No evidence of HTLV-I proviral integration in lymphoproliferative disorders associated with cutaneous T-cell lymphoma

Several recent studies have reported detection of HTLV-I genetic sequences in patients with cutaneous T-cell lymphoma (CTCL) including mycosis fungoides and Sezary syndrome. The purpose of this study was to determine whether HTLV-I was detectable in lesional tissues of patients suffering from diseases known to be associated with CTCL. Thirty-five cases were obtained from diverse geographical locations including Ohio, California, Switzerland, and Japan. Six of them had concurrent CTCL. Cases were analyzed using a combination of genomic polymerase chain reaction (PCR)/ Southern blot, dot blot, and Southern blot analyses. All assays were specific for HTLV-I provirus. Sensitivity ranged from approximately 10(-6) for PCR-based studies to 10(-2) for unamplified genomic blotting. Lesional DNA from patients with lymphomatoid papulosis (fourteen cases), Hodgkin's disease (twelve cases), and CD30+ large-cell lymphoma (nine cases) was tested for the HTLV-I proviral pX region using a genomic PCR assay followed by confirmatory Southern blot analysis with a nested oligonucleotide pX probe. All cases were uniformly negative. All of the Hodgkin's disease cases, eight of the large-cell lymphoma cases, and six of the lymphomatoid papulosis cases were then subjected to dot blot analysis of genomic DNA using a full-length HTLV-I proviral DNA probe that spans all regions of the HTLV-I genome. Again, all cases were negative. Finally, eleven of the Hodgkin's disease cases were also subjected to Southern blot analysis of EcoRI-digested genomic DNA using the same full-length HTLV-I probe. Once again, all cases were negative. These findings indicated that, despite utilization of a variety of sensitive and specific molecular biological methods, HTLV-I genetic sequences were not detectable in patients with CTCL-associated lymphoproliferative disorders. These results strongly suggest that the HTLV-I retrovirus is not involved in the pathogenesis of these diseases.     

Detection of the t(2;5)-associated NPM/ALK fusion cDNA in peripheral blood cells of healthy individuals

The translocation t(2;5), which leads to the fusion of the nucleophosmin gene (NPM) on chromosome 5q35 to the receptor kinase ALK on chromosome 2p23, is found in CD30+ anaplastic large cell lymphomas and some cases of B-cell lymphoma. Hodgkin's disease (HD) is a malignant lymphoma characterized by large multinucleated tumour cells, Hodgkin and Reed-Sternberg (H&RS) cells, surrounded by a dense lymphohistiocytic infiltrate. Our group recently demonstrated NPM/ALK fusion cDNAs by single-cell RT-PCR in < 3% of CD30+ tumour cells in 2/9 cases of HD. To further delineate the relevance of this finding for HD, we studied the occurrence of NPM/ALK fusion genes in peripheral blood cells of healthy donors by RT-PCR. NPM/ALK fusion cDNAs were found by RT-PCR in 14/29 healthy individuals and confirmed by hybridization with a breakpoint-specific oligonucleotide. Due to the low rate of NPM/ALK-positive cells in the peripheral blood of positive individuals, an assignment to a defined cellular subpopulation was not possible. We conclude that NPM/ALK fusion genes are present in peripheral blood cells of healthy donors. After t(14;18) and t(9;22), t(2;5) represents the third example of tumour-associated translocation products in blood cells of apparently healthy donors. The implications of this finding are discussed.     

ALK gene products in anaplastic large cell lymphomas and Hodgkin's disease

The translocation t(2;5)(p23;q35), discovered in CD30+ anaplastic large cell (ALC) lymphomas, creates a potentially oncogenic fusion gene, part of which is contributed by a novel tyrosine kinase, ALK. Absence of ALK expression from normal hematolymphoid cells provides a basis for the morphologic assessment of t(2;5). The distribution of the t(2;5) in ALC lymphomas and Hodgkin's disease (HD), as assayed by nonmorphologic methods, is controversial. We used in situ hybridization and/or immunohistology to show ALK gene products in 85 ALC lymphomas, 82 HD cases, 40 other lymphoproliferations, as well as in 6 HD- and 4 ALC lymphoma-derived cell lines. ALK gene products were restricted to t(2;5)-positive ALC lymphoma cell lines and tumor cells of 16 primary non-B cell, common-type ALC lymphomas. These were mainly from young patients with initial lymphonodal disease. ALK expression was not detectable in any other specimen, including all cases of HD and HD-like type ALC lymphoma as well as secondary ALC lymphomas. Full congruence was noted for labeling results obtained with both methods. In agreement with cytogenetic analyses, but at variance with recently published studies, ALK gene expression distinguishes a subset of ALC lymphomas from other CD30+ lymphomas, including HD. The results do not support concepts attributing a significant role to the t(2;5) in the development of HD.     

