Germline and somatic testing for ovarian Cancer: An SGO clinical practice statement.

Germline and somatic genetic testing have become critical components of care for people with ovarian cancer. The identification of germline and somatic pathogenic variants as well as homologous recombination deficiency can contribute to the prediction of treatment response, prognostic outcome, and suitability for targeted agents (e.g. poly (ADP-ribose) polymerase (PARP) inhibitors). Furthermore, identifying germline pathogenic variants can prompt cascade genetic testing for at-risk relatives. Despite the clinical benefits and consensus recommendations from several organizations calling for universal genetic testing in ovarian cancer, only about one third of patients complete germline or somatic genetic testing. The members of the Society of Gynecologic Oncology (SGO) Clinical Practice Committee have composed this statement to provide an overview of germline and somatic genetic testing for patients with epithelial ovarian cancer, focusing on available testing modalities and options for care delivery.

The risk of breast cancer in BRCA1 and BRCA2 mutation carriers without a first-degree relative with breast cancer.

The objective of this study was to estimate the lifetime risk of breast cancer in women with a BRCA1 or BRCA2 mutation with and without at least 1 first-degree relative with breast cancer. A total of 2835 women with a BRCA1 or BRCA2 mutation were followed. Age- and gene-specific breast cancer rates were calculated. The relative risks of breast cancer for subjects with a family history of breast cancer, compared to no family history were calculated. The mean age at baseline was 41.1 years, and they were followed for a mean of 6.0 years. The estimated penetrance of breast cancer to age 80 years was 60.8% for BRCA1 and 63.1% for BRCA2. For all BRCA carriers, the penetrance of breast cancer to age 80 for those with no first-degree relative with breast cancer was 60.4% and 63.3% for those with at least 1 first-degree relative with breast cancer. The risk of breast cancer for BRCA carriers with no first-degree relative with breast cancer is substantial, and as a result, clinical management for these women should be the same as those for women with an affected relative.

Anti-thyroglobulin antibodies do not significantly increase the risk of finding iodine avid metastases on post-radioactive iodine ablation scan in low-risk thyroid cancer patients.

OBJECTIVE: Post-operative thyroglobulin (Tg) levels can predict the likelihood of residual cancer, including distant metastases, thereby influencing postsurgical treatment strategies even in patients with low-risk disease. Circulating anti-thyroglobulin antibodies (anti-Tg Abs) interfere with Tg measurement preventing this clinical use. It is not known if the presence of anti-Tg Abs predicts metastatic disease on post-therapy scan in patients with low-risk disease or if they should influence the use or dose of I-131 therapy. In the present study, we compare post-therapy scans in low-risk patients with and without anti-Tg Abs. METHODS: This is a single-institution retrospective study. The study population (Group A) included all patients with low-risk differentiated thyroid cancer (DTC) who underwent total thyroidectomy and RAI between 1/1/2006 to 9/11/2015 with intrathyroidal T1-T2, Nx, N0 or N1a (≤5 nodes all measuring, when reported, <2 mm) that had anti-thyroglobulin antibodies. Patients were excluded if they had known distant metastases and/or extensive vascular invasion. A second group of patients (Group B) treated during the same period but without anti-Tg antibodies was selected to match group A by propensity core matching with a logistic regression model. RESULTS: Each group included 37 patients. In group A: Median age was 40 years, 86% female and 76% PTC. Median tumor size was 2 cm (0.2-3.8), 32% had multifocal disease, 16% were N1a and 4% had vascular invasion. Parameters in group B were not statistically different from Group A, as expected based on the selection criteria, except being less likely to have Hashimoto's thyroiditis on pathology (p < 0.001). Post-therapy scan results were compared by Chi-square test with 86% negative post therapy scan frequency in group A and 92% in group B without evidence of a difference (p = 0.45). CONCLUSION: In patients with low-risk DTC, anti-Tg Abs did not significantly predict metastatic disease on post-therapy scan. If confirmed, these data suggest that the presence of anti-Tg Abs alone should not influence initial therapy in patients with low-risk DTC.

Linking distant relatives with BRCA gene mutations: potential for cost savings.

Thousands of individuals have undergone mutational analysis of BRCA1 and BRCA2. The Ohio State University Clinical Cancer Genetics program has identified 466 individuals from 289 families with a mutation in BRCA1 or BRCA2. Excluding Ashkenazi Jewish founder mutations, we observed 9 deleterious BRCA mutations five or more times in ostensibly unrelated families and another 13 mutations in 3-4 families. We hypothesized that some of the rarer recurrent mutations observed in our population were due to different branches of the same family being tested independently without knowledge of previous testing of relatives. We examined 90 pedigrees for individuals with the same mutations that were seen three or more times for shared reported family medical history or surnames. Familial links were made in four instances out of a total of 22 shared mutations despite the fact that individuals were not aware that another family member had been tested. As more individuals undergo BRCA testing, we propose that this phenomenon will become more common. Being unaware of previous testing in a family not only affects the risk assessment but also likely increases the costs associated with the genetic testing and subsequent cancer screening in many cases.

