Emmanouil Saloustros

Germline and somatic SMARCA4 mutations characterize small cell carcinoma of the ovary, hypercalcemic type

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy in women under 40 years of age. We sequenced the exomes of six individuals from three families with SCCOHT. After discovering segregating deleterious germline mutations in SMARCA4 in all three families, we tested DNA from a fourth affected family, which also carried a segregating SMARCA4 germline mutation. All the familial tumors sequenced harbored either a somatic mutation or loss of the wild-type allele. Immunohistochemical analysis of these cases and additional familial and non-familial cases showed loss of SMARCA4 (BRG1) protein in 38 of 40 tumors overall. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. Additionally, the SCCOHT cell line BIN-67 had biallelic deleterious mutations in SMARCA4. Our findings identify alterations in SMARCA4 as the major cause of SCCOHT, which could lead to improvements in genetic counseling and new treatment approaches.

Cytokeratin-19 mRNA-positive circulating tumor cells during follow-up of patients with operable breast cancer: prognostic relevance for late relapse

BackgroundThe detection of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTC) before and/or after adjuvant chemotherapy in patients with operable breast cancer is associated with poor clinical outcome. Reliable prognostic markers for late disease relapse are not available. In this study we investigated the value of CTC detection during the first five years of follow-up in predicting late disease relapse.MethodsBlood was analyzed from 312 women with operable breast cancer who had not experienced disease relapse during the first two years of follow-up. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) for CK-19 mRNA was used to detect CTC three months after the completion of adjuvant chemotherapy and every six months thereafter for a follow-up period of five years.ResultsEighty patients (25.6% of the study population) remained CTC free throughout the five-year period. A change in CTC status was observed in 133 patients (42.6%); 64 patients (20.5%) with initially CK-19 mRNA-positive CTC during the first 24 months turned CTC-negative afterwards while 69 (22.1%) who were initially CTC-negative became CTC-positive. Ninety-nine patients (31.7%) remained persistently CK-19 mRNA-positive. After a median follow-up period of 107 months (range: 38 to 161 months), the persistently CTC-positive patients with either hormonal receptor positive or negative tumors, had a higher risk of late-disease relapse compared to the persistently CTC-negative patients (36.4% versus 11.2%, P <0.001). Multivariate analysis revealed that persistently CTC-positive patients also had a shorter disease-free (P = 0.001) and overall survival (P = 0.001).ConclusionsPersistent detection of CK-19 mRNA-positive CTC during the first five years of follow-up is associated with an increased risk of late relapse and death in patients with operable breast cancer and indicates the presence of chemo-and hormonotherapy-resistant residual disease. This prognostic evaluation may be useful when deciding on subsequent adjuvant systemic therapy.

KCNJ5 mutations in the National Institutes of Health cohort of patients with primary hyperaldosteronism: an infrequent genetic cause of Conn's syndrome.

KCNJ5 mutations were recently described in primary hyperaldosteronism (PH or Conns syndrome). The frequency of these mutations in PH and the way KCNJ5 defects cause disease remain unknown. A total of 53 patients with PH have been seen at the National Institutes of Health over the last 12 years. Their peripheral and tumor DNAs (the latter from 16 that were operated) were screened for KCNJ5 mutations; functional studies on the identified defects were performed after transient transfection. Only two mutations were identified, and both in the tumor DNA only. There were no germline sequencing defects in any of the patients except for known synonymous variants of the KCNJ5 gene. One mutation was the previously described c.G451C alteration; the other was a novel one in the same codon: c.G451A; both lead to the same amino acid substitution (G151R) in the KCNJ5 protein. Functional studies confirmed previous findings that both mutations caused loss of channel selectivity and a positive shift in the reversal potential. In conclusion, the KCNJ5 protein was strongly expressed in the zona glomerulosa of normal adrenal glands but showed variable expression in the aldosterone-producing adenomas with and without mutation. The rate of KCNJ5 mutations among patients with PH and/or their tumors is substantially lower than what was previously reported. The G151R amino acid substitution appears to be the most frequent one so far detected in PH, despite additional nucleotide changes. The mutation causes loss of this potassium channels selectivity and may assist in the design of new therapies for PH.