Cutaneous CD30+ lymphoproliferative disorders: expression of bcl-2 and proteins of the tumor necrosis factor receptor superfamily

The spectrum of CD30+ cutaneous lymphoproliferative disorders is characterized by the histology of a high-grade lymphoma but frequent clinical regression of skin lesions in lymphomatoid papulosis (LyP) and occasional regression in CD30+ large cell lymphomas (LCLs). A recent study shows that apoptosis may be a significant mechanism of regression of LyP (Arch Dermatol 133:828-833, 1997). Therefore, we studied expression of proteins that induce apoptosis, including CD27, CD40, CD95, and nerve growth factor receptor (NGF-R), as well as anti-apoptotic protein bcl-2 in skin lesions from 25 patients within the spectrum of CD30+ cutaneous lymphoma. Our results show consistent expression of CD95 (APO-1/Fas), but rare or absent expression of CD27, CD40, and NGF-R on tumor cells from both regressing LyP lesions and nonregressing CD30+ lymphomas. Bcl-2 was expressed at low levels in LyP and at high levels in pleomorphic CD30+ lymphomas. These results indicate that, in addition to CD30, CD95 expression is preferentially expressed at high levels in all cutaneous CD30+ lymphomas and suggest that CD95 may play a role in the regression of CD30+ skin lesions. Expression of bcl-2 appears to protect tumor cells from apoptosis in CD30+ lymphoproliferative disorders.     

Clinical experience with nephron-sparing surgery in the presence of a normal contralateral kidney

Lymphomatoid papulosis is a rare cutaneous lymphoproliferative disorder with nodular, papulonecrotic or plaque-like lesions. Although it is clinically benign, the histology shows large, atypical lymphoid cells that display antigenic markers of activated T-helper lymphocytes and express CD30. There is a close relationship to Hodgkin's disease and to Ki-1-positive anaplastic large-cell lymphoma of the skin. For therapy, various modalities such as PUVA, steroids and acyclovir have been used. We report on a patient with a 10-year history of disease. Treatment with interferon alfa-2a, 3 MU 3 times/week for 4 weeks, and etretinate, 50 mg/day for 5 months, was initially successful, but lesions further relapsed 5 months after cessation of the therapy.     

ALK expression defines a distinct group of T/null lymphomas ("ALK lymphomas") with a wide morphological spectrum

The t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffm sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma "common" type (75%), "lymphohistiocytic" (10%), "small cell" (8.3%), "giant cell" (3.3%), and "Hodgkin's like" (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin's-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and "Hodgkin's-like" features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype ("ALK lymphomas"), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past.     

NPM/ALK gene fusion transcripts identify a distinct subgroup of null type Ki-1 positive anaplastic large cell lymphomas

The chromosomal aberration t(2:5) resulting in the juxtaposition of NPM and ALK genes is a well-known feature of several Ki-1+ anaplastic large cell lymphomas (ALCL) of the T-cell type. However, conflicting results have been reported concerning the presence of this gene rearrangement in other ALCL and Hodgkin's disease (HD), respectively. We performed NPM/ALK RT-PCR on 14 cases of ALCL expressing distinct myelomonocytic markers, e.g. CD11c, CD13, CD14 or CD68, but neither T-cell nor B-cell associated antigens (null cell phenotype). The specific translocation was found exclusively in six childhood tumours previously diagnosed as malignant histiocytosis (MH), whereas all adult lymphomas (three ALCL without characteristics of MH, three secondary ALCL following HD) and two paediatric cases of secondary ALCL following HD did not show NPM/ALK gene fusion products. By Southern blotting, the status of T-cell receptor (TCR) and immunoglobulin heavy chain genes (IgH) were investigated; two patients with initially diagnosed MH had the TCRdelta-chain gene rearranged (Ddelta2-Ddelta3 and Vdelta1-Jdelta1, respectively). IgH rearrangements were detected in only one patient with secondary ALCL. Our data indicate a high association of previously diagnosed MH and NPM/ALK gene rearrangements. In one case, this specific translocation was demonstrated at an early stage of development; in another, a mature TCRdelta-chain gene rearrangement was detected. These data support the hypothesis of a lymphoid origin of this subgroup of Ki-1 positive ALCL previously diagnosed as MH.     