Incidence of colorectal cancer in BRCA1 and BRCA2 mutation carriers: results from a follow-up study.

BACKGROUND: The BRCA1 and BRCA2 genes confer increased susceptibility to breast and ovarian cancer and to a spectrum of other cancers. There is controversy regarding the risk of colorectal cancer conferred by germline mutations in these two genes. METHODS: We followed 7015 women with a BRCA mutation for new cases of colorectal cancer. Incidence rates in carriers were compared with population-specific incidence rates, and standardised incidence ratios (SIRs) were estimated. The expected numbers of cancers were computed by multiplying person-years at risk by the appropriate age-, sex- and country-specific incidence rates from the five countries. RESULTS: Twenty-one incident colorectal cancer cases were observed among all mutation carriers, compared with 23.6 cases expected. The SIR for BRCA1 carriers was 0.92 (95% confidence interval (CI), 0.54-1.40, P=0.7) and for BRCA2 carriers was 0.82 (95% CI, 0.30-1.81, P=0.7). The SIR for colon cancer was 3.81 (95% CI 1.77-7.23) for women below the age of 50 years (both genes combined) and was 0.60 (95% CI 0.33-1.00) for women aged 50 years and above. CONCLUSION: The risk of colorectal cancer is increased in female carriers of BRCA1 mutations below the age of 50 years but not in women with BRCA2 mutations or in older women.

Recurrent and founder mutations in the PMS2 gene.

Germline mutations in PMS2 are associated with Lynch syndrome (LS), the most common known cause of hereditary colorectal cancer. Mutation detection in PMS2 has been difficult due to the presence of several pseudogenes, but a custom-designed long-range PCR strategy now allows adequate mutation detection. Many mutations are unique. However, some mutations are observed repeatedly across individuals not known to be related due to the mutation being either recurrent, arising multiple times de novo at hot spots for mutations, or of founder origin, having occurred once in an ancestor. Previously, we observed 36 distinct mutations in a sample of 61 independently ascertained Caucasian probands of mixed European background with PMS2 mutations. Eleven of these mutations were detected in more than one individual not known to be related and of these, six were detected more than twice. These six mutations accounted for 31 (51%) ostensibly unrelated probands. Here, we performed genotyping and haplotype analysis in four mutations observed in multiple probands and found two (c.137G>T and exon 10 deletion) to be founder mutations and one (c.903G>T) a probable founder. One (c.1A>G) could not be evaluated for founder mutation status. We discuss possible explanations for the frequent occurrence of founder mutations in PMS2.

The incidence of pancreatic cancer in BRCA1 and BRCA2 mutation carriers.

BACKGROUND: Germline mutations in BRCA1 and BRCA2 predispose to pancreatic cancer. We estimated the incidence of pancreatic cancer in a cohort of female carriers of BRCA1 and BRCA2 mutation. We also estimated survival rates in pancreatic cancer cases from families with a BRCA mutation. METHODS: We followed 5149 women with a mutation for new cases of pancreatic cancer. The standardised incidence ratios (SIR) for pancreatic cancer were calculated based on age group and country of residence. We also reviewed the pedigrees of 8140 pedigrees with a BRCA1 or a BRCA2 mutation for those with a case of pancreatic cancer. We recorded the year of diagnosis and the year of death for 351 identified cases. RESULTS: Eight incident pancreatic cancer cases were identified among all mutation carriers. The SIR for BRCA1 carriers was 2.55 (95% CI=1.03-5.31, P=0.04) and for BRCA2 carriers was 2.13 (95% CI=0.36-7.03, P=0.3). The 5-year survival rate was 5% for cases from a BRCA1 family and 4% for cases from a BRCA2 family. CONCLUSION: The risk of pancreatic cancer is approximately doubled in female BRCA carriers. The poor survival in familial pancreatic cancer underscores the need for novel anti-tumoural strategies.

A frame-shift mutation of PMS2 is a widespread cause of Lynch syndrome.

