Ronald P. Cleveland

Immunohistochemical expression patterns of germinal center and activation B-cell markers correlate with prognosis in diffuse large B-cell lymphoma.

Recent studies with cDNA microarrays showed that diffuse large B-cell lymphoma (DLBCL) cases with gene expression profiles similar to germinal center (GC) B cells had much better prognosis than DLBCL cases with gene expression profiles resembling activated B cells. The goal of the current study is to evaluate if using a panel of GC B-cell (CD10 and Bcl-6) and activation (MUM1/IRF4 and CD138) markers by immunohistochemistry defines prognosis in patients with de novo DLBCL. Immunohistochemical stains for the above markers were performed on paraffin-embedded tissues from 42 de novo DLBCL patients. Median follow-up in all patients was 41 months (range, 1–103 months) and in surviving patients was 65 months (range, 14–103 months). These cases could be classified into three expression patterns: GC B-cell pattern (pattern A) expressing CD10 and/or Bcl-6 but not activation markers; activated GC B-cell pattern (pattern B) expressing at least one of GC B-cell markers and one of activation markers; and activated non-GC B-cell pattern (pattern C) expressing MUM1/IRF4 and/or CD138 but not GC B-cell markers. Patients with pattern A had much better overall survival than those with the other two patterns (Kaplan-Meier survival analysis, P < 0.008, log rank test). Using multivariate Cox proportional hazards regression analysis, the international prognostic index scores and the expression pattern of these markers were independent prognostic indicators. Our results suggest that expression patterns of this panel of GC B-cell and activation markers by immunohistochemistry correlate with the prognosis of patients with DLBCL. Immunohistochemical analysis on paraffin-embedded tissues is more readily available than gene expression profiling by cDNA microarray and may provide similar prognostic information.

Expression of c-Myc and p53 Correlates With Clinical Outcome in Diffuse Large B-Cell Lymphomas

We performed a retrospective immunohistochemical study of the relationships between clinical manifestations and outcomes of diffuse large B-cell lymphoma (DLBCL) and expression of oncogenic proteins in 21 cases of DLBCL at various clinical stages. Cases of nodal origin expressed p53 more often and presented with high clinical stage more frequently than those of extranodal origin. Expression of c-Myc or p53, but not Bcl-6, Bcl-2, or Bcl-1, showed a statistically significant positive correlation with high clinical stage at presentation and with high or high-intermediate risk. Coexpression of c-Myc and p53 occurred in 7 of 12 patients with high clinical stage but was absent in patients with low clinical stage; coexpression was more frequent in patients with high or high-intermediate risk than in patients with low or low-intermediate risk. Four patients with this coexpression pattern demonstrated an unusually aggressive clinical course (median survival, 7 months). Coexpression of c-Myc and p53 seems to be a better indicator than the MIB1 proliferative index for identification of a cohort of aggressive disease in patients with DLBCL.

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.

Relative importance of CD38 expression over myeloid-associated markers expression in predicting the clinical course of B-CLL patients

Expression of CD38 or myeloid-associated markers has been reported to be important in predicting prognosis in B-cell chronic lymphocytic leukemia (B-CLL) in separate studies but the impact of combining these markers on prognosis has not been examined. The current study aimed to evaluate the relative contribution of expression of CD38 and/or myeloid-associated markers (CD11b, CD13, CD15 and CD33) by flow cytometry (FCM) on the clinical course of 24 B-CLL patients. B-CLL patients with high levels of CD38 expression, defined as greater than or equal to 30% of neoplastic lymphocytes expressing CD38, had a significantly poorer OS than those with low levels of CD38 expression (54% cumulative survival: 51 months vs. 103 months, Kaplan-Meier survival analysis, p <0.005, Logrank test). High levels of expression of myeloid-associated markers showed no statistically significant impact on OS in these patients. Ten of 11 patients (91%) with high levels of CD38 expression required chemotherapy. In contrast, only 5 of 13 patients (38%) with low levels of CD38 expression required chemotherapy (p <0.009, Chi Square). There was no significant difference in the requirement for chemotherapy between patients with high levels of expression of myeloid-associated marker and those without (5/8 or 63% vs. 10/16 or 63%). Thus, our results suggest that CD38 is superior to myeloid-associated markers in predicting the prognosis of patients with B-CLL. Further studies with a larger sample size are indicated to confirm our observation.

Cytologic detection of Strongyloides stercoralis in a 55-year-old Hispanic man with routine rectal/anal pap smear.

The anal–rectal cytology is introduced recently to evaluate human‐papillomavirus related cellular changes in the cells of anal canal. It is especially useful in high risk patients such as HIV patients. Very few reports were published regarding cytomorphological findings in anal cytology. Strongyloides stercoralis is an enteric helminthic parasite with particular significance in immunocompromised patients. The infection is asymptomatic or manifests as mild gastrointestinal symptoms in normal hosts. The infection can be devastating in immunocompromised persons, and carries a high mortality rate. The presence of S. stercoralis larva in anal–rectal pap smears is rare. We report a case in a routine anal–rectal Pap smear with Strongyloides, and discuss the clinical significance and life cycle of S. stercoralis. Diagn. Cytopathol. 2009.