Clinicopathologic manifestations of Epstein-Barr virus-associated cutaneous lymphoproliferative disorders

OBJECTIVE: To elucidate clinicopathologic manifestations of cutaneous lymphoproliferative disorders associated with Epstein-Barr virus (EBV) infection. DESIGN: Retrospective survey of case series. SETTING: University hospital medical center. PATIENTS: Sixty-five patients with cutaneous lymphomas and related disorders. MAIN OUTCOME MEASURES: Detection of EBV genes and EBV-encoded small nuclear RNAs. RESULTS: Evidence of latent EBV infection was demonstrated in 15 patients: 3 had malignant lymphoma with clinical features mimicking cytophagic histiocytic panniculitis, 6 had facial vesiculopapular eruptions mimicking hydroa vacciniforme, 4 had angiocentric lymphoma, 1 had histiocytoid lymphoma associated with hemophagocytosis, and 1 had plasmacytoma. Hypersensitivity to mosquito bites was noted in a patient with hydroa vacciniforme-like eruptions and another with histiocytoid lymphoma. Angiocentric infiltration of atypical lymphoid cells was a common histological feature in the patients with hydroa vacciniforme-like eruptions and angiocentric lymphoma. No evidence of EBV infection was apparent in 19 patients with mycosis fungoides or Sézary syndrome, 7 with adult T-cell leukemia or lymphoma, 3 with lymphomatoid papulosis (type A), and 2 with lymphocytoma cutis. CONCLUSION: Patients with EBV-associated cutaneous lymphoproliferative disorders present with unique and diagnostic clinicopathologic features distinct from those of mycosis fungoides or Sézary syndrome.     

CD30 antigen expression in florid immunoblastic proliferations. A clinicopathologic study of 14 cases

Reactive immunoblastic proliferations may be confused with certain types of non-Hodgkin's lymphoma and Hodgkin's disease on morphologic grounds. In addition, a characteristic antigen, CD30 (Ki-1; BerH2) on these neoplastic entities may be observed in morphologically atypical yet reactive florid immunoblastic proliferations such as those associated with acute infectious mononucleosis. Although it has been documented, a large series determining the frequency and extent of CD30 antigen expression on a variety of nonneoplastic immunoblastic proliferations is lacking. The authors studied 14 florid immunoblastic proliferations (9 in lymph nodes and 5 in tonsils) for CD30 antigen expression and for B- and T-cell paraffin markers. In situ hybridization to determine the presence of Epstein-Barr virus (EBV) genomes also was performed. Cases were classified into monospot-positive acute infectious mononucleosis (4 cases), EBV-related lymphoproliferative disorder suggestive of acute infectious mononucleosis (5 cases), and other etiologies (5 cases). CD30 Antigen expression was found on the immunoblasts in cases from all three categories and overall in 9 (64%) of 14 specimens. CD30 reactivity in the positive cases varied from occasional to numerous positive cells; 4 samples (3 EBV-related lymphoid proliferations and 1 vaccine-related lymphadenopathy) had numerous CD30-reactive immunoblasts. Expression of CD30 antigen on B or T cells and prominence of B or T cells within a proliferation were variable. Significant "atypia" of immunoblasts was found only in EBV-related disorders and correlated with B-cell prominence of the infiltrate. Appropriate clinical correlation and ancillary laboratory data are necessary to assist in differentiating these CD30(+)-reactive disorders from similar-appearing malignant lymphomas. Most important, a fresh tissue sample should be procured and adequately processed to allow for comprehensive determination of clonality and cellular lineage.     

