct patterns of cutaneous involvement. Multiple erythematous dermal papules and/or subcutaneous nodules, with or without ulceration, were present in 17 patients (85%). These lesions demonstrate a marked angiocentric lymphohistiocytic infiltrate, composed predominantly of CD4-positive T-cells, with a high propensity for involving the subcutaneous tissues, and exhibiting angiodestruction, necrosis, and cytologic atypia. EBV-positive B-cells were detected in the nodules from five patients; clonal immunoglobulin heavy chain gene (IgH) rearrangements were detected by polymerase chain reaction in two patients. Multiple indurated, erythematous to white plaques were present in three patients (15%). The plaque lesions were negative for EBV and clonal IgH gene rearrangements in all cases studied. The clinical course of overall disease was variable, ranging from spontaneous regression without treatment (1 of 13; 7%), resolution with chemo/immunomodulatory therapy (8 of 13; 62%), and progression (4 of 13; 31%). The clinical and histopathologic features of cutaneous LYG are extremely diverse. However, the majority (85%) of the cutaneous lesions mirrors to some extent LYG in the lung, although EBV+ cells are less frequently identified. This subset of cases shows the histopathologic triad of angiodestruction with associated necrosis, panniculitis, and in some cases atypical lymphoid cells. The commonality of the histologic features in this group suggests a common pathophysiologic basis, possibly mediated by cytokines and chemokines induced by EBV. A small percentage of the lesions (15%) presented as indurated and atrophic plaques, and EBV was not identified in the small number of cases studied. The relationship of the plaque-like lesions to LYG remains uncertain. Whereas some cases of LYG regress spontaneously, most require therapy.

Fatal EBV-related post-transplant lymphoproliferative disorder (LPD) after matched related donor nonmyeloablative peripheral blood progenitor cell transplant.

A 39-year-old male underwent a nonmyeloablative stem cell transplant (NMAPBPCT) from his HLA-matched sister for recurrent anaplastic large cell lymphoma in CR-2, receiving fludarabine, cyclophosphamide, and rabbit antithymocyte globulin for the preparative therapy. The patient was readmitted on day+33 for persistent culture-negative fevers. He rapidly developed marked elevations of alkaline phosphatase and bilirubin. Liver biopsy showed a periportal infiltrate of large immunoblastic appearing cells. The tumor cells did not stain for CD3/CD20/CD30 and alk protein, but did stain for CD79a/LCA and CD43. In situ hybridization for Epstein-Barr virus (EBV) RNA (EBER 1) was strongly positive in the periportal infiltrating lymphocytes. Fluorescence in situ hybridization (FISH) studies revealed female (XX) cells in the tumor cells and male (XY) in the surrounding hepatic parenchymal cells. The patient developed severe lactic acidosis, oliguric renal failure and expired on day+44. Both donor and patient had positive IgG serologies for EBV VCA and EBNA pretransplant. The donor also had a positive IgM titer for EBV VCA in the pretransplant specimen. The LPD may have been related to the intense immunosuppression of the preparative therapy and the presence of recent EBV infection in the donor.

High-dose therapy and autologous stem cell transplant does not result in long-term disease-free survival in patients with recurrent chemotherapy-sensitive ALK-negative anaplastic large-cell lymphoma.

Primary systemic anaplastic lymphoma kinase (ALK)-negative anaplastic large-cell lymphoma (ALCL) has a poor prognosis. This study sought to determine if high-dose therapy and ASCT results in long-term disease-free survival (DFS) in patients with recurrent, chemotherapy-sensitive ALK-negative ALCL. All patients with non-Hodgkin's lymphoma (NHL) who underwent ASCT at Wake Forest University and Upstate Medical University from 1 January 1990 to 12 December 2002 were reviewed to determine if they had T-, B- or null-cell NHL that was CD30+/CD15-/ALK negative. In all, 16 patients were thus identified as having ALK-negative ALCL. All 16 patients underwent ASCT at the time of first relapse and form the basis of this report. Median age of the 16 patients was 51 years. There were 11 males and five females. International prognostic index scores in 12 patients at the time of relapse were: low 3, LI 6 and HI 3. Of 15 patients, 13 relapsed after ASCT; one patient was lost to follow-up. Median progression-free survival for the 15 patients was 12 weeks (3-212+ weeks). Of 15 patients, 10 have died; nine of recurrent disease. Median overall survival for the 15 evaluable patients was 72 weeks. In our experience, high-dose therapy and ASCT does not produce long-term DFS in patients with recurrent chemotherapy-sensitive ALK-negative ALCL.

