Kevin M. Elias

Impaired T H 17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome

The autosomal dominant hyper-IgE syndrome (HIES, ‘Job’s syndrome’) is characterized by recurrent and often severe pulmonary infections, pneumatoceles, eczema, staphylococcal abscesses, mucocutaneous candidiasis, and abnormalities of bone and connective tissue. Mutations presumed to underlie HIES have recently been identified in stat3, the gene encoding STAT3 (signal transducer and activator of transcription 3) (refs 3, 4). Although impaired production of interferon-γ and tumour-necrosis factor by T cells, diminished memory T-cell populations, decreased delayed-type-hypersensitivity responses and decreased in vitro lymphoproliferation in response to specific antigens have variably been described, specific immunological abnormalities that can explain the unique susceptibility to particular infections seen in HIES have not yet been defined. Here we show that interleukin (IL)-17 production by T cells is absent in HIES individuals. We observed that ex vivo T cells from subjects with HIES failed to produce IL-17, but not IL-2, tumour-necrosis factor or interferon-γ, on mitogenic stimulation with staphylococcal enterotoxin B or on antigenic stimulation with Candida albicans or streptokinase. Purified naive T cells were unable to differentiate into IL-17-producing (TH17) T helper cells in vitro and had lower expression of retinoid-related orphan receptor (ROR)-γt, which is consistent with a crucial role for STAT3 signalling in the generation of TH17 cells. TH17 cells have emerged as an important subset of helper T cells that are believed to be critical in the clearance of fungal and extracellular bacterial infections. Thus, our data suggest that the inability to produce TH17 cells is a mechanism underlying the susceptibility to the recurrent infections commonly seen in HIES.

IL-21 is produced by Th17 cells and drives IL-17 production in a STAT3-dependent manner.

CD4+ helper T cells can differentiate into several possible fates including: Th1, Th2, T regulatory, and Th17 cells. Although, cytokine production by non-T cells is an important factor in helper T cell differentiation, a characteristic feature of both Th1 and Th2 lineages is their ability to secrete cytokines that promote their respective differentiation. However, cytokines produced by T cells that help to sustain Th17 cells have not yet been identified. Here we show that IL-21 is a product of Th17 cells, which is induced in a Stat3-dependent manner. Additionally, Stat3 can directly bind the Il21 promoter. IL-21 also induces IL-17 production and expression of the transcription factor, RORγt. Furthermore, generation of Th17 cells in the conventional manner is attenuated by blocking IL-21. IL-21 is known to activate Stat3 and its ability to induce Th17 differentiation is abrogated in the absence of Stat3. These data argue that IL-21 serves as an autocrine factor secreted by Th17 cells that promotes or sustains Th17 lineage commitment.

Mesenchymal gene program-expressing ovarian cancer spheroids exhibit enhanced mesothelial clearance.

Metastatic dissemination of ovarian tumors involves the invasion of tumor cell clusters into the mesothelial cell lining of peritoneal cavity organs; however, the tumor-specific factors that allow ovarian cancer cells to spread are unclear. We used an in vitro assay that models the initial step of ovarian cancer metastasis, clearance of the mesothelial cell layer, to examine the clearance ability of a large panel of both established and primary ovarian tumor cells. Comparison of the gene and protein expression profiles of clearance-competent and clearance-incompetent cells revealed that mesenchymal genes are enriched in tumor populations that display strong clearance activity, while epithelial genes are enriched in those with weak or undetectable activity. Overexpression of transcription factors SNAI1, TWIST1, and ZEB1, which regulate the epithelial-to-mesenchymal transition (EMT), promoted mesothelial clearance in cell lines with weak activity, while knockdown of the EMT-regulatory transcription factors TWIST1 and ZEB1 attenuated mesothelial clearance in ovarian cancer cell lines with strong activity. These findings provide important insights into the mechanisms associated with metastatic progression of ovarian cancer and suggest that inhibiting pathways that drive mesenchymal programs may suppress tumor cell invasion of peritoneal tissues.

Efficacy and safety of imatinib mesylate (Gleevec®) and immunohistochemical expression of c-Kit and PDGFR-β in a Gynecologic Oncology Group Phase Il Trial in women with recurrent or persistent carcinosarcomas of the uterus