The gray zone between malignant and reactive processes in lymphoproliferative diseases

The differences between reactive and malignant processes are sometimes blurred. Homogeneity is no longer a requisite for the diagnosis of lymphoma, as witnessed in mucosa-associated lymphatic tissue lymphoma and T-cell-rich B cell lymphoma, which are composed of an admixture of neoplastic clonal B cells and reactive T cells which occasionally are very prominent in the histological picture. Infectious mononucleosis, anaplastic large cell lymphoma, composite lymphoma and Hodgkin's disease, all share many similarities and may actually represent a continuous spectrum of pathological conditions. Immunodeficiency states, whether primary or acquired, are commonly associated with clonal lymphatic malignancies preceded by a polyclonal lymphoproliferative stage, which is usually reversible by reducing immunosuppression. The distinction between these stages is sometimes difficult to assess. Immunologists have so far failed to find a lymphatic tumor-specific antigen, hence, monoclonality is usually based on a constellation of factors, namely homogeneity of the phenotypic expression of few antigens, aberrant expression of antigens and restricted expression of kappa- or lambda-chains in malignancies expressing surface immunoglobulins. Nonrandom chromosomal translocations as well as other aberrations, usually important in the diagnosis of malignancy, are sometimes of limited value. This is mainly due to the existence of translocations [like t(14;18) and t(2;5)] in nonmalignant states, and their non-specificity [the existence of t(8;14) in Burkitt's lymphoma and large cell lymphoma, t(2;5) in Hodgkin's disease and anaplastic large cell lymphoma, and t(14;18) in large cell lymphoma evolving from follicular lymphoma and Burkitt's lymphoma]. The diagnostic tools available in 1995, although usually sufficient, are sometimes unable to distinguish between malignancy and reactivity. Some problematic cases will be more accurately defined as lying in the gray zone, or as belonging to a spectrum ranging between reactivity and malignancy.     

The safety and efficacy of intrabursal oxycodone and bupivacaine in analgesia after shoulder surgery

We experienced three patients with CD30+ diffuse large cell lymphoma having chromosomal abnormalities. The first patient was an 8-year-old girl with bilateral cervical lymphadenopathy. A biopsy of a cervical lymph node revealed diffuse large cell lymphoma (stage III), positive for CD30 and a chromosomal abnormality, t(2;5). She attained a remission and is now in complete remission 108 months after diagnosis, despite frequent relapses. The second patient was a 13-year-old boy with right axillar and supraclavicular lymph-node adenopathy. A biopsy of a cervical lymph node revealed diffuse large cell lymphoma (stage III), positive for CD30 and a chromosomal abnormality, t(2;5). He attained remission and was in continuous first remission 112 months after diagnosis. The third patient was an 11-year-old boy with fever and bilateral cervical lymph node revealed diffuse large cell lymphoma (stage III), positive for CD30 and chromosomal abnormality without t(2;5). He showed a very aggressive clinical course. Only the patients with Ki-1 lymphoma having t(2;5) survived over 100 months from the diagnosis, despite the advanced stage of the disease. These findings and a review of the literature showed that the presence or absence of t(2;5) may influence the outcome of Ki-1 lymphoma.     

Laparoscopic biopsy for suspected abdominal lymphoma

This study retrospectively reviewed the findings in laparoscopic biopsy specimens from 51 consecutive patients with suspected abdominal lymphoproliferative disorders. Histologic evaluation was supplemented (as necessary) by paraffin-section or frozen-section immunohistochemical analysis or by Southern blot hybridization. The laparoscopic procedure was diagnostic of a lymphoproliferative disorder in 24 patients (47%), of other neoplasms in 5 patients (10%), and of reactive tissue in 11 patients (22%); no tissue could be obtained for technical reasons (adhesions and inaccessible lesions) in 11 patients (22%). The 24 patients with lymphoproliferative disorders diagnosed by laparoscopic techniques included 14 patients with a new diagnosis of lymphoma and 10 patients with recurrent disease; pathologic findings were diagnostic of diffuse large cell lymphoma (11 patients), follicular lymphoma (11 patients), chronic lymphocytic leukemia (1 patient), and lymphocyte-predominant Hodgkin's disease (1 patient). Previous abdominal cytologic or core-needle biopsy specimens from 11 lymphoma patients did not yield an unequivocal diagnosis or subtype of lymphoma. The 11 patients (22%) in whom laparoscopic techniques did not produce a tissue sample needed laparotomy (10 patients) or femoral lymph node biopsy (1 patient) to document the diagnosis of large cell lymphoma (2 patients), follicular lymphoma (5 patients), composite lymphoma (1 patient), myeloma (1 patient), neurofibroma (1 patient), and reactive lymph nodes (1 patient). In the majority of patients with suspected abdominal lymphoma, laparoscopic techniques provide sufficient tissue for the diagnosis and classification of lymphoma and for the diagnosis of other causes of abdominal lymphadenopathy.     