Pancreatic Ductal and Acinar Cell Neoplasms in Carney Complex: A Possible New Association

CONTEXT Carney complex (CNC) is a rare disease inherited as an autosomal dominant trait, associated with various tumors, and caused most frequently by inactivation of the PRKAR1A gene. OBJECTIVES In our recent investigation of a large cohort of CNC patients, we identified several cases of pancreatic neoplasms. This possible association and PRKAR1As possible involvement in pancreatic tumor have not been reported previously. PATIENTS AND METHODS Nine patients (2.5%) with CNC and pancreatic neoplasms in an international cohort of 354 CNC patients were identified; we studied six of them. Immunohistochemistry and PRKAR1A sequencing were obtained. RESULTS Three men and three women with a mean age of 49 yr (range 34-75 yr) had acinar cell carcinoma (n = 2), adenocarcinoma (n = 1), and intraductal pancreatic mucinous neoplasm (n = 3). Five patients had a germline PRKAR1A mutation, including two patients with acinar cell carcinoma, for whom mutations were found in a hemizygous state in the tumor, suggesting loss of heterozygosity. PRKAR1A expression was not detected in five of the six pancreatic neoplasms from CNC patients, whereas the protein was amply expressed on other sporadic pancreatic tumors and normal tissue. CONCLUSION An unexpectedly high prevalence of rare pancreatic tumors was found among CNC patients. Immunohistochemistry and loss-of-heterozygosity studies suggest that PRKAR1A could function as a tumor suppressor gene in pancreatic tissue, at least in the context of CNC. Clinicians taking care of CNC patients should be aware of the possible association of CNC with a potentially aggressive pancreatic neoplasm.

Large-cell calcifying Sertoli cell tumors of the testes in pediatrics.

Purpose of review The aim of this review is to describe the clinical, biochemical, radiographic, histological, and functional characteristics of large-cell calcifying Sertoli cell tumors of the testes (LCCSCTs). We describe the two main syndromes associated with these tumors: Peutz–Jeghers syndrome (PJS) caused mainly by mutations in the STK11 (aka LKB1) gene, which encodes a serine–threonine kinase, and Carney complex (CNC), which is most often caused by PRKAR1A mutations, the gene encoding regulatory subunit type 1 of protein kinase A. Recent findings Relatively few patients have been reported in the literature with LCCSCTs. In children they often present as prepubertal and/or peripubertal gynecomastia. Although these tumors are very rare, they occur with higher frequency among patients with PJS and CNC. Orchiectomy was often performed in the past; however, these tumors are overwhelmingly benign and, unless there are significant hormonal changes or pain and/or mass effects, there is no need for surgery. Tumors that lead to hyperestrogenemia may be treated efficiently with aromatase inhibitors; any change in appearance should prompt evaluation for malignancy. Summary The detection of LCCSCTs may point to an underlying genetic multiple neoplasia syndrome such as PJS or CNC. Surgery is rarely indicated and aromatase inhibitors constitute an effective treatment for those cases that are associated with gynecomastia and/or advanced skeletal age.

The influence of clinical and genetic factors on patient outcome in small cell carcinoma of the ovary, hypercalcemic type

OBJECTIVE Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is an aggressive tumor, with long term survival at ~30% in early stage disease. SCCOHT is caused by germline and somatic SMARCA4 mutations, but the effect of the mutation type on patients remains unknown. Furthermore, the rarity of SCCOHT has resulted in varied treatment, with no standardized protocols. We analyzed 293 cases to determine the effect of treatment modalities and SMARCA4 mutations on patient diagnosis and outcome. METHODS In 293 SCCOHT patients we collected information on age and stage at diagnosis, treatment modality (surgery, chemotherapy, radiotherapy, and/or high-dose chemotherapy with autologous stem cell rescue (HDC-aSCR)), SMARCA4 mutation origin (germline/somatic), and overall survival. Cox analysis and log-rank tests were performed on 257 cases with available survival data. RESULTS The strongest prognostic factors were stage at diagnosis (p=2.72e-15) and treatment modality (p=3.87e-13). For FIGO stages II-IV, 5-year survival was 71% for patients who received HDC-aSCR, compared to 25% in patients who received conventional chemotherapy alone following surgery (p=0.002). Patients aged ≥40 had a worse outcome than younger patients (p=0.04). Twenty-six of 60 tested patients carried a germline SMARCA4 mutation, including all patients diagnosed <15years; carriers presented at a younger age than non-carriers (p=0.02). CONCLUSIONS Stage at diagnosis is the most significant prognostic factor in SCCOHT and consolidation with HDC-aSCR may provide the best opportunity for long-term survival. The large fraction of SMARCA4 germline mutations carriers warrants genetic counseling for all patients.