Differential distribution of (Na,K)-ATPase alpha isoform mRNAs in the peripheral nervous system.

mRNA transcripts for 3 isoforms of the alpha subunit of (Na,K)-ATPase have been previously identified in the nervous system (designated alpha 1, alpha 2, and alpha 3). In order to study the localization and expression of the different alpha isoforms in the peripheral nervous system, we prepared probes from the unique 3' untranslated region of alpha 1 cDNA, and from the translated region of alpha 3 cDNA. These probes were used in dot blot and in situ hybridization assays of rat spinal cord, dorsal root ganglia (DRG), and sciatic nerve. Within the ventral horn of lumbar spinal cord, alpha 1 mRNA was found in a discrete set of laterally placed motor neurons, while alpha 3 was found in all the identified neurons of the spinal cord, including those motor neurons containing alpha 1. In the lumbar DRG, alpha 3 was uniformly distributed in DRG neurons, while alpha 1 was abundant in some neurons but little or none was found in other neurons. Satellite cells contained neither isoform. Schwann cells in sciatic nerve were labeled with the alpha 1 probe in a perinuclear distribution, but contained no detectable alpha 3. Dot blot analysis showed alpha 1 and alpha 3 in spinal cord and DRG, but only alpha 1 in peripheral nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential distribution of (Na, K)-ATPase alpha isoforms in the central nervous system.

1. mRNA transcripts for three isoforms of the alpha subunit of (Na,K)-ATPase have been previously identified in the rat nervous system and designated alpha 1, alpha 2 and alpha 3. 2. In order to study the localization and expression of the different alpha isoform mRNAs on a regional and cellular level in the brain, we prepared probes from the unique 3' untranslated region of rat alpha 1 cDNA and from a segment containing a portion of the translated region of rat alpha 3 cDNA. These probes were used in dot blot and in situ hybridization assays of rat brain. 3. alpha 1 mRNA was found predominantly in cerebral cortex, dentate gyrus of hippocampus, and specific isolated brain-stem nuclei such as locus ceruleus and motor nuclei V and VII. In contrast alpha 3 mRNA was found predominantly in pyramidal neurons in the deep layers of cerebral cortex, in both pyramidal and dentate gyrus neurons of the hippocampus, and in neurons of most subcortical structures of the thalamus, basal ganglia, and brain-stem nuclei. 4. In the cerebellum, Purkinje cells showed predominantly alpha 3, as did stellate and basket cells. The granule cells contained predominantly alpha 1. 5. These experiments show that mRNAs for both alpha 1 and alpha 3 isoforms of (Na,K)-ATPase are found in neurons of the CNS. The isoforms have unique cellular and regional distributions, which in some cases overlap.

Effusion cytology of malignant melanoma. A morphologic and immunocytochemical analysis including application of the MART-1 antibody.

BACKGROUND: Malignant effusions are complications of metastatic malignant melanoma (MM). Differential diagnosis often involves distinguishing MM from adenocarcinoma and reactive mesothelial cells. Descriptions in the literature of the morphologic and immunocytochemical (IM) staining characteristics of MM in effusions are sparse. A combination of morphology and immunocytochemistry should yield the most accurate diagnostic results. The MART-1 antigen, a transmembrane protein, is specifically expressed in melanocytes and MM. A recently developed monoclonal antibody to the MART-1 antigen may represent a useful marker for the identification of MM in effusions. METHODS: The authors conducted a retrospective review of 32 effusion samples diagnosed as MM. The review consisted of morphologic and IM analyses of the effusion samples with antibodies to MART 1, HMB45, S-100, and cytokeratins (AE1/ AE3). IM stains were performed on cell block or cytospin material, depending on availability. In the morphologic review, emphasis was placed on Diff Quik-stained material, due to its enhanced cytoplasmic volume and detail. RESULTS: Predominant cytologic features noted were lack of cellular cohesion (in 100% of cases), large eccentric nuclei with prominent nucleoli (in 100%), multinucleation (in 84%), variable cytoplasmic vacuolization (in 75%), pigment (in 72%), and cell-in-cell engulfment (in 47%). All immunoreactive cases with sufficient material stained with at least one of the markers used. Tumor cells were positive with IM stains to MART-1 in 78% of cases, HMB45 in 81%, and S-100 in 81%. Coexpression of MART-1, HMB45, and S-100 was noted in 63% of cases. Of cases that showed expression for only 1 of the 3 antigens, the MART-1 was positive in 1 case, and HMB45 and S-100 were positive in 2 cases each. Three cases showed immunoreactivity for cytokeratins in the melanoma cells. CONCLUSIONS: The diagnosis of MM in effusions can be made reliably through a combination of morphologic and IM features. Differential diagnosis often involves distinguishing MM from adenocarcinoma or reactive mesothelial cells. Cytoplasmic vacuolization, multinucleation, prominent nucleoli, and cell-in-cell engulfment are cytologic features common to all three. The lack of IM staining for cytokeratins alone cannot reliably distinguish MM; 11% of cases showed positive staining with this antibody in the melanoma cells. The use of a panel of antibodies increases the accuracy of diagnosing MM. In this study, MART-1 proved a useful adjunct to the HMB45/S-100/cytokeratin panel for the diagnosis of MM in effusions, staining 78% of the immunoreactive cases, with positivity in 1 case that was negative for HMB45 and S-100.

