Expression of apoptosis-related proteins in the pathogenesis of endometrial clear cell carcinoma.

BACKGROUND: The balance of pro- and anti-apoptotic proteins plays a critical role in the regulation of cell death, and a disruption of this delicate balance may eventuate in carcinogenesis through a net reduction in apoptosis. Numerous chemotherapeutic strategies directly or indirectly target apoptotic pathways. However, a thorough assessment of apoptosis-related proteins has not previously been performed in endometrial clear cell carcinoma (CCC). This study aims to determine the significance of 9 apoptosis-related proteins in the pathogenesis of CCC as compared to non-neoplastic endometrium (NNE), low- and high-grade endometrial endometrioid carcinomas (LG-EEC, HG-EEC), and endometrial serous carcinoma (ESC). MATERIALS AND METHODS: Expression of 6 anti-apoptotic proteins (Bcl-2, Bcl-xL, cFLIPL, MCL-1, survivin, NFκB/p65) and three pro-apoptotic proteins (Bax, caspase-3, caspase-8) was assessed by immunohistochemistry on 49 CCC, 37 LG-EEC, 12 HG-ECC, 16 ESC, and 25 NNE in a tissue microarray. Objective IHC scores were assigned by an automated image capture system. Scores were then correlated with clinicopathologic values and each other. RESULTS: Most notably, CCC showed significantly reduced expression of cFLIPL relative to ESC, LG-EEC, HG-EEC, and NNE. CCC also showed significantly reduced expression of both Caspase 8 and NF-κB/p65 relative to ESC, HG-EEC, and NNE, but not LG-EEC. Bcl-2 and Bcl-xL showed reduced expression in CCC relative to all groups except ESC for Bcl-2 and NNE for Bcl-xL. There was no significant correlation between the proteins regarding expression levels. Within the CCC group, none of the proteins showed any significant association with patient age, myometrial invasion, final stage, lymphovascular invasion, disease-free or overall survival. CONCLUSION: Our analysis of the expression and correlation patterns of a panel of apoptosis-related proteins suggests that the downregulation of cFLIPL in CCC is significant relative to almost all other tissues, NNE, HG-EEC, and ESC. Other proteins, including Caspase 8, NF-κB/p65, Bcl-2 and Bcl-xL may also be significant. The regulation of apoptosis-related proteins in CCC may be important and may provide insight into chemoresistance in this enigmatic histotype. However, the paradoxical downregulation of both pro- and anti-apoptotic mediators suggests that additional study is needed to clarify the role of apoptotic mechanisms in CCC.

Neurosarcoidosis Presenting as Aseptic Meningitis in an Immunosuppressed Renal Transplant Recipient.

BACKGROUND: Sarcoidosis is a presumptive autoimmune disorder characterized by the presence of noncaseating granulomas and is usually treated successfully with immunosuppression. METHODS AND RESULTS: Here, we describe the case of a 63-year-old male renal transplant recipient with a remote history of pulmonary sarcoidosis on chronic immunosuppression who developed recurrent aseptic meningitis and underwent brain biopsy revealing a diagnosis of neurosarcoidosis. CONCLUSIONS: This case highlights the possibility of recurrence of sarcoidosis in the setting of maintenance immunosuppression, the need for heightened awareness of alternative sites of recurrence of autoimmune disease, and future studies to determine the underlying mechanism of recurrence in organ transplant recipients.

Melanoma-specific MHC-II expression represents a tumour-autonomous phenotype and predicts response to anti-PD-1/PD-L1 therapy.

Anti-PD-1 therapy yields objective clinical responses in 30-40% of advanced melanoma patients. Since most patients do not respond, predictive biomarkers to guide treatment selection are needed. We hypothesize that MHC-I/II expression is required for tumour antigen presentation and may predict anti-PD-1 therapy response. In this study, across 60 melanoma cell lines, we find bimodal expression patterns of MHC-II, while MHC-I expression was ubiquitous. A unique subset of melanomas are capable of expressing MHC-II under basal or IFNγ-stimulated conditions. Using pathway analysis, we show that MHC-II(+) cell lines demonstrate signatures of 'PD-1 signalling', 'allograft rejection' and 'T-cell receptor signalling', among others. In two independent cohorts of anti-PD-1-treated melanoma patients, MHC-II positivity on tumour cells is associated with therapeutic response, progression-free and overall survival, as well as CD4(+) and CD8(+) tumour infiltrate. MHC-II(+) tumours can be identified by melanoma-specific immunohistochemistry using commercially available antibodies for HLA-DR to improve anti-PD-1 patient selection.

ERBB activation modulates sensitivity to MEK1/2 inhibition in a subset of driver-negative melanoma.