BACKGROUND: When compared to the other mismatch repair genes involved in Lynch syndrome, the identification of mutations within PMS2 has been limited (<2% of all identified mutations), yet the immunohistochemical analysis of tumour samples indicates that approximately 5% of Lynch syndrome cases are caused by PMS2. This disparity is primarily due to complications in the study of this gene caused by interference from pseudogene sequences. METHODS: Using a recently developed method for detecting PMS2 specific mutations, we have screened 99 patients who are likely candidates for PMS2 mutations based on immunohistochemical analysis. RESULTS: We have identified a frequently occurring frame-shift mutation (c.736_741del6ins11) in 12 ostensibly unrelated Lynch syndrome patients (20% of patients we have identified with a deleterious mutation in PMS2, n = 61). These individuals all display the rare allele (population frequency <0.05) at a single nucleotide polymorphism (SNP) in exon 11, and have been shown to possess a short common haplotype, allowing us to calculate that the mutation arose around 1625 years ago (65 generations; 95% confidence interval 22 to 120). CONCLUSION: Ancestral analysis indicates that this mutation is enriched in individuals with British and Swedish ancestry. We estimate that there are >10 000 carriers of this mutation in the USA alone. The identification of both the mutation and the common haplotype in one Swedish control sample (n = 225), along with evidence that Lynch syndrome associated cancers are rarer than expected in the probands' families, would suggest that this is a prevalent mutation with reduced penetrance.

Ecto-ATPase activity of alpha-sarcoglycan (adhalin).

alpha-Sarcoglycan is a component of the sarcoglycan complex of dystrophin-associated proteins. Mutations of any of the sarcoglycan genes cause specific forms of muscular dystrophies, collectively termed sarcoglycanopathies. Importantly, a deficiency of any specific sarcoglycan affects the expression of the others. Thus, it appears that the lack of sarcoglycans deprives the muscle cell of an essential, yet unknown function. In the present study, we provide evidence for an ecto-ATPase activity of alpha-sarcoglycan. alpha-Sarcoglycan binds ATP in a Mg2+-dependent and Ca2+-independent manner. The binding is inhibited by 3'-O-(4-benzoyl)benzoyl ATP and ADP. Sequence analysis reveals the existence of a consensus site for nucleotide binding in the extracellular domain of the protein. An antibody against this sequence inhibits the binding of ATP. A dystrophin.dystrophin-associated protein preparation demonstrates a Mg-ATPase activity that is inhibited by the antibody but not by inhibitors of endo-ATPases. In addition, we demonstrate the presence in the sarcolemmal membrane of a P2X-type purinergic receptor. These data suggest that alpha-sarcoglycan may modulate the activity of P2X receptors by buffering the extracellular ATP concentration. The absence of alpha-sarcoglycan in sarcoglycanopathies leaves elevated the concentration of extracellular ATP and the persistent activation of P2X receptors, leading to intracellular Ca2+ overload and muscle fiber death.

Increased expression of dystrophin, beta-dystroglycan and adhalin in denervated rat muscles.

To evaluate a potential regulatory role of the nerve, the distribution and expression of dystrophin, of beta-dystroglycan (43DAG) and adhalin (50DAG), two of the dystrophin-associated proteins and utrophin (dystrophin related protein or DRP) were studied in rat muscles after 2 weeks of denervation. We found that dystrophin, beta-dystroglycan and adhalin were overexpressed in denervated muscle, whereas utrophin did not increase and was found only in the post-synaptic membrane. The study of the distribution of dystrophin in the sarcolemma of single muscle fibres indicates that the molecular organization of dystrophin was maintained after denervation. Dystrophin in addition of forming a scaffold around the fibre was found around the clusters of AChR that reappeared in the extra-synaptic membrane after denervation. Also beta-dystroglycan colocalises at these clusters. These results suggest that the increase in dystrophin, beta-dystroglycan and adhalin is correlated with the reappearance of AChRs in the extra synaptic membrane.

Assessment of full-thickness wounds in the genetically diabetic mouse for suitability as a wound healing model.

While using the diabetic C57BL/KsJ db/db mouse as a wound healing model, we encountered several repair patterns which affect its suitability as a predictive screening model for certain indications. For example, wound contraction, albeit impaired, was found to be particularly dependent on bandaging technique and vehicle type. Wounds which had been continuously occluded with Opsite dressings had a high relative variability in contraction, and there was a tendency toward reduced contraction, suggesting that the dressings were acting as a splint. Viscous dosing vehicles inhibited contraction of occluded wounds but appeared to enhance contraction of nonoccluded wounds. In contrast to many other models, occlusion in these studies did not enhance reepithelialization when compared with air exposure (the rate of reepithelialization in db/db mice appeared normal, typically growing 2 mm from each edge in 10 days). Also in contrast to other wound healing models, viscous dosing vehicles when used under occlusion inhibited reepithelialization. However, as seen in other wound healing models, granulation tissue thickness was reliably increased in response to treatment with recombinant human platelet-derived growth factor-BB. Our experience with the db/db diabetic mouse model has led us to recommend the use of this animal model only after its limitations have been identified and accepted.

Phosphorylation of dystrophin:effects on actin binding.