The effect of antibiotic therapy on recovery of intracellular bacteria from bronchoalveolar lavage in suspected ventilator-associated nosocomial pneumonia

Several recent reports have suggested that nodular lymphocyte predominance Hodgkin's disease (NLPHD) may be distinct from other forms of Hodgkin's disease and may be more closely related to B-cell non-Hodgkin's lymphoma. This is primarily based on immunophenotypic studies that have shown that the L & H cells in NLPHD demonstrate a B-cell phenotype. In 1989, Poppema reported that the T cells in NLPHD differ from T cells in other forms of Hodgkin's disease in that they demonstrate reactivity for Leu 7 (CD57). In this study we tested the hypothesis that Leu 7 (CD57) reactivity of small lymphocytes in NLPHD is an immunophenotypic feature that distinguishes NLPHD from nodular sclerosing Hodgkin's disease and from certain B-cell lymphomas that may histologically simulate NLPHD, namely T-cell-rich B-cell lymphoma and follicular lymphoma. Using an image analysis method, we found Leu 7 (CD57) reactivity in an average of 18.9% of the small lymphocytes in the nodules of NLPHD compared with 3.9% in nodular sclerosing Hodgkin's disease, 4.3% in T-cell-rich B-cell lymphoma, and 2.1% in follicular lymphoma. Moreover, Leu 7 (CD57)-reactive small lymphocytes often showed a distinctive pattern in NLPHD, forming a ring of cells around the large L & H cells. While scattered Leu 7 (CD57)-reactive lymphocytes were found in the other disorders, the percentage of reactive cells and the pattern of reactivity were significantly different in NLPHD. These results suggest that Leu 7 (CD57) reactivity may be used as an additional immunophenotypic criterion in distinguishing NLPHD from nodular sclerosing Hodgkin's disease, T-cell-rich B-cell lymphoma, and follicular lymphoma. The clinical and biological significance of Leu 7 (CD57) reactivity of small lymphocytes in NLPHD merits further investigation.     

Detection of Epstein-Barr virus in transformations of low-grade B-cell lymphomas after fludarabine treatment

Fludarabine is a highly effective chemotherapeutic agent for chronic lymphocytic leukemia/small lymphocytic lymphoma and is also active in other B-cell lymphoproliferative disorders. Although highly efficacious in destroying the malignant B-cells, fludarabine also causes T-cell lymphopenia and immunosuppression. We present five patients given fludarabine for low-grade B-cell lymphoproliferative disorders who showed transformation of the primary neoplasm to a higher grade tumor. Immunohistologic antibody studies were performed on paraffin-embedded tissue sections of the initial tissue (when available) and on the follow-up biopsy specimens for CD20, CD3, CD45RO, CD43, CD30, CD15, and latent membrane protein (LMP-1) for Epstein-Barr virus (EBV). The initial diagnoses in these five patients included chronic lymphocytic leukemia/small lymphocytic lymphoma (three cases), follicle center lymphoma (one case), and Waldenstrom's macroglobulinemia (one case). All of the follow-up biopsy specimens showed scattered Hodgkin's-like cells, and two of the five also showed foci of large-cell transformation. The Hodgkin's-like cells showed CD30 immunoreactivity in four of the five cases and CD15 immunoreactivity in three of the five. Strong immunoreactivity of the large, atypical, Hodgkin's-like cells for LMP-1 of EBV was noted in four cases; in the remaining case, this finding was equivocal. In situ hybridization for EBV-encoded RNA was positive in four of the five cases. Molecular studies by polymerase chain reaction (PCR) showed the presence of EBV in three of the five cases. PCR for detection of immunoglobulin heavy chain demonstrated identical monoclonal rearrangements in the original lymphoma and transformation in one case with available material. The CD4 lymphocyte count in each patient was less than 550/microL, indicating cellular dysfunction. Transformation of low-grade non-Hodgkin's lymphomas after fludarabine therapy might be associated with EBV and severe immunosuppression.