PURPOSE This multi-institutional phase II trial assessed the activity and toxicity of imatinib mesylate and explored c-Kit and platelet-derived growth factor receptor (PDGFR)-beta in recurrent or persistent uterine carcinosarcoma. METHODS Women with measurable uterine carcinosarcoma, who had a performance status of 0, 1, or 2 and had received up to two prior treatment regimens, were eligible and treated with a 600-mg daily oral dose of imatinib mesylate until disease progression or unacceptable toxicity. Endpoints included progression-free survival (PFS) >or=6 months, overall toxicity, PFS, overall survival (OS), and response. c-Kit and PDGFR-beta were evaluated by immunohistochemistry in archival tumor. RESULTS Twenty-six women were enrolled, 23 were eligible/evaluable, 1 was ineligible (wrong primary), and 2 were inevaluable (never treated). One subject had a PFS time >or=6 months, yielding the only patient with stable disease. All other patients had progressive disease (n=17) or were inevaluable for tumor response (n=5). Adverse events included grade 4 hypocalcemia (n=1) and grade 3 fatigue, dehydration and anorexia, genitourinary/renal, lymphatic, metabolic, and ocular toxicity (n=1 each, 4%). Positive expression of c-Kit or PDGFR-beta was observed in 88% (14/16) or 40% (6/15) and in 56% (9/16) or 73% (11/15) of cases in the sarcoma and carcinoma component of the tumor, respectively. Expression of c-Kit in the carcinoma component appeared to be associated with worse OS. No other associations were notable. CONCLUSION Imatinib mesylate was generally well tolerated but had minimal activity as a single agent in unscreened patients. The potential association between carcinomatous c-Kit and OS requires validation.

Beyond genomics: Critical evaluation of cell line utility for ovarian cancer research

OBJECTIVE Comparisons of The Cancer Genome Atlas (TCGA) with high grade serous ovarian cancer (HGSOC) cell lines used in research reveal that many common experimental models lack defining genomic characteristics seen in patient tumors. As cell lines exist with higher genomic fidelity to TCGA, this study aimed to evaluate the utility of these cell lines as tools for preclinical investigation. METHODS We compared two HGSOC cell lines with supposed high genomic fidelity to TCGA, KURAMOCHI and OVSAHO, with the most commonly cited ovarian cancer cell line, SKOV3, which has poor genomic fidelity to TCGA. The lines were analyzed for genomic alterations, in vitro performance, and growth in murine xenografts. RESULTS Using targeted next generation sequencing analyses, we determined that each line had a distinct mutation profile, including alterations in TP53, and copy number variation of specific genes. KURAMOCHI and OVSAHO better recapitulated serous carcinoma morphology than SKOV3. All lines expressed PAX8 and stathmin, but KURAMOCHI and OVSAHO did not express CK7. KURAMOCHI was significantly more platinum sensitive than OVSAHO and SKOV3. Unlike SKOV3, KURAMOCHI and OVSAHO engrafted poorly in subcutaneous xenografts. KURAMOCHI and OVSAHO grew best after intraperitoneal injection in SCID mice and recapitulated miliary disease while SKOV3 grew in all murine systems and formed oligometastatic disease. CONCLUSIONS The research utility of HGSOC cell line models requires a comprehensive assessment of genomic as well as in vitro and in vivo properties. Cell lines with closer genomic fidelity to human tumors may have limitations in performance for preclinical investigation.

Derivation and validation of the acute organ failure score to predict outcome in critically ill patients: a cohort study.

Objectives:Prediction models for ICU mortality rely heavily on physiologic variables that may not be available in large retrospective studies. An alternative approach when physiologic variables are absent stratifies mortality risk by acute organ failure classification. Design:Retrospective cohort study. Setting:Two large teaching hospitals in Boston, MA. Subjects:Ninety-two thousand eight hundred eighty-six patients aged 18 years old or older admitted between November 3, 1997, and February 25, 2011, who received critical care. Interventions:None. Measurements and Main Results:The derivation cohort consisted of 35,566 patients from Brigham and Women’s Hospital, and the validation cohort comprised 57,320 patients from Massachusetts General Hospital. Acute organ failure was determined for each patient based on International Classification of Diseases, 9th Revision, Clinical Modification code combinations. The main outcome measure was 30-day mortality. A clinical prediction model was created based on a logistic regression model describing the risk of 30-day mortality as a function of age, medical versus surgical patient type, Deyo-Charlson index, sepsis, and type acute organ failure (respiratory, renal, hepatic, hematologic, metabolic, and neurologic) after ICU admission. We computed goodness-of fit statistics and c-statistics as measures of model calibration and 30-day mortality discrimination, respectively. Thirty-day mortality occurred in 5,228 of 35,566 patients (14.7%) assigned to the derivation cohort. The clinical prediction model was predictive for 30-day mortality. The c-statistic for the clinical prediction model was 0.7447 (95% CI, 0.74–0.75) in the derivation cohort and 0.7356 (95% CI, 0.73–0.74) in the validation cohort. For both the derivation and validation cohorts, the Hosmer-Lemeshow chi-square p values indicated good model fit. In a smaller cohort of 444 patients with Acute Physiologic and Chronic Health Evaluation II scores determined, differences in model discrimination of 30-day mortality between the clinical prediction model and Acute Physiologic and Chronic Health Evaluation II were not significant (chi-square = 0.76; p =0.38). Conclusions:An acute organ failure–based clinical prediction model shows good calibration and discrimination for 30-day mortality in the critically ill. The clinical prediction model compares favorably to Acute Physiologic and Chronic Health Evaluation II score in the prediction of 30-day mortality in the critically ill. This score may be useful for severity of illness risk adjustment in observational studies where physiologic data are unavailable.

