Howard Ratech

Neutrophil-rich, Ki-1-positive anaplastic large-cell malignant lymphoma

The presence of neutrophils, in the absence of necrosis, is uncommon in malignant lymphoma (ML). We identified a subgroup of Ki-1-positive anaplastic large cell ML (Ki-1 ALCL) in which neutrophils were a prominent component. Six of 20 cases of Ki-1 ALCL had a significant neutrophil infiltrate that varied from 5 to 10% to >50% of cells per high power field. Neutrophils were not seen in 100 cases of other types of ML reviewed. Patients were first seen with skin lesions (four), localized lymphadenopathy (three), generalized lymphadenopathy (one), and localized extranodal disease (one). All had primary disease. Two patients had peripheral neutrophilia. Three of six patients had clinical stage IV disease. Four patients are currently in clinical remission; one died of recurrent disease; and one patient with acquired immunodeficiency syndrome (AIDS) died of Pneumocystis carinii pneumonia. Four cases demonstrated a T-cell phenotype, one of which arose in a patient with AIDS. Two had a B-cell phenotype. All cases were positive for CD30 (Ki-1). These observations expand the morphologic spectrum of Ki-1 ALCL to include a neutrophil-rich variant. We conclude that the presence of neutrophils is another morphologic feature shared by some cases of Ki-1 ALCL, lymphomatoid papulosis, and Hodgkins disease, which suggests a possible pathogenetic link among them.

Pilot trial of infusional cyclophosphamide, doxorubicin, and etoposide plus didanosine and filgrastim in patients with human immunodeficiency virus- associated non-Hodgkin's lymphoma

PURPOSE To determine the following: (1) the feasibility of combining the antiretroviral didanosine (ddl) with a 96-hour continuous intravenous (IV) infusion of cyclophosphamide (800 mg/m2), doxorubicin (50 mg/m2), and etoposide (240 mg/m2) (CDE) plus filgrastim in patients with non-Hodgkins lymphoma (NHL) associated with human immunodeficiency virus (HIV) infection; (2) the effect of ddl on CDE-induced myelosuppression and CD4 lymphopenia; and (3) the effect of CDE on serum p24 antigen and quantitative HIV blood cultures. METHODS Twenty-five patients with HIV-related NHL received CDE every 28 or more days. Consecutive patients were assigned in an alternating fashion to group A (ddl given at a standard dose during cycles one, two, five, and six) or group B (ddl given during cycles three, four, five, and six). RESULTS ddl use was associated with less leukopenia (mean nadir, 3.33 v 1.49 x 10(3)/microL; p = .03), neutropenia (2.38 v 1.07 x 10(3)/microL; p = .03), and thrombocytopenia (76 v 48 x 10(3)/microL; p = .059), and fewer RBC (1.6 v 3.1 per cycle; p < .01) and platelet transfusions (0.7 v 1.5 per cycle; p < .01), but had no significant effect on CD4 lymphopenia. Furthermore, lymphomatous bone marrow involvement and low CD4 count were associated with significantly greater myelosuppression. Although there was no substantial change in serum p24 antigen, the HIV blood culture became quantitatively more positive or converted from negative to positive in seven patients (64%). Complete response (CR) occurred in 58% of patients (95% confidence interval, 38% to 78%), median CR duration exceeded 18 months, tumor-related mortality was 20%, and median survival was 18.4 months. CONCLUSION Our results suggest that the CDE and filgrastim regimen is tolerable and effective for patients with HIV-associated NHL, and that combination with ddl is feasible and may result in less myelosuppression.

Atypical clonal T-cell proliferation in infectious mononucleosis

An atypical case of infectious mononucleosis characterized by fever, acute tonsillitis, and bilateral cervical adenopathy is reported in a previously healthy young man. Although serology was positive for the Epstein-Barr virus, the patient did not display peripheral blood lyrnphocytosis or atypical, reactive lymphocytes. The patient’s tonsilar tissue revealed an expanded T-zone of diffuse, monomorphous lymphocytes suggestive of lymphoma. Immunophenotypic analysis of the tonsilar tissue demonstrated more than 90% expression of pan-T markers, while pan-B markers were positive in 5-10% of the interfollicular T-zone cells and in 90% of germinal centre cells. In situ hybridization with a probe specific for EBER1 demonstrated positive staining in approximately 1% of the interfollicular tonsilar lymphocytes. Finally, Southern blot analysis of tonsilar tissue demonstrated a clonal rearrangement of the T-cell receptor gene. The patient recovered from his infection and remains in good health years after presenting with his illness. This case illustrates that T-cell clonality must be evaluated with caution in the setting of a viral infection and can occur in association with benign, self-limited infectious mononucleosis.