Melanomas are characterized by activating "driver" mutations in BRAF, NRAS, KIT, GNAQ, and GNA11. Resultant mitogen-activated protein kinase (MAPK) pathway signaling makes some melanomas susceptible to BRAF (BRAF V600 mutations), MEK1/2 (BRAF V600, L597, fusions; NRAS mutations), or other kinase inhibitors (KIT), respectively. Among driver-negative ("pan-negative") patients, an unexplained heterogeneity of response to MEK1/2 inhibitors has been observed. Analysis of 16 pan-negative melanoma cell lines revealed that 8 (50%; termed Class I) are sensitive to the MEK1/2 inhibitor, trametinib, similar to BRAF V600E melanomas. A second set (termed Class II) display reduced trametinib sensitivity, paradoxical activation of MEK1/2 and basal activation of ERBBs 1, 2, and 3 (4 lines, 25%). In 3 of these lines, PI3K/AKT and MAPK pathway signaling is abrogated using the ERBB inhibitor, afatinib, and proliferation is even further reduced upon the addition of trametinib. A potential mechanism of ERBB activation in Class II melanomas is minimal expression of the ERK1/2 phosphatase, DUSP4, as ectopic restoration of DUSP4 attenuated ERBB signaling through potential modulation of the ERBB ligand, amphiregulin (AREG). Consistent with these data, immunohistochemical analysis of patient melanomas revealed a trend towards lower overall DUSP4 expression in pan-negative versus BRAF- and NRAS-mutant tumors. This study is the first to demonstrate that differential ERBB activity in pan-negative melanoma may modulate sensitivity to clinically-available MEK1/2 inhibitors and provides rationale for the use of ERBB inhibitors, potentially in combination with MEK1/2 inhibitors, in subsets of this disease.

Preliminary results from a prospective trial of preoperative combined BRAF and MEK-targeted therapy in advanced BRAF mutation-positive melanoma.

BACKGROUND: We conducted a prospective trial of BRAF and mitogen-activated protein kinase kinase (MEK) targeted therapy in advanced, operable BRAF mutation-positive melanoma to determine feasibility, tumor response rates, and biomarkers of response and resistance. STUDY DESIGN: Thirteen patients with locally or regionally advanced BRAF mutation-positive melanoma received dabrafenib 150 mg po bid for 14 days, followed by dabrafenib plus trametinib 2 mg po daily for 14 days before operation. Biopsies and tumor measurements were obtained at baseline and days 14 and 28. Formalin-fixed paraffin embedded specimens were analyzed with hematoxylin and eosin, Ki-67, cleaved caspase-3, CD8, phosphorylated extracellular signal-regulated kinase (ERK), and phosphorylated MEK immunostains. RESULTS: Therapy was tolerated well, with toxicity ≥ grade 3 in 2 of 13 (15%) patients. All 12 patients receiving >14 days of therapy had substantial reduction in tumor volume (65% at day 14 and 78% at day 28) and underwent resection. After 14 days of dabrafenib therapy, there was a marked reduction in viable melanoma cells and a CD8 T-cell--rich infiltrate. Proliferation of the residual melanoma cells was reduced and apoptosis was increased. The cells continued to express phosphorylated ERK and phosphorylated MEK consistent with incomplete mitogen-activated protein kinase pathway inhibition. CONCLUSIONS: Preoperative targeted therapy of advanced BRAF-mutant melanoma is feasible, well tolerated, induces brisk tumor responses, and facilitates correlative science. A CD8 T-cell-rich infiltrate indicates a potential immune-mediated mechanism of action. Both proliferation and apoptosis were inhibited, but the mitogen-activated protein kinase pathway remained activated, suggesting intrinsic resistance in a subset of tumor cells. Additional investigation of the anti-tumor immune response during targeted therapy and the mechanisms of intrinsic resistance can yield novel therapeutic strategies.

Warm autoimmune hemolytic anemia secondary to Plasmodium ovale infection: a case report and review of the literature.

A three year old male from the Democratic Republic of the Congo was admitted to Monroe Carell Jr. Children's Hospital at Vanderbilt with a 10-day history of fever, emesis, and diarrhea. Examination demonstrated scleral icterus, splenomegaly, and anemia. By peripheral blood smear, the patient was diagnosed with Plasmodium ovale. Immunohematology demonstrated a positive direct antiglobulin test (DAT) for IgG and C3d with pan-agglutination on eluate. These findings, in combination with hemolytic labs, signified presence of an autoimmune hemolytic anemia (AIHA). We believe this to be the first reported case of P. ovale infection-mediated AIHA.

Carbachol-mediated pigment granule dispersion in retinal pigment epithelium requires Ca2+ and calcineurin.

BACKGROUND: Inside bluegill (Lepomis macrochirus) retinal pigment epithelial cells, pigment granules move in response to extracellular signals. During the process of aggregation, pigment motility is directed toward the cell nucleus; in dispersion, pigment is directed away from the nucleus and into long apical processes. A number of different chemicals have been found to initiate dispersion, and carbachol (an acetylcholine analog) is one example. Previous research indicates that the carbachol-receptor interaction activates a Gq-mediated pathway which is commonly linked to Ca2+ mobilization. The purpose of the present study was to test for involvement of calcium and to probe calcium-dependent mediators to reveal their role in carbachol-mediated dispersion. RESULTS: Carbachol-induced pigment granule dispersion was blocked by the calcium chelator BAPTA. In contrast, the calcium channel antagonist verapamil, and incubation in Ca2+-free medium failed to block carbachol-induced dispersion. The calcineurin inhibitor cypermethrin blocked carbachol-induced dispersion; whereas, two protein kinase C inhibitors (staurosporine and bisindolylmaleimide II) failed to block carbachol-induced dispersion, and the protein kinase C activator phorbol 12-myristate 13-acetate failed to elicit dispersion. CONCLUSION: A rise in intracellular calcium is necessary for carbachol-induced dispersion; however, the Ca2+ requirement is not dependent on extracellular sources, implying that intracellular stores are sufficient to enable pigment granule dispersion to occur. Calcineurin is a likely Ca2+-dependent mediator involved in the signal cascade. Although the pathway leads to the generation of diacylglycerol and calcium (both required for the activation of certain PKC isoforms), our evidence does not support a significant role for PKC.