Platinum and PARP Inhibitor Resistance Due to Overexpression of MicroRNA-622 in BRCA1-Mutant Ovarian Cancer

High-grade serous ovarian carcinomas (HGSOCs) with BRCA1/2 mutations exhibit improved outcome and sensitivity to double-strand DNA break (DSB)-inducing agents (i.e., platinum and poly(ADP-ribose) polymerase inhibitors [PARPis]) due to an underlying defect in homologous recombination (HR). However, resistance to platinum and PARPis represents a significant barrier to the long-term survival of these patients. Although BRCA1/2-reversion mutations are a clinically validated resistance mechanism, they account for less than half of platinum-resistant BRCA1/2-mutated HGSOCs. We uncover a resistance mechanism by which a microRNA, miR-622, induces resistance to PARPis and platinum in BRCA1 mutant HGSOCs by targeting the Ku complex and restoring HR-mediated DSB repair. Physiologically, miR-622 inversely correlates with Ku expression during the cell cycle, suppressing non-homologous end-joining and facilitating HR-mediated DSB repair in S phase. Importantly, high expression of miR-622 in BRCA1-deficient HGSOCs is associated with worse outcome after platinum chemotherapy, indicating microRNA-mediated resistance through HR rescue.

Genomic aberrations in cervical adenocarcinomas in Hong Kong Chinese women.

Although the rates of cervical squamous cell carcinoma have been declining, the rates of cervical adenocarcinoma are increasing in some countries. Outcomes for advanced cervical adenocarcinoma remain poor. Precision mapping of genetic alterations in cervical adenocarcinoma may enable better selection of therapies and deliver improved outcomes when combined with new sequencing diagnostics. We present whole‐exome sequencing results from 15 cervical adenocarcinomas and paired normal samples from Hong Kong Chinese women. These data revealed a heterogeneous mutation spectrum and identified several frequently altered genes including FAT1, ARID1A, ERBB2 and PIK3CA. Exome sequencing identified human papillomavirus (HPV) sequences in 13 tumors in which the HPV genome might have integrated into and hence disrupted the functions of certain exons, raising the possibility that HPV integration can alter pathways other than p53 and pRb. Together, these provisionary data suggest the potential for individualized therapies for cervical adenocarcinoma based on genomic information.

Biomarker discovery in epithelial ovarian cancer by genomic approaches.

Ovarian cancer is the fifth most common form of cancer in women in the United States. It is a complex disease composed of different histological grades and histological types. Most of epithelial ovarian cancer cases are detected at an advanced stage. Patients usually respond to primary treatment with surgery and chemotherapy. However, the disease usually recurs and is ultimately fatal. So far, a satisfactory screening procedure and regime to treat the recurrence disease are not available. High-throughput genomic analyses have the potential to change the detection and the treatment of ovarian neoplasms. They can help diagnose subtypes of disease and predict patient survival. New diagnostic and prognostic markers for ovarian cancer are emerging. One day, profiling may influence treatment decisions, informing both which patients should receive chemotherapy and what type of chemotherapeutic agents should be employed. As greater numbers of tumor samples are analyzed, the power of these profiling studies will increase, raising the possibility that novel molecular targets and less toxic therapies will be identified. These powerful techniques hold the potential to unravel the genetic origins of ovarian cancer. Hopefully, this will translate into earlier diagnosis and better patient outcome from disease.

Elafin drives poor outcome in high-grade serous ovarian cancers and basal-like breast tumors.

High-grade serous ovarian carcinoma (HGSOC) and basal-like breast cancer (BLBC) share many features including TP53 mutations, genomic instability and poor prognosis. We recently reported that Elafin is overexpressed by HGSOC and is associated with poor overall survival. Here, we confirm that Elafin overexpression is associated with shorter survival in 1000 HGSOC patients. Elafin confers a proliferative advantage to tumor cells through the activation of the MAP kinase pathway. This mitogenic effect can be neutralized by RNA interference, specific antibodies and a MEK inhibitor. Elafin expression in patient-derived samples was also associated with chemoresistance and strongly correlates with bcl-xL expression. We extended these findings into the examination of 1100 primary breast tumors and six breast cancer cell lines. We observed that Elafin is overexpressed and secreted specifically by BLBC tumors and cell lines, leading to a similar mitogenic effect through activation of the MAP kinase pathway. Here too, Elafin overexpression is associated with poor overall survival, suggesting that it may serve as a biomarker and therapeutic target in this setting.