Primary malignant lymphoma of uterine corpus: case report and review of the literature.

We describe a patient presenting with postmenopausal vaginal bleeding and a uterine mass subjected to endometrial biopsy that showed a high-grade non-Hodgkin's lymphoma, consistent with a diffuse large B-cell lymphoma. Staging computed tomography (CT) scans of the chest, abdomen, and pelvis revealed three lung nodules in addition to the uterine mass. Fine needle aspirate of one lung lesion showed lymphomatous involvement. She was treated with intensive chemotherapy alone and has remained in complete remission 21 months after diagnosis. The literature on primary lymphoma of the uterine corpus is reviewed.

Fine-needle aspiration of metastatic clear cell carcinoma of the kidney: employment of microdissection and the polymerase chain reaction as a potential diagnostic tool.

BACKGROUND: The differential diagnosis of metastatic clear cell carcinoma is broad. To date, there are no specific immunohistochemical markers for renal cell carcinoma (RCC) in general use. Loss of heterozygosity (LOH) at 3p25.5, the von Hippel-Lindau (VHL) gene locus, is frequent in sporadic clear cell RCC. The authors compared LOH in primary and metastatic RCC through microdissection and the polymerase chain reaction (PCR) to evaluate these techniques as potential diagnostic tools. METHODS: The authors identified 14 patients with known clear cell RCC who underwent fine-needle aspiration (FNA) evaluation of presumed metastatic lesion. Direct-visualization microdissection was performed from archival histologic glass slides of the primary neoplasm and the adjacent normal kidney parenchyma. Malignant cell clusters were microdissected from archival FNA slides of metastatic lesions. The cytology slides were previously stained with either Diff-Quik or Papanicolaou stain. This was followed by a single-step DNA extraction and PCR amplification for evaluation of LOH using polymorphic markers, D3S1038 and D3S1110, flanking the VHL gene. RESULTS: Thirteen of the 14 cases contained DNA suitable for PCR in both the paraffin embedded and the FNA material. Eight of the 13 cases were heterozygous (informative) for the above markers, and 6 of these showed identical allelic loss in the primary and metastatic tumor for either one or both of the markers used. The remaining two cases did not show LOH at the VHL locus with the two polymorphic markers used. CONCLUSIONS: DNA from archival cytologic material stained with Papanicolaou stain or Diff-Quik is reliable for PCR amplification. Visually directed microdissection in combination with PCR has the potential to be a useful technique for confirmatory identification and diagnosis of metastatic clear cell RCC in cytologic material, because a specific genetic abnormality is present in the primary tumor. As characteristic genetic abnormalities are identified in various neoplasms, the use of this technique has the potential for conclusive evaluation of metastatic disease with FNA material, when used in comparison with surgical or cytologic material from the primary tumor. The utility of this combination of techniques has the potential for the molecular diagnosis of morphologically ambiguous cell populations.

Composite low grade B-cell lymphomas with two immunophenotypically distinct cell populations are true biclonal lymphomas. A molecular analysis using laser capture microdissection.

Low grade B-cell lymphomas comprise several well defined, clinically and immunophenotypically distinct disease entities. Composite lymphomas showing phenotypic characteristics of more than one of these tumor subtypes in the same site are rare, and both common and separate clonal origins of the two tumor parts have been reported for cases studied by molecular methods. We describe the detailed immunohistochemical and molecular findings in three cases with features of composite low grade B-cell non-Hodgkin's lymphoma (B-NHL). All three neoplasms contained morphologically distinct but interwoven compartments of different cell types, which exhibited discordant expression of several markers, including CD5, CD10, CD43, and cyclin D1. According to their morphology and phenotypes, they were classified as mantle cell lymphoma and follicular lymphoma (Case 1), follicular lymphoma and small lymphocytic lymphoma (Case 2), and mantle cell lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma (Case 3). PCR analysis of DNA obtained from whole tissue sections failed to reveal evidence for biclonality in any of the cases. We therefore isolated cell populations with different antigen expression patterns by laser capture microdissection and analyzed them by polymerase chain reaction amplification and sequencing of clonal immunoglobulin heavy chain gene rearrangements and oncogene rearrangements. Sequence analysis revealed unrelated clonal rearrangements in each of the two tumor parts in all three cases, suggesting distinct clonal origins. In addition, Case 1 showed a bcl-2 rearrangement present only in the follicular lymphoma part. Our findings suggest that low grade B-NHL with two distinct morphological and immunophenotypic patterns in the same anatomical site are frequently biclonal. This is in keeping with current classification schemes, which recognize subtypes of low grade B-NHL as separate disease entities. Furthermore, our analysis demonstrates the power of laser capture microdissection in revealing molecular microheterogeneity in complex neoplasms.