Simultaneous hepatosplenic T-cell lymphoma and myelofibrosis

Hepatosplenic T-cell lymphoma (HSTL) is a rare T-cell neoplasm of the lymphoid system. This type of lymphoma is characterized by sinusoidal infiltration of spleen, liver, bone marrow and lymph nodes by neoplastic lymphocytes. Here, we discuss a patient who had a left axillary lymph node biopsy with characteristic histological and immunohistochemical features of HSTL. In addition, infiltrating neoplastic T-cells and simultaneous characteristic features of myelofibrosis (MF) were also present in the bone marrow biopsy specimen. In contrast to secondary MF, primary MF is a progressive disease and may significantly affect the prognosis of coexisting HSTL. There are few reports in the literature talking about mild bone marrow fibrosis in association with T cell lymphoma, however marked increase in bone marrow fibrosis and HSTL never being reported. This case is shedding light on HSTL and marked increase in bone marrow fibrosis.

Coinfusion of irradiated splenocytes with low titer tumor-infiltrating lymphocytes augments antitumor efficacy in adoptive immunotherapy

We hypothesized that adoptively increasing the density of antigen-presenting cells (APCs) at a tumor site would improve tumor-infiltrating lymphocyte (TIL) in vivo antitumor efficacy. Irradiated splenocytes were used as crude APCs. Alone, they did not have in vitro antitumor activity nor did they augment TIL efficacy in vitro. Pulmonary metastases were established by intravenous (i.v.) injection of 5 x 10(5) MC-38 tumor into irradiated C57B1/6 mice (500 cGy). After 3 days, MC-38 TIL (0.1, 0.5, and 1 x 10(6) cells) +/- irradiated splenocytes (5,000 cGy) as APCs were administered intravenously (0.25, 0.5, and 1 x 10(6) cells) to each group (n = 5/group). Interleukin-2 (60,000 IU) was injected intraperitoneally three times daily for 3 days. Mice were sacrificed 9 days later and metastases elaborated in blinded fashion. A titer of 1 x 10(6) TIL, completely eradicated pulmonary metastases. In two consecutive experiments, when increasing titers of irradiated splenocytes were coinfused with a constant titer of TIL that did not completely eradicate pulmonary metastases, a moderate reduction in pulmonary metastases was observed. The contribution of splenocytes to an improvement in TIL antitumor efficacy was not altered when irradiated splenocytes derived from mice bearing 10-day subcutaneous MC-38 tumors were used. The coinfusion of nonirradiated splenocytes did not improve TIL antitumor in vivo activity. Activated B cells (expressing ICAM-1, B7.1, and B7.2) had no effect on in vitro tumor lysis and did not augment in vivo TIL efficacy. The results show a modest but statistically significant improvement in adoptive immunotherapy antitumor efficacy with fewer TIL by coinfusion of irradiated splenocytes. Further studies to characterize the active potential APC cell subpopulation and to clarify the mechanism(s) responsible for in vivo augmentation of TIL antitumor efficacy are in progress.

Monocyte Aggregates: A Clue to an Unsuspected Monoclonal B‐cell Lymphoproliferative Disorder

*Correspondence to: Linda M.S. Resar, Johns Hopkins University School of Medicine, 720 Rutland Ave, Ross Building #1025, Baltimore, MD 21205. E-mail: lresar@jhmi.edu Additional Supporting Information may be found in the online version of this article. Received for publication: 20 May 2016; Revised: 25 May 2016; Accepted: 31 May 2016 Published online: 2 June 2016 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ajh.24440

Human immunodeficiency virus type 1 infection alters enzymatic and ultrastructural features of peripheral blood monocytes

INTRODUCTION: Human immunodeficiency virus-1 (HIV-1) infected monocytes are now believed to serve as a reservoir for HIV-1 infection, and to play a role in viral rebound phenomena in certain groups of patients who failed or stopped highly active antiretroviral therapy (HAART). Data characterizing the morphological changes of peripheral blood monocytes in HIV-1-infected individuals are limited. MATERIALS AND METHODS: In this study, we collected monocytes from 21 asymptomatic HIV-1-infected individuals with CD4 count more than 500 cells/mm3 and healthy individuals. The monocytes ultrastructural morphologic changes and α-naphthyl butyrate esterase (ANBE) activity were compared between the two groups. RESULTS: In monocytes from patients infected with HIV-1, activity of α-naphthyl butyrate esterase (ANBE) was markedly increased compared with normal monocytes. In both light microscopic and ultrastructural studies, the cytoplasm of monocytes from HIV-1-infected patients contained a haphazard appearing network of thin fibrils. Cell surface expression of the activation marker HLA-DR molecule was upregulated. There were no discernible differences between the cell surface expression of CD4, CD14, and CD16 molecules comparing normal monocytes to those from HIV-1-infected patients. α-naphthyl butyrate esterase (ANBE) was markedly increased compared with normal monocytes. In both light microscopic and ultrastructural studies, the cytoplasm of monocytes from HIV-1-infected patients contained a haphazard appearing network of thin fibrils. Cell surface expression of the activation marker HLA-DR molecule was upregulated. There were no discernible differences between the cell surface expression of CD4, CD14, and CD16 molecules comparing normal monocytes to those from HIV-1-infected patients. CONCLUSIONS: Possibly, changes in the activity of ANBE along with a disrupted appearing cytoplasmic fibril network contribute to monocyte dysfunction in HIV-1-infected patients.