Comparison of BIOMED-2 Versus Laboratory-Developed Polymerase Chain Reaction Assays for Detecting T-Cell Receptor-γ Gene Rearrangements

Detecting clonal T-cell receptor (TCR)-gamma gene rearrangements (GRs) is an important adjunct test for diagnosing T-cell lymphoma. We compared a recently described assay (BIOMED-2 protocol), which targets multiple variable (V) gene segments in two polymerase chain reaction (PCR) reactions (multi-V), with a frequently referenced assay that targets a single V gene segment in four separate PCR reactions (mono-V). A total of 144 consecutive clinical DNA samples were prospectively tested for T-cell clonality by PCR using laboratory-developed mono-V and commercial multi-V primer sets for TCR-gamma GR. The combination of TCR-beta, mono-V TCR-gamma and multi-V TCR-gamma detected more clonal cases (68/144, 47%) than any individual PCR assay. We detected clonal TCR-beta GR in 47/68 (69%) cases. Using either mono-V or multi-V TCR-gamma primers, the sensitivities for detecting clonality were 52/68 (76%) or 51/68 (75%). Using both mono-V and multi-V TCR-gamma primers improved the sensitivity for detecting clonality, 60/68 (88%). Combining either mono-V or multi-V TCR-gamma primers with TCR-beta primers also improved the sensitivity, 64/68 (94%). Significantly, TCR-gamma V11 GRs could only be detected using the mono-V-PCR primers. We conclude that using more than one T-cell PCR assay can enhance the overall sensitivity for detecting T-cell clonality.

Rapid and Accurate Detection of Monoclonal Immunoglobulin Heavy Chain Gene Rearrangement by DNA Melting Curve Analysis in the LightCycler System

The detection of immunoglobulin heavy chain gene rearrangement (IgH-R) is a standard tool for distinguishing polyclonal from monoclonal B-cell populations. Current DNA-based polymerase chain reactions (PCR) strategies can diagnose monoclonal IgH-R either by measuring the length of the amplicon or by detecting gel mobility variations owing to sequence-dependent conformational changes. However, amplification and analysis remain sequential operations usually requiring manual transfer. We have developed a novel PCR strategy for detecting monoclonal IgH-R that monitors fluorescence of the specific double-stranded DNA binding dye SYBR Green I during melting curve analysis using the LightCycler System. We compared polyacrylamide gel electrophoresis (PAGE) versus melting curve analysis in 130 clinical DNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues (mostly skin biopsies) of 128 patients. The identical FR3 primers were used to amplify the IgH variable region for both analytic techniques. We detected IgH-R in 24 DNA samples from FFPE tissue of 22 patients. Melting curve analysis, compared to PAGE, revealed no false negative and no false positive results, yielding both sensitivity and specificity equal to 100%. We also compared Southern blot analysis versus melting curve analysis in 23 clinical DNA samples from fresh-frozen lymph nodes of 23 patients. We detected IgH-R by melting curve analysis in 7 DNA samples from fresh-frozen lymph nodes. Melting curve analysis, compared to Southern blot analysis, revealed sensitivity equal to 58.3% (7 of 12) and specificity equal to 100% (11 of 11). We conclude that continuous fluorescence monitoring of PCR products with DNA melting curve analysis can rapidly and reproducibly distinguish polyclonal from monoclonal B-cell populations.

Eccrine gland infiltration by mycosis fungoides

After identifying prominent eccrine infiltration by atypical lymphocytes in a biopsy of tumor stage mycosis fungoides (MF), we sought to determine the pattern of eccrine epithelial infiltration in MF. The frequency, intensity, and distribution of infiltration of eccrine gland structures, including acrosyringium, duct and coil epithelium, was studied by examining 71 biopsy specimens from 42 patients with MF in which eccrine structures were present. These were obtained from a retrospective review of pathologic specimens from Duke University Medical Center from 1992 and 1993. At least focal eccrine infiltration was noted in 23 of the 71 biopsy specimens (32%). Immunohistochemical confirmation of T-lymphocyte phenotype was performed in the 23 cases with positive reaction to antibodies CD3 and CD45RO and negative reaction with CD20. Folliculosebaceous units were present in 22 of the 71 biopsy specimens and were at least focally involved by MF in 11 (50%) in this series. A control group of biopsy specimens of reactive dermatoses were characterized by more superficial location of lymphocytes, with more spongiosis and epithelial degenerative changes. These findings further illustrate the epitheliotropic behavior of MF.

AIDS and non-AIDS diffuse large B-cell lymphomas express different antigen profiles

Based on gene expression profiling, diffuse large B-cell lymphomas arising in immunocompetent patients can be divided into germinal center and activated B-cell types. Since little is known about acquired immunodeficiency syndrome associated diffuse large B-cell lymphomas, we tested whether the protein expression of germinal center and activated B-cell markers differed between acquired immunodeficiency syndrome (AIDS) vs non-AIDS diffuse large B-cell lymphomas. We immunohistochemically stained tissue microarrays of 39 de novo diffuse large B-cell lymphomas: 12 AIDS associated and 27 non-AIDS, with germinal center (BCL6, CD10, CyclinH) and activated B-cell markers (MUM1, CD138, PAK1, CD44, BCL2). We scored each case for percent positive cells (0–19%=0; 20–49%=1; 50–100%=2). The activated B-cell and germinal center summation scores of each case were used as (x, y) coordinate data points to construct two-dimensional contour-frequency plots. The contour plot of non-AIDS diffuse large B-cell lymphomas showed two distinct clusters: a cluster with a high germinal center phenotype (cluster 1) and a cluster with a high activated B-cell phenotype (cluster 3). In contrast, the AIDS-related diffuse large B-cell lymphomas formed a single aggregate (cluster 2) (P=0.02, Fisher exact test). When the contour plots of the AIDS-related and the non-AIDS cases were superimposed, cluster 2 of the AIDS cases expressed an intermediate germinal center/activated B-cell phenotype compared to clusters 1 and 3 of the non-AIDS diffuse large B-cell lymphomas. Our results confirm that non-AIDS diffuse large B-cell lymphomas segregate into two groups with either germinal center or activated B-cell phenotype. We report the new finding that the AIDS status of the patient predicts the immunophenotype of the diffuse large B-cell lymphomas.

