Optimizing assessment of CD30 expression in Hodgkin lymphoma by controlling for low expression.

Since the approval of brentuximab vedotin (BV), assessment of CD30 status by immunohistochemistry gained increasing importance in the clinical management of patients diagnosed with CD30-expressing lymphomas, including classical Hodgkin lymphoma (CHL). Paradoxically, patients with low or no CD30 expression respond to BV. This discrepancy may be due to lack of standardization in CD30 staining methods. In this study, we examined 29 cases of CHL and 4 cases of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) for CD30 expression using a staining protocol that was designed to detect low CD30 expression levels, and an evaluation system similar to the Allred scoring system used for breast cancer evaluation. For CHL, 10% of cases had low scores and 3% were CD30 negative, with 3 cases in which the majority of tumor cells showed very weak staining. Unexpectedly, one of four cases of NLPHL was positive. We demonstrate intra-patient heterogeneity in CD30 expression levels and staining patterns in tumor cells. Three CHL cases with weak staining may have been missed without the use of control tissue for low expression. Thus, standardization of CD30 immunohistochemical staining with use of known low-expressing controls may aid in proper CD30 assessment and subsequent therapeutic stratification of patients.

The AIDS and Cancer Specimen Resource (ACSR): HIV malignancy specimens and data available at no cost.

The goal of the AIDS and Cancer Specimen Resource (ACSR) is to play a major role in the advancement of HIV/AIDS cancer-related research/treatment by providing richly annotated biospecimens and data to researchers at no cost. The ACSR acquires, stores, and equitably distributes these samples and associated clinical data to investigators conducting HIV/AIDS-related research, at no costs. Currently, it is the only biorepository of human biospecimens from people with HIV and cancer available to eligible researchers globally who are studying HIV associated malignancies.This review describes the history and organizational structure of the ACSR, its types of specimens in its inventory, and the process of requesting specimens. In addition, the review provides an overview of research that was performed over the last 5 years with its support and gives a summary of important new findings acquired by this research into the development of cancers in people with HIV, including both Aids-related and non-Aids-related malignancies.

Biorepository best practices for research and clinical investigations.

Translational research requires good quality specimens to ensure the integrity of research results. Clinical research must rely not only on quality specimens, but as well on clinical annotation for consistent, accurate and verifiable scientific and clinical outcomes. In laboratory research performed on a specimen by a single investigator, quality control is easily maintained. In a multi-site clinical research network, the numerous steps for biospecimens from procurement through transport, processing, storage and ultimately testing requires strict standardization of operational workflows and procedures. The practices of a central biorepository can inform and contribute to best practices regarding clinical research specimen integrity for multi-site clinical research.

A Plan for Emergency Shutdown and Reopening for a Consortium of Biobanks.

Background: The AIDS and Cancer Specimen Resource (ACSR) is a network of four regional biospecimen repositories and a technical core in the United States and South Africa. Its mission is to acquire, store, and distribute HIV-associated malignancy specimens and related clinical data to support translational research. At the outset of the COVID-19 pandemic, it became apparent that existing ACSR Standard Operating Procedures (SOPs) were not sufficient to ensure long-term maintenance and integrity of inventories during periods of extended shutdown. The ACSR needed an administrative SOP for situations pertaining to epidemics/pandemics. The ACSR Quality Working Group (QWG), comprised of representatives from each of the five ACSR sites and an external member who directs a large university medical center biorepository, addressed the issue. Methods: To understand the individual problems the sites faced, questions were developed to query each of the six QWG sites' contingency plans to cover this type of emergency, the amount of work allowed onsite and by whom, the challenges sites experienced, and the lessons learned to assist with future similar situations, while remaining consistent with the existing IRB protocols. Results: Reported challenges spanned all activities of classical biobanks and differed within the geographical locations of the sites and the local COVID-19 infection rate. Review of the responses to the questions revealed that the general shutdown of society external to the biorepositories presented them with a homogeneous collection of problems, limitations, and needs. This led to creating an SOP that addresses planning for pandemic emergencies, scaling down of activities, shutting down, and reopening plans. Conclusions: The ACSR QWG sites now have a structured response SOP for their sites, including guidance on how to develop and implement an emergency shutdown and reopening plan. The complete SOP is publicly available on the ACSR website.

