Improved immunohistochemical evaluation of micrometastases in sentinel lymph nodes of cutaneous melanoma with 'MCW melanoma cocktail'--a mixture of monoclonal antibodies to MART-1, Melan-A, and tyrosinase.

BACKGROUND: MART-1, Melan-A, and Tyrosinase have shown encouraging results for evaluation of melanoma micrometastases in sentinel lymph nodes, as compared to conventionally used S-100 protein and HMB-45. To achieve higher sensitivity, some studies recommend evaluation of three sections, each at intervals of 200 micron. This would mean, routine staining of three adjacent sections in each of the three clusters at intervals of 200 micron, requiring nine slides resulting in added expense. If a cocktail of these antibodies could be used, only one section would be required instead of three generating significant cost savings. METHODS: We prepared a combination of monoclonal antibodies to these three immunomarkers in optimized dilutions (MART-1, clone M2-7C10, dilution 1:500; Melan-A, clone A103, dilution 1:100; and Tyrosinase, clone T311, dilution 1:50) and designated it as 'MCW melanoma cocktail'. Formalin-fixed paraffin-embedded tissue sections of sentinel lymph nodes from patients with cutaneous melanoma, without macro-metastases were evaluated with this cocktail. RESULTS: Melanoma micrometastases were easily detectable with the cocktail in 41 out of 188 slices (8/24 cases). The diagnostic accuracy amongst five pathologists did not show statistically significant difference. Out of 188 slices, 78 had adjacent sections immunostained individually with MART-1 and Melan-A during our previous study. Of these 78 slices, 21 were positive for melanoma micrometastases with MART-1 and Melan-A individually. However, the adjacent section of these slices immunostained with the cocktail detected metastases in four additional slices. Thus, MART-1 and Melan-A could not detect melanoma micrometastases individually in 16% (4/25) of slices positive with the cocktail. Benign capsular nevi were immunoreactive for the cocktail in 4.8% (9/188) slices. All 81 slices of negative test controls (sentinel lymph nodes of mammary carcinoma) were interpreted correctly as negative for melanoma micrometastases. CONCLUSIONS: The melanoma cocktail facilitated easy interpretation of melanoma micrometastases in sentinel lymph nodes with high interobserver agreement. There was improvement in detection rate with the cocktail as compared to MART-1 and Melan-A individually. Furthermore, this approach facilitates cost savings.

Phenotypic heterogeneity of B cells in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma.

Although some studies have examined the expression of aberrant markers such as CD2, CD7, CD10, CD13, CD33, and CD34 on B cells in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), a uniform multiparametric analysis of the frequency of expression of these markers using stringent criteria is lacking. By using 3-color flow cytometry, we analyzed 117 cases (bone marrow, 71; blood, 31; lymph nodes, 15) for coexpression of aberrant markers with CD19. Marker expression was considered positive when present on at least 20% of CD19+ cells. Of 117 cases, 40 (34.2%) showed expression of 1 or more aberrant markers. Expression of 4 aberrant markers was seen in 1 case, 3 in 4 cases, 2 in 15 cases, and 1 in 20 cases. Kaplan-Meier survival curves and the log-rank test revealed that the group with aberrant markers showed significantly shortened overall survival compared with the group without aberrant markers (P < .001). There is considerable phenotypic heterogeneity in CLL/SLL, and expression of aberrant markers indicates aggressiveness.

Rituximab-induced remission of a gastric MALT lymphoma.

Gastric MALT lymphoma is usually associated with H. pylori infection, and responds to treatment with antibiotics and a proton pump inhibitor. We report a case of H. pylori negative gastric MALT lymphoma. The patient was followed conservatively for 2 years until she developed gastrointestinal bleeding with significant anemia. She was treated with rituximab 375 mg/m2 weekly for four doses, which resulted in a biopsy proven complete remission. Rituximab therapy is a reasonable, well tolerated treatment alternative for MALT lymphomas not associated with H. pylori.

Mantle cell lymphoma in lymph nodes with metastatic small cell carcinoma of lung: a diagnostic and treatment dilemma.