Rapid Detection of Clonal T-Cell Receptor-β Gene Rearrangements in T-Cell Lymphomas Using the LightCycler-Polymerase Chain Reaction with DNA Melting Curve Analysis

Various molecular methods have been developed to diagnose clonal T-cell receptor (TCR) gene rearrangements in clinical samples. Most polymerase chain reaction strategies for detecting clonal TCR gene rearrangements rely on either gel or capillary electrophoresis. However, a cumbersome manual transfer step separates amplification from analysis. Recently, we developed a novel polymerase chain reaction assay using the LightCycler system to detect clonal immunoglobulin heavy chain gene rearrangement. In the current study, we extend this work to include the TCR. We report that clonal TCR-beta (TCR-beta) gene rearrangements can be detected in less than 1 hour after preparing the DNA by measuring DNA melting immediately after amplification in a single closed capillary tube. We retrospectively studied 52 fresh-frozen tissue samples from patients clinically suspected of T-cell malignancy. A clonal TCR-beta gene rearrangement was detected in 14 samples by DNA melting curve analysis. When DNA melting was compared to the gold standard methods of Southern blot or denaturing gradient gel electrophoresis, it achieved a sensitivity equal to 71% and a specificity equal to 94%. We also compared melting curve analysis and polyacrylamide gel electrophoresis: melting curve analysis reached a sensitivity equal to 100% and a specificity equal to 97%. We conclude that DNA melting curve analysis in the LightCycler system has potential for clinical use as a new, ultra-fast method for the initial diagnosis of clonal TCR-beta gene rearrangements.

Rapid diagnosis of clonal immunoglobulin heavy chain gene rearrangements in cutaneous B-cell lymphomas using the lightcycler-polymerase chain reaction with DNA melting curve analysis

We have recently developed a novel Immunoglobulin heavy chain gene rearrangement (IgH-R) assay that combines polymerase chain reaction (PCR) amplification and analysis in the same closed capillary tube using the LightCycler System. IgH-R can be identified by DNA melting curve analysis within 40 minutes after DNA preparation and amplification. To test the clinical utility of this new IgH-R assay for rapidly diagnosing cutaneous B-cell lymphomas, we prospectively analyzed 44 formalin-fixed, paraffin-embedded tissues suspected of B-cell malignant lymphoma: skin (n = 31), lymph node (n = 7), stomach (n = 3), spleen (n = 1), colon (n = 1), and soft tissue (n = 1). We detected IgH-R in 12 DNA samples, including 8 skin biopsies, with the following diagnoses: B-cell chronic lymphocytic leukemia (n = 4), extranodal marginal zone B-cell lymphoma (n = 4), diffuse large B-cell lymphoma (n = 2), Burkitt lymphoma (n = 1), and precursor B-lymphoblastic lymphoma (n = 1). DNA melting curve analysis, compared with polyacrylamide gel electrophoresis, achieved a sensitivity equal to 92.3% and a specificity equal to 100%. There was a single false negative result because DNA melting curve analysis could not detect less than 10.0% clonal B-cells. We conclude that this new, rapid PCR assay for detecting IgH-R based on DNA melting curve analysis can be clinically useful for confirming the initial diagnosis of B-cell malignant lymphoma.

Distribution of Bcl-2 and Bax in embryonic and fetal human skin: antiapoptotic and proapoptotic proteins are differentially expressed in developing skin.

The Bcl-2 protein is involved in the regulation of apoptosis. Bax has an antagonistic effect and enhances cell death. We report that in early gestation, Bcl-2 and Bax colocalize to the epidermal portion of the hair follicle. In the more advanced stages, Bax is located in the compartments where a hair canal is excavated and keratinization and holocrine secretion are initiated, in contrast to Bcl-2, which is expressed in the follicular papilla, preventing apoptosis and underscoring its role as a permanent and stable population of specialized fibroblasts. Scattered dendritic cells located in the basal and immediate suprabasal interfollicular epidermis as well as in the outer root sheath of the developing hair follicle, including the bulge, strongly express Bcl-2 and label for HMB-45, identifying them as melanocytes. The spatial and temporal expression pattern of Bcl-2 and Bax during human hair follicle development underscores their importance for hair biology and most likely is disturbed in the evolution of follicular tumors.