Phase I Study of the Investigational Aurora A Kinase Inhibitor Alisertib plus Rituximab or Rituximab/Vincristine in Relapsed/Refractory Aggressive B-cell Lymphoma.

PURPOSE: The aurora A kinase inhibitor alisertib demonstrated single-agent clinical activity and preclinical synergy with vincristine/rituximab in B-cell non-Hodgkin lymphoma (B-NHL). This phase I study aimed to determine the safety and recommended phase II dose (RP2D) of alisertib in combination with rituximab ± vincristine in patients with relapsed/refractory aggressive B-NHL. PATIENTS AND METHODS: Patients with relapsed/refractory, diffuse, large, or other aggressive B-NHL received oral alisertib 50 mg b.i.d. days 1 to 7, plus i.v. rituximab 375 mg/m2 on day 1, for up to eight 21-day cycles (MR). Patients in subsequent cohorts (3 + 3 design) received increasing doses of alisertib (30 mg starting dose; 10 mg increments) b.i.d. days 1 to 7 plus rituximab and vincristine [1.4 mg/m2 (maximum 2 mg) days 1, 8] for 8 cycles (MRV). Patients benefiting could continue single-agent alisertib beyond 8 cycles. Cell-of-origin and MYC/BCL2 IHC was performed on available archival tissue. RESULTS: Forty-five patients participated. The alisertib RP2D for MR was 50 mg b.i.d. For MRV (n = 32), the RP2D was determined as 40 mg b.i.d. [1 dose-limiting toxicity (DLT) at 40 mg; 2 DLTs at 50 mg]. Drug-related adverse events were reported in 89% of patients, the most common was neutropenia (47%). Seven patients had complete responses (CR), 7 had partial responses (PRs); 9 of 20 (45%) patients at the MRV RP2D responded (4 CRs, 5 PRs), all with non-germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL). CONCLUSIONS: The combination of alisertib 50 mg b.i.d. plus rituximab or alisertib 40 mg b.i.d. plus rituximab and vincristine was well tolerated and demonstrated activity in non-GCB DLBCL.

A Phase II Exploratory Study of PXD-101 (Belinostat) Followed by Zevalin in Patients with Relapsed Aggressive High-Risk Lymphoma.

OBJECTIVE: Aggressive lymphomas (aNHL) including diffuse large B-cell lymphoma (DLBCL) have poor outcomes in relapsed refractory patients. Prior studies have demonstrated that loss of major histocompatibility complex class II (MHCII) expression in DLBCL is associated with poor survival. The objective of this single-arm phase II study was to evaluate if PXD-101 would increase MHCII expression, synergize with Zevalin, and improve clinical outcomes. METHODS: This was a single-center open-label phase II trial (NCT01686165) geared toward heavily pretreated patients with CD20-positive aNHL. The primary endpoint was overall response rate (ORR) in aNHL patients treated with 2 cycles of PXD-101 followed by restaging CT and 1 cycle of Zevalin. RESULTS: Five patients were enrolled, and all were heavily pretreated. Therapy was well tolerated, with nausea and vomiting being the most frequent adverse events. All patients progressed after receiving therapy; the study did not achieve the required ORR to proceed to the next stage. CONCLUSION: The pleotropic effects of histone deacetylase inhibition and lack of clinical biomarkers have precluded a priori identification of responding patients. Thus, while we report a negative trial of PXD-101 in combination with Zevalin, this study highlights the importance of a clinically feasible biomarker.

Pak2 regulates myeloid-derived suppressor cell development in mice.