We report a rare case of small cell carcinoma (SCC) of lung, metastatic to ipsilateral hilar and peribronchial lymph nodes with synchronous mantle cell lymphoma (MCL), in a 58-year-old female. She was treated with Cisplatin, Etoposide, and Rituximab, and remained in complete remission for approximately two and a half years following the initial diagnosis. To the best of our knowledge, synchronous SCC and MCL or SCC metastatic to lymph nodes involved by MCL has not been previously reported. In this case, the features of MCL were very inconspicuous in the lymph nodes with extensive metastases of SCC. The presence of MCL was confirmed by immunohistochemistry and fluorescence in situ hybridization (FISH). The co-existence of lymphoma and metastatic carcinoma in the same lymph node, as seen in this case, highlights the significance of analyzing subtle lymphoid architectural changes, and applying ancillary studies such as immunohistochemistry and molecular analysis in suspicious cases. The management of synchronous SCC and MCL requires consideration of their respective biologic behavior, and cumulative toxicity of treatment regimens of both tumors. In such cases an optimum treatment strategy should be adopted to cover both malignancies with minimal toxic effect.

Colon biopsies for evaluation of acute graft-versus-host disease (A-GVHD) in allogeneic bone marrow transplant patients.

BACKGROUND: Proper histomorphological interpretation of intestinal acute graft versus host disease (A-GVHD) associated with allogeneic bone marrow transplantation (BMT) is critical for clinical management. However, studies methodically evaluating different histomorphological features of A-GVHD are rare. METHODS: Colonic biopsies from 44 allogeneic BMT patients having biopsy-proven cutaneous A-GVHD were compared with colon biopsies from 48 negative controls. RESULTS: A-GVHD showed intra-cryptal apoptosis in 91% and pericryptal apoptosis in adjacent lamina propria in 70% (p < 0.002). Nonspecific apoptosis along the surface epithelium was observed in all groups with comparable frequency. The number of apoptotic cells in mucosa were approximately four times (5.3 per 10 HPF) the negative controls (p < 0.002) in A-GVHD group. 48% of cases with A-GVHD showed decreased number of lymphocytes in lamina propria. Some features, including intraepithelial lymphocytes in surface or crypt epithelium; and neutrophils, eosinophils, and edema in lamina propria, did not demonstrate significant difference in A-GVHD and negative controls. Pericryptal apoptosis, dilated crypts, irregular distribution of crypts, decreased lymphocytes, increased microvessel network, focal fibrosis, presence of muciphages, reactive changes in surface epithelium with mucin depletion, mucosal ulceration, and/or reduced mucosal thickness showed higher association with A-GVHD group. CONCLUSIONS: Intracyptal apoptosis is a reliable indicator of A-GVHD. Its diagnostic significance was improved if intracyptal apoptosis was associated with features which were observed more frequently in A-GVHD group as mentioned above.

Monocytes with altered phenotypes in posttrauma patients.

CONTEXT: Posttrauma patients show impaired immune responsiveness and increased susceptibility to infections. Although monocytes in these patients have been known to express decreased HLA-DR, induction of HLA-DR using interferon gamma failed to reduce susceptibility to infection, suggesting additional factors also may be involved in the impaired immune responsiveness. CD4 plays an integral role in most of the functions of HLA-DR. In newborn infants, who have impaired immune responsiveness, we found a concomitant reduction of CD4 on monocytes with decreased HLA-DR expression. OBJECTIVE: Because monocytes in posttrauma patients have not been previously studied for morphology, coexpression of CD4 and HLA-DR, and activity of alpha-naphthyl butyrate esterase, the purpose of this study was to analyze these factors in this population. DESIGN: Monocyte morphology; expression of CD4, CD11b, CD13, CD16, and HLA-DR by 3-color flow cytometry; and analysis of alpha-naphthyl butyrate esterase activity by cytochemical staining were studied in 27 posttrauma patients and 20 control subjects. RESULTS: Monocytes in posttrauma patients showed significant differences in the following characteristics compared with controls: (1) increase of subsets displaying the phenotypes CD4-/CD14+/HLA-DR- and CD4-/CD14+/CD16-, (2) decrease in mean fluorescence intensity of CD4 and HLA-DR expression in monocytes that were positive for these markers, (3) decrease in alpha-naphthyl butyrate esterase activity, and (4) decreased amount of cytoplasm and cytoplasmic vacuoles.Conclusion.-Our study suggests that in posttrauma patients, as in newborns, there is a marked increase of monocytes with decreased expression of CD4 and HLA-DR, as well as decreased alpha-naphthyl butyrate esterase activity. Concomitant reduction in CD4 and HLA-DR expression on monocytes may contribute to impaired immune responsiveness in these patients.