Prophylactic and therapeutic strategies in chemotherapy-induced neutropenia

Introduction: Neutropenia poses a serious threat to patients on chemotherapy. It exposes them to the risk of infection – including potentially fatal infections – and also leads to delays in treatment and reductions in dose intensity, which can compromise the possibility of a favorable outcome. Areas covered: The use of granulocyte colony-stimulating factors (G-CSF) and antibiotics to prevent febrile neutropenia (FN) and to ameliorate cancer chemotherapy-induced myelosuppression is discussed, based on a systematic search of Pubmed for clinical trials, reviews and meta-analysis published in the last 20 years. We consider that the treatment of FN, with the emphasis on careful attention to the patient, prompts antibiotic therapy and good hospital care. Expert opinion: We would argue that antibiotic prophylaxis should be offered routinely to patients receiving cytotoxic chemotherapy for acute leukemia and for patients with solid tumors and lymphoma receiving high-dose chemotherapy. In patients undergoing cyclical standard-dose myelosuppressive chemotherapy, we believe that prophylaxis is indicated during the first cycle of chemotherapy in which there is an expectation of grade 4 neutropenia (< 500 neutrophils). However, although the use of antibiotics and haematopoietic growth factors may improve quality of life by reducing the risk and consequences of FN, further study of the magnitude of their effects is needed.

Candidate DNA repair susceptibility genes identified by exome sequencing in high-risk pancreatic cancer

The genetic basis underlying the majority of hereditary pancreatic adenocarcinoma (PC) is unknown. Since DNA repair genes are widely implicated in gastrointestinal malignancies, including PC, we hypothesized that there are novel DNA repair PC susceptibility genes. As germline DNA repair gene mutations may lead to PC subtypes with selective therapeutic responses, we also hypothesized that there is an overall survival (OS) difference in mutation carriers versus non-carriers. We therefore interrogated the germline exomes of 109 high-risk PC cases for rare protein-truncating variants (PTVs) in 513 putative DNA repair genes. We identified PTVs in 41 novel genes among 36 kindred. Additional genetic evidence for causality was obtained for 17 genes, with FAN1, NEK1 and RHNO1 emerging as the strongest candidates. An OS difference was observed for carriers versus non-carriers of PTVs with early stage (≤IIB) disease. This adverse survival trend in carriers with early stage disease was also observed in an independent series of 130 PC cases. We identified candidate DNA repair PC susceptibility genes and suggest that carriers of a germline PTV in a DNA repair gene with early stage disease have worse survival.

Cytokeratin 19-positive circulating tumor cells in early breast cancer prognosis

In spite of the heterogeneity of breast cancer at the molecular level, circulating tumor cells (CTCs) may provide a novel prognostic marker. Approximately 20-40% of early breast cancer patients have detectable CTCs using reverse transcription PCR for CK19. The detection of CTCs before adjuvant chemotherapy or during tamoxifen administration has been demonstrated to be an independent adverse prognostic factor in women with early-stage breast cancer. The prognostic value of CTC detection is of great significance in subgroups of patients with estrogen receptor-negative and human EGF receptor 2-positive tumors. Prospective clinical trials are warranted in order to validate the use of CTCs as predictive and/or prognostic markers and assess their utility in individualizing therapy of patients with early breast cancer.

Celecoxib reduces glucocorticoids in vitro and in a mouse model with adrenocortical hyperplasia.

Primary pigmented nodular adrenocortical disease (PPNAD), whether in the context of Carney complex (CNC) or isolated, leads to ACTH-independent Cushings syndrome (CS). CNC and PPNAD are caused typically by inactivating mutations of PRKAR1A, a gene coding for the type 1a regulatory subunit (R1α) of cAMP-dependent protein kinase (PKA). Mice lacking Prkar1a, specifically in the adrenal cortex (AdKO) developed CS caused by bilateral adrenal hyperplasia (BAH), which is formed from the abnormal proliferation of fetal-like adrenocortical cells. Celecoxib is a cyclooxygenase 2 (COX2) inhibitor. In bone, Prkar1a inhibition is associated with COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of bone stromal cells. We hypothesized that COX2 inhibition may have an effect in PPNAD. In vitro treatment of human cell lines, including one from a patient with PPNAD, with celecoxib resulted in decreased cell viability. We then treated AdKO and control mice with 1500 mg/kg celecoxib or vehicle. Celecoxib treatment led to decreased PGE2 and corticosterone levels, reduced proliferation and increased apoptosis of adrenocortical cells, and decreased steroidogenic gene expression. We conclude that, in vitro and in vivo, celecoxib led to decreased steroidogenesis. In a mouse model of PPNAD, celecoxib caused histological changes that, at least in part, reversed BAH and this was associated with a reduction of corticosterone levels.