Myeloid-derived suppressor cells (MDSCs) are CD11b+Gr1+ cells that induce T-cell hyporesponsiveness, thus impairing antitumor immunity. We have previously reported that disruption of Pak2, a member of the p21-activated kinases (Paks), in hematopoietic stem/progenitor cells (HSPCs) induces myeloid lineage skewing and expansion of CD11bhighGr1high cells in mice. In this study, we confirmed that Pak2-KO CD11bhighGr1high cells suppressed T-cell proliferation, consistent with an MDSC phenotype. Loss of Pak2 function in HSPCs led to (1) increased hematopoietic progenitor cell sensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, (2) increased MDSC proliferation, (3) decreased MDSC sensitivity to both intrinsic and Fas-Fas ligand-mediated apoptosis, and (4) promotion of MDSCs by Pak2-deficient CD4+ T cells that produced more interferon γ, tumor necrosis factor α, and GM-CSF. Pak2 disruption activated STAT5 while downregulating the expression of IRF8, a well-described myeloid transcription factor. Together, our data reveal a previously unrecognized role of Pak2 in regulating MDSC development via both cell-intrinsic and extrinsic mechanisms. Our findings have potential translational implications, as the efficacy of targeting Paks in cancer therapeutics may be undermined by tumor escape from immune control and/or acceleration of tumorigenesis through MDSC expansion.

Belinostat and vincristine demonstrate mutually synergistic cytotoxicity associated with mitotic arrest and inhibition of polyploidy in a preclinical model of aggressive diffuse large B cell lymphoma.

Diffuse Large B-cell lymphoma (DLBCL) is an aggressive malignancy that has a 60 percent 5-year survival rate, highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics being clinically-evaluated in combination with a variety of other drugs. However, rational selection of companion therapeutics for HDACi is difficult due to their poorly-understood, cell-type specific mechanisms of action. To address this, we developed a pre-clinical model system of sensitivity and resistance to the HDACi belinostat using DLBCL cell lines. In the current study, we demonstrate that cell lines sensitive to the cytotoxic effects of HDACi undergo early mitotic arrest prior to apoptosis. In contrast, HDACi-resistant cell lines complete mitosis after a short delay and arrest in G1. To force mitotic arrest in HDACi-resistant cell lines, we used low dose vincristine or paclitaxel in combination with belinostat and observed synergistic cytotoxicity. Belinostat curtails vincristine-induced mitotic arrest and triggers a strong apoptotic response associated with downregulated MCL-1 expression and upregulated BIM expression. Resistance to microtubule targeting agents (MTAs) has been associated with their propensity to induce polyploidy and thereby increase the probability of genomic instability that enables cancer progression. Co-treatment with belinostat effectively eliminated a vincristine-induced, actively cycling polyploid cell population. Our study demonstrates that vincristine sensitizes DLBCL cells to the cytotoxic effects of belinostat and that belinostat prevents polyploidy that could cause vincristine resistance. Our findings provide a rationale for using low dose MTAs in conjunction with HDACi as a potential therapeutic strategy for treatment of aggressive DLBCL.

A phase II study of belinostat (PXD101) in relapsed and refractory aggressive B-cell lymphomas: SWOG S0520.

Recent advances in diffuse large B-cell lymphomas (DLBCL) have underscored the importance of tumor microenvironment in escaping host anti-tumor responses. One mechanism is loss of major histocompatibility Class II antigens (MHCII) associated with decreased tumor infiltrating T lymphocytes (TIL) and poor survival. Transcription of MHCII is controlled by CIITA which in turn is regulated by histone acetylation. In this study, we hypothesized that HDAC inhibition with belinostat increases MHCII, CIITA expression, TIL and improves patient outcomes. Primary objective was evaluation of toxicity and response. Twenty-two patients were enrolled for the study. Belinostat was well tolerated with mild toxicity. Two partial responses were observed at 5, 13 months after registration for an overall response rate (ORR) (95% CI) of 10.5% (1.3-33.1%), and three patients had stable disease for 4.7, 42.3+, and 68.4 + months with minimum 3-year follow-up. Included correlative studies support the hypothesis and serve as the basis for SWOG S0806 combining vorinostat with R-CHOP.