CD5+ T-cell/histiocyte-rich large B-cell lymphoma.

CD5 expression in neoplastic large B-cells in T-cell/histiocyte-rich large B-cell lymphoma has not been reported, to the best of our knowledge. Here we describe the first case of CD5+ T-cell/histiocyte-rich large B-cell lymphoma that is well documented by histomorphology, immunohistochemistry, flow cytometry immunophenotyping and sorting, and immunoglobulin heavy-chain gene rearrangement study by polymerase chain reaction. The expression of CD5 in large neoplastic B-cells was demonstrated by immunohistochemistry and multicolor flow cytometry. The clonal nature of the CD5+ neoplastic B-cells was confirmed by rearranged immunoglobulin heavy (IgH) chain with polymerase chain reaction (PCR) of flow cytometry-sorted CD5+/CD19+/kappa+ cells. The CD5+ neoplastic large B-cells expressed bcl-6 and MUM1/IRF4 but not CD138 by immunohistochemistry. This suggests that the neoplastic cells may be of late germinal-center B-cell/ early post-germinal center B-cell origin. The patient responded to chemotherapy, CHOP (Cytoxan, doxorubicin, vincristine, and prednisone), and Rituxan very well and is currently in complete remission clinically. We propose that the current case, CD5+ T-cell/histiocyte-rich large B-cell lymphoma, represents a variant of recently reported de novo CD5+ diffuse large B-cell lymphomas. Our patient has had an excellent response to treatment; however, the clinical and biologic significance of CD5 expression in T-cell/histiocyte-rich large B-cell lymphoma requires further studies. Awareness of the CD5+ T-cell/histiocyte-rich large B-cell lymphoma variant will prompt pathologists to perform CD5 immunohistochemical stain in cases of T-cell/histiocyte-rich large B-cell lymphoma. This will lead to identifying more cases to understand the clinical and biologic characteristics of this variant.

A unique collision tumor in breast: invasive ductal carcinoma and mucosa-associated lymphoid tissue lymphoma.

We report an extraordinary case of a collision tumor consisting of invasive ductal carcinoma with adjacent malignant lymphoma presenting as a single mass in the breast. A 79-year-old woman presented with a breast mass. A core biopsy performed at an outside hospital was interpreted as medullary carcinoma. On review of the breast core biopsy, a diagnosis of a synchronous malignant lymphoma and invasive ductal carcinoma was rendered. The patient underwent lumpectomy and axillary dissection. The excised specimen revealed a 2.1-cm, moderately differentiated invasive ductal carcinoma, partially surrounded by malignant lymphoma with areas where both tumors were intermixed. All 27 axillary lymph nodes were extensively involved by lymphoma, and 1 lymph node demonstrated metastatic carcinoma. The morphology and results of immunohistochemistry, flow cytometry, and cytogenetic analysis were consistent with extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue.

Inhibition of nitrosylation, nitration, lymphocyte proliferation, and gene expression in acute and delayed cardiac allograft rejection by an orally active dithiocarbamate.

Dithiocarbamate derivatives sequester metals such as iron and may have benefits in inflammatory diseases. We examined the actions of a new dithiocarbamate-based oral formulation, NOX-700, on protein modification by nitric oxide (NO), gene expression, and lymphocyte proliferation in a model of acute and delayed cardiac rejection. Chronic treatment with NOX-700 prolonged graft survival. In combination with low-dose cyclosporine (CsA), NOX-700 produced a synergistic action to prolong graft survival. NOX-700 decreased myocardial heme nitrosylation. A single bolus injection with NOX-700 in untreated recipients did not decrease heme nitrosylation but normalized NO metabolites and caused the formation of a mononitrosyl iron complex indicating NO scavenging in vivo. NOX-700 alone given with CsA inhibited protein nitration. NOX-700 or CsA each alone decreased intragraft inflammatory cell infiltration. NOX-700 also potentiated the CsA-induced inhibition of splenocyte proliferation ex vivo stimulated by concanavalin A. In splenocytes derived from treated rats but stimulated ex vivo in a mixed lymphocyte response (MLR), interferon-gamma and cyclin D3 gene expression was inhibited by NOX-700 suggesting down-regulation of lymphocyte activation and proliferation by in vivo treatment. These studies suggest that NOX-700 is protective in cardiac rejection, in part, by scavenging of NO and by limiting lymphocyte activation infiltration.