Langerhans Cell Histiocytosis Shows Distinct Cytoplasmic Expression of Major Histocompatibility Class II Antigens.

OBJECTIVES: Langerhans cell histiocytosis (LCH) is a monoclonal proliferation of antigen presenting cells (APC). In benign APCs, antigen loading occurs in the Major Histocompatibility class II (MHCII)-lysosomal compartment of the endocytic pathway followed by transport to the cell surface upon antigen stimulation. The pattern of MHC II expression in LCH is not well characterized. METHODS: The cellular localization of MHCII was determined using immunohistochemisty (IHC). Staining pattern for the representative MHCII molecule, HLA-DR, (cell surface, cytoplasmic granular, or cytoplasmic globular) and intensity (0 to 3+) were recorded for normal tissues and 44 LCH samples along with available clinicopathologic features. Results were confirmed with a different antibody to confirm the appearance. RESULTS: In the normal tissue survey, strong HLA-DR cell surface expression was present on APCs, benign B cells, some T cells, and pulmonary macrophages. A granular cytoplasmic staining pattern (without surface expression) was seen in benign Langerhans cells (LCs) in the skin and histiocytes. Strikingly, all 44 LCH samples demonstrated both cytoplasmic granular and an unusual "globular" staining pattern with no surface staining. CONCLUSION: This is the first report of a highly specific HLA-DR staining pattern in LCH detected by IHC. The cytoplasmic perinuclear globular localization of MHCII may possibly be useful in diagnostics and may result from an immature/antigen-naïve differentiation state of the neoplastic cell.

Prognostic criteria for squamous cell cancer of the skin.

BACKGROUND: Non-well-differentiated cutaneous squamous cell carcinomas may display a more aggressive behavior. It is important to better define prognostic criteria for these tumors. METHODS: This was a retrospective case-control analysis of a squamous cell carcinoma database. Patients with non-well-differentiated and well-differentiated tumors were matched based on site of tumor, age, and immunocompromised status. Comparisons included demographics, histology, immunohistochemical protein expressions (Ki-67, p53, E-cadherin, cyclin D1), and clinical outcomes. RESULTS: Demographic features were similar between cases (n=30) and controls (n=30). Non-well-differentiated tumors were larger (1.8 cm versus 1.3 cm, P=0.08), deeper (0.81 cm versus 0.32 cm, P<0.0001), and had greater recurrence (P=0.003). Non-well-differentiated tumors showed increased proliferation rate, Ki-67 index (77% versus 61%, P=0.001); no significant difference in activity of p53, E-cadherin, and cyclin D1 between the two groups. CONCLUSIONS: Tumor differentiation and depth are important pathologic and prognostic criteria for cutaneous squamous cell carcinoma. Immunohistochemistry helps describe patterns of biomarker protein expression and may exemplify aggressive subtypes.

Implantation of a three-dimensional fibroblast matrix improves left ventricular function and blood flow after acute myocardial infarction.

This study was designed to determine if a viable biodegradable three-dimensional fibroblast construct (3DFC) patch implanted on the left ventricle after myocardial infarction (MI) improves left ventricular (LV) function and blood flow. We ligated the left coronary artery of adult male Sprague-Dawley rats and implanted the 3DFC at the time of the infarct. Three weeks after MI, the 3DFC improved LV systolic function by increasing (p < 0.05) ejection fraction (37 +/- 3% to 62 +/- 5%), increasing regional systolic displacement of the infarcted wall (0.04 +/- 0.02 to 0.11 +/- 0.03 cm), and shifting the passive LV diastolic pressure volume relationship toward the pressure axis. The 3FDC improved LV remodeling by decreasing (p < 0.05) LV end-systolic and end-diastolic diameters with no change in LV systolic pressure. The 3DFC did not change LV end-diastolic pressure (LV EDP; 25 +/- 2 vs. 23 +/- 2 mmHg) but the addition of captopril (2mg/L drinking water) lowered (p < 0.05) LV EDP to 12.9 +/- 2.5 mmHg and shifted the pressure-volume relationship toward the pressure axis and decreased (p < 0.05) the LV operating end-diastolic volume from 0.49 +/- 0.02 to 0.34 +/- 0.03 ml. The 3DFC increased myocardial blood flow to the infarcted anterior wall after MI over threefold (p < 0.05). This biodegradable 3DFC patch improves LV function and myocardial blood flow 3 weeks after MI. This is a potentially new approach to cell-based therapy for heart failure after MI.

Nuclear labeling of Coccidioides posadasii with green fluorescent protein.

Coccidioidomycosis is a mild to life-threatening disease in otherwise healthy humans and other mammals caused by the fungus Coccidioides spp. Understanding the development of the unique dimorphic life cycle of Coccidioides spp. and its role in pathogenesis has been an area of research focus. However, nuclear behavior during the saprobic and parasitic life cycle has not been studied intensively. In this study, green fluorescent protein (GFP) was fused to histone H1 and introduced into Coccidioides posadasii (C. posadasii) strain Silveira to monitor the nuclear behavior of the fungus during the saprobic and parasitic stages of the life cycle. We constructed an Agrobacterium tumefaciens-mediated transformation (ATMT) vector that had in its T-DNA region a hygromycin-resistance gene as well as the fused histone H1-GFP gene under the control of the histone H3 promoter of C. posadasii. More than 30 hygromycin-resistant transformants were obtained and 23 were purified to homozygosity through multiple passages of the original transformants on hygromycin-containing media. One strain (VFC1420) transformed with a single copy of the fusion histone H1-GFP gene was selected for cytological studies. Strong nuclear-localized GFP signals were observed in arthroconidia, hyphae, as well as in spherules and endospores developed in vitro. Thus GFP can be used to study the expression pattern of potential virulence genes identified in serial analysis of gene expression (SAGE) or expressed sequence tags (EST) libraries, and could be a useful tool to monitor disease development in the murine model.

Integrin-dependent amplification of the G2 arrest induced by ionizing radiation.

The progressive loss of laminin 5 and the alpha6beta4 integrin is a characteristic of the transition of prostatic intraepithelial neoplasia (PIN) to invasive human prostate cancer. Our objective was to determine if the loss of the interaction with laminin 5 would influence the ability of human epithelial cells to respond to DNA damage. Three cellular damage responses to ionizing radiation (IR) were analyzed including G2 progression, cdc2 phosphorylation, and cell survival. The adhesion of normal human prostate epithelial cells to laminin 5 amplified the G2 arrest induced by IR, and depends on a known cell binding domain of laminin 5. The alteration of G2 arrest was confirmed by an inhibition of phospho-cdc2 nuclear translocation. In contrast, a prostate epithelial cancer cell line blocked in G2 independent of adhesion to laminin 5. The survival of these cell lines in response to IR was unaffected by adhesion to laminin 5. These results suggest that cell adhesion to laminin 5 in normal cells will amplify the IR induced G2 cell cycle progression block without altering cell survival. The loss of laminin 5 and the alpha6beta4 integrin in PIN lesions may contribute to the selection and progression of genetically unstable cell types via attenuation of a DNA damage induced G2 arrest.

Cellular immune suppressor activity resides in lymphocyte cell clusters adjacent to granulomata in human coccidioidomycosis.