Expression of p53, c-Myc, or Bcl-6 suggests a poor prognosis in primary central nervous system diffuse large B-cell lymphoma among immunocompetent individuals.

CONTEXT: Primary central nervous system (CNS) diffuse large B-cell lymphoma (DLBCL) in immunocompetent individuals, although rare, has been rising in incidence. Currently, no reliable prognostic markers are available for these individuals. OBJECTIVE: To study the implications of expression of a panel of oncogenic proteins (Bcl-2, Bcl-6, and c-Myc) and p53 for predicting clinical outcome, particularly overall survival, in immunocompetent individuals with primary CNS DLBCL. DESIGN: Fourteen primary CNS DLBCL cases were retrospectively studied by immunohistochemistry on formalin-fixed, paraffin-embedded sections for the expression of c-Myc, Bcl-2, Bcl-6, and p53. RESULTS: The overall frequencies of expression for p53, c-Myc, Bcl-2, and Bcl-6 in these cases were 29%, 50%, 71%, and 57%, respectively. Cases with expression of p53, c-Myc, or Bcl-6 had a poorer overall survival than those without (Kaplan-Meier survival analysis: 50% cumulative overall survival, 2 months vs 30-60 months, P =.02, log-rank test; 9-16 months vs 21-60 months, P =.03, log-rank test; and 9-16 months vs 21-60 months, P =.16, log-rank test, respectively). The expression of Bcl-2 or proliferation activity by MIB-1 showed no correlation with overall survival. Likewise, the clinical parameters, including age, location of tumors, multiplicity of tumor lesions, and lactase dehydrogenase levels revealed no impact on overall survival. CONCLUSION: Our results suggest that patients with expression of p53, c-Myc, or Bcl-6 have a poorer overall survival than those without. Since traditional prognostic markers in non-CNS DLBCL, such as staging and International Prognostic Index scores, are not applicable to primary CNS DLBCL, evaluation of p53, c-Myc, and Bcl-6 by immunohistochemistry may be warranted as part of prognostic evaluation in immunocompetent patients with primary CNS DLBCL. Further studies are indicated to confirm our observations.

Expression of MUM1/IRF4 correlates with clinical outcome in patients with B-cell chronic lymphocytic leukemia.

In this study, we evaluated the prognostic significance of multiple myeloma-1/interferon regulatory factor-4 (MUM1/IRF4) expression in B-cell chronic lymphocytic leukemia (B-CLL). Our results demonstrated that the absence of MUM1/IRF4 expression showed the highest relative risk among the factors analyzed in determining the probability for death in patients with B-CLL using univariate and multivariate Cox regression analysis. Patients without MUM1/IRF4 expression had significantly worse overall survival than did those with MUM1/IRF4 expression (52% cumulative survival, 63 months vs not reached, Kaplan-Meier survival analysis; P <.03, log-rank test). Patients with MUM1/IRF4 expression were more likely to have disease at low Rai stage and interstitial/nodular marrow involvement. Furthermore, only 1 of 11 patients with MUM1/IRF4 expression and interstitial/nodular marrow involvement died during a 100-month follow-up. Our results suggest that B-CLL with expression of MUM1/IRF4, indicative of postgerminal center origin, has a more favorable clinical course and that MUM1/IRF4 is an important prognostic marker in B-CLL.

A ruthenium (III) polyaminocarboxylate complex, a novel nitric oxide scavenger, enhances graft survival and decreases nitrosylated heme protein in models of acute and delayed cardiac transplant rejection.