The in situ immunologic response in human coccidioidomycosis remains undefined. To explore this further, pulmonary necrotizing coccidioidal granulomata were examined using immunohistochemical staining for lymphocyte subsets and for the cytokines interleukin-10 (IL-10) and gamma interferon (IFN-gamma). Discrete perigranulomatous lymphocytic clusters were seen in eight of nine tissues examined. In these tissues, T lymphocytes (CD3+) significantly outnumbered B lymphocytes (CD20+) in the mantle area of the granulomata (P = 0.028), whereas the clusters were composed of roughly equal numbers of T and B lymphocytes. While the number of cells in the mantle expressing IL-10 was similar to those in the perigranulomatous clusters, there were significantly more cells expressing IFN-gamma in the mantle than in the clusters (P = 0.037). Confocal microscopy revealed that CD4+ T lymphocytes and B lymphocytes are associated with IL-10 production. CD4+CD25+ T lymphocytes were identified in the perigranulomatous clusters but were not associated with IL-10 production. This is the first report noting perigranulomatous lymphocyte clusters and IL-10 in association with human coccidioidal granulomata and suggests that down-regulation of the cellular immune response is occurring within coccidioidal granulomata.

Identification of a stem cell candidate in the normal human prostate gland.

Stem cells of the human prostate gland have not yet been identified utilizing a structural biomarker. We have discovered a new prostatic epithelial cell phenotype-expressing cytokeratin 6a (Ck6a+ cells). The Ck6a+ cells are present within a specialized niche in the basal cell compartment in fetal, juvenile and adult prostate tissue, and within the stem cell-enriched urogenital sinus. In adult normal prostate tissue, the average abundance of Ck6a+ cells was 4.9%. With proliferative stimuli in the prostate organ culture model, in which the epithelial-stromal interaction was maintained, a remarkable increase of Ck6a expression was noticed to up to 64.9%. The difference in cytokeratin 6a expression between the normal adult prostate and the prostate organ culture model was statistically significant (p<0.0001). Within the prostate organ culture model the increase of cytokeratin 6a-expressing cells significantly correlated with increased proliferation index (r = 0.7616, p = 0.0467). The Ck6a+ cells were capable of differentiation as indicated by their expression of luminal cell markers such as ZO-1 and prostate specific antigen (PSA). Our data indicate that Ck6a+ cells represent a prostatic epithelial stem cell candidate possessing high potential for proliferation and differentiation. Since the development of benign prostatic hyperplasia and prostate carcinogenesis are disorders of proliferation and differentiation, the Ck6a+ cells may represent a major element in the development of these diseases.

Immunohistochemical comparison of vascular and sinusoidal adherens junctions in cavernosal endothelium.

OBJECTIVES: To characterize endothelial cell-to-cell junctions in the sinusoids and microvasculature of the corpus cavernosum. METHODS: Corporal tissue was obtained from 6 potent human subjects, cut into 5-microm cryosections, and double-labeled with consecutive applications of primary and secondary antibodies. Laser scanning confocal microscopy identified subcellular localization of endothelial anchoring and adhesion molecules. Fluorescence intensity was rated as strong, weak, or absent by two observers. RESULTS: The cavernosal endothelial adherens junction was composed of vascular endothelial cadherin, alpha-catenin, plakoglobin, vinculin, and the regulatory proteins beta-catenin and ZO-1. Adherens junctions in sinusoids were elongated, redundant, and narrow versus short, dense, linear cell-to-cell contacts in small arterioles and venules. Vinculin expression along the basal interface of the endothelium and stroma was weak in the sinusoids and strong in the arterioles. Definite sinusoidal expression of CD31 and CD34 was noted. P-selectin was only expressed within the cavernosal microvessels. CONCLUSIONS: The regulatory and structural proteins extending from vascular endothelial cadherin provide immunohistochemical evidence of a role for adherens junctions in cavernosal endothelial barrier function and cellular homeostasis. The sinusoidal endothelium has a unique junctional phenotype consistent with its blood trapping function. Differential expression of functional proteins in sinusoidal and microvascular endothelium may reflect segmental variation in hemodynamic exposure to pressure, stretch, or flow.