Nitric oxide (NO) derived from the up-regulation of inducible NO synthase (iNOS) is believed to play an important role in organ rejection. In experimental models of acute cardiac transplant rejection (i.e., without immunosuppression), treatment using NOS inhibitors to prevent acute rejection have yielded conflicting results. This is most likely due to potential inhibition of constitutive NOS. Accordingly, agents that trap NO directly may have some advantage. In the current study, we evaluated the actions of a ruthenium-based NO scavenger, AMD6221, to inhibit the nitrosylation of myocardial protein and to prolong cardiac allograft survival in a model of acute cardiac transplant rejection (without immunosuppression). In addition, we evaluated the efficacy of AMD6221 used in combination with low-dose cyclosporine (CsA) (i.e., a model of delayed graft rejection). Heterotopic abdominal cardiac transplantation was performed using rat strains with disparities at major and minor histocompatibility loci. Grafts were harvested on postoperative day 6 for histologic examination or analysis of myocardial protein nitrosylation using electron paramagnetic resonance (EPR) spectroscopy. Other animals were monitored twice daily to determine rejection times. Plasma was also taken at postoperative day 6 for determining the concentration of NO by-products (nitrate plus nitrite). Treatment with AMD6221 either prolonged graft survival and/or caused a marked decrease in myocardial nitrosylprotein formation as determined by EPR spectroscopy. In vivo scavenging of NO by AMD6221 was verified by high-performance liquid chromatography analysis of nitrosylated-drug in plasma samples. Low-dose CsA given alone or in combination with AMD6221 completely blocked formation of myocardial nitrosylprotein complexes. Whereas low-dose CsA alone prolonged graft survival, combination therapy with CsA plus AMD6221 produced a synergistic effect on graft survival. These studies indicate that treatment with a ruthenium-based NO scavenger, such as AMD6221, may be an effective regimen used alone or in combination with CsA to protect myocardial proteins from posttranscriptional modification and to prolong cardiac graft survival.

Mechanisms of the protective action of diethyldithiocarbamate-iron complex on acute cardiac allograft rejection.

In this study, we examined the actions of diethyldithiocarbamate-iron (DETC-Fe) complex in acute graft rejection heterotopically transplanted rat hearts. Chronic treatment with DETC-Fe inhibited the increase in plasma nitric oxide (NO) metabolites and nitrosylation of myocardial heme protein as determined by electron paramagnetic resonance (EPR) spectroscopy. Pulse injection with DETC-Fe normalized NO metabolites. We verified intragraft trapping of NO in vivo by pulse injection with DETC-Fe by the detection within allografts of an anisotropic triplet EPR signal for DETC-Fe-NO adduct with resonance positions (g tensor factors for perpendicular and parallel components, respectively g( perpendicular ) = 2.038 and g( parallel ) = 2.02; hyperfine coupling of 12.5 G). DETC-Fe prolonged graft survival and decreased histological rejection scores. DNA binding activity for nuclear factor (NF)-kappaB and activator protein-1 was increased in allografts and prevented by DETC-Fe. Abrogation of the activation of NF-kappaB by DETC-Fe was associated with increased IkappaBalpha inhibitory protein. Western blotting and RT-PCR analysis revealed that DETC-Fe inhibited inducible NO synthase protein and gene expression. Gene expression for the proinflammatory cytokine interferon-gamma was also decreased by DETC-Fe. Thus DETC-Fe limits NF-kappaB-dependent gene expression and possesses significant immunosuppressive properties.

Variable efficacy of N6-(1-iminoethyl)-L-lysine in acute cardiac transplant rejection.

We examined the efficacy and mechanism of action of N(6)-(1-iminoethyl)-L-lysine (L-NIL), a highly selective inhibitor of inducible nitric oxide (NO) synthase (iNOS), on acute cardiac transplant rejection. L-NIL produced a concentration-dependent attenuation of plasma NO by-products and a decrease in nitrosylation of heme protein without altering protein levels of iNOS. At postoperative day 4, L-NIL did not alter the increased binding activities for transcription factors nuclear factor-kappaB and activator protein-1. Whereas L-NIL decreased inflammatory cell infiltration, graft survival was only prolonged at the dose of 1.0 microg/ml that incompletely blocked NO production. Higher L-NIL concentrations (30 and 60 microg/ml) ablated the increased NO production but failed to improve graft survival and even potentiated NF-kappaB binding activity examined at day 6. Alloimmune activation indicated by increased cytokine gene expression for interferon-gamma, interleukin-6, and interleukin-10 was inhibited in grafts only by treatment with 1.0 microg/ml L-NIL. These findings suggest a complex role of NO in acute cardiac allograft rejection. Partial inhibition of iNOS is beneficial to graft survival, whereas total ablation may oppose any benefits to graft survival. These studies have important implications in understanding the dual role of NO in acute rejection and help to reconcile discrepancies in the literature.