Dystrophin is phosphorylated by several protein kinases. In this work, we have studied the effects of dystrophin phosphorylation on the binding to actin. Purified dystrophin was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (PKA), casein kinase II (CK-II), and protein kinase c (PKC). The results demonstrate that phosphorylation of dystrophin by PKA phosphorylation caused a three fold increase in dystrophin binding to actin. In contrast, phosphorylation by CK-II or PKC inhibited the binding to actin. These results indicate that phosphorylation of dystrophin modulates its interaction with the actin cytoskeleton. It is suggested that phosphorylation may be one mechanism for regulating protein turnover in muscle membrane-skeleton.

Dystrophin is phosphorylated by endogenous protein kinases.

Dystrophin, the protein coded by the gene missing in Duchenne muscular dystrophy, is assumed to be a component of the membrane cytoskeleton of skeletal muscle. Like other cytoskeletal proteins in different cell types, dystrophin bound to sarcolemma membranes was found to be phosphorylated by endogenous protein kinases. The phosphorylation of dystrophin was activated by cyclic AMP, cyclic GMP, calcium and calmodulin, and was inhibited by cyclic AMP-dependent protein kinase peptide inhibitor, mastoparan and heparin. These results suggest that membrane-bound dystrophin is a substrate of endogenous cyclic AMP- and cyclic GMP-dependent protein kinases, calcium/calmodulin-dependent kinase and casein kinase II. The possibility that dystrophin could be phosphorylated by protein kinase C is suggested by the inhibition of phosphorylation by staurosporin. On the other hand dystrophin seems not to be a substrate for protein tyrosine kinases, as shown by the lack of reaction of phosphorylated dystrophin with a monoclonal antiphosphotyrosine antibody. Sequence analysis indicates that dystrophin contains seven potential phosphorylation sites for cyclic AMP- and cyclic GMP-dependent protein kinases (all localized in the central rod domain of the molecule) as well as several sites for protein kinase C and casein kinase II. Interestingly, potential sites of phosphorylation by protein kinase C and casein kinase II are located in the proximity of the actin-binding site. These results suggest, by analogy with what has been demonstrated in the case of other cytoskeletal proteins, that the phosphorylation of dystrophin by endogenous protein kinases may modulate both self assembly and interaction of dystrophin with other cytoskeletal proteins in vivo.

Interaction of dystrophin with cytoskeletal proteins: binding to talin and actin.

Dystrophin, the protein product of the Duchenne gene, is thought to be a member of muscle membrane cytoskeleton. In this work we studied the interactions of purified dystrophin from rabbit skeletal muscle sarcolemma membranes with other cytoskeletal proteins. The interaction of dystrophin with purified talin from chicken gizzard was tested by solid phase immunoassay. Under these conditions dystrophin bound talin with high affinity (Kd 3.5 nM). Vinculin purified from chicken gizzard did not bind dystrophin, but it inhibited the binding of dystrophin to talin. Furthermore, co-sedimentation and solid phase immunoassay experiments both demonstrated that native dystrophin binds purified actin from rabbit skeletal muscle. In conclusion, our results show that dystrophin can interact in vitro with proteins that are members of muscle membrane cytoskeleton. These proteins may represent additional sites for anchoring dystrophin to sarcolemma.

Evaluation of PDGF-BB, PDGF-AA, bFGF, IL-1, and EGF dose responses in polyvinyl alcohol sponge implants assessed by a rapid histologic method.

Polyvinyl alcohol sponge implants were used in rats, mice, and guinea pigs to determine dose responses of growth factors. Eight differently treated sponges per rat or guinea pig (4/mouse) were injected with test material on alternate days and evaluated at day 8. Much of the observed response occurred in and around the capsule and was manifest as densely cellular granulation tissue. Including this capsular response in a single histologic slide ranking system provided a more sensitive and faster method of assessing growth factor effects than measurement of connective tissue ingrowth alone. Clear dose responsive effects were seen with recombinant human PDGF-BB, PDGF-AA, bFGF, and IL-1 beta, while EGF gave a lesser response. Lipopolysaccharide did not affect the connective tissue response, alone or in combination with PDGF-BB. PDGF-BB was tested in each species, and the dose response characteristics were qualitatively and quantitatively similar across species.

Prevalence of dystrophin-positive fibers in 85 Duchenne muscular dystrophy patients.

The prevalence of dystrophin-positive fibers in Duchenne muscular dystrophy (DMD) muscle was estimated by direct counting on immunostained sections in a series of biopsy specimens from 85 patients, 42 of which were also screened for intragenic deletions by cDNA probes. Dystrophin-positive fibres are normotrophic and occur in muscle sections at a frequency between 0.01 and 6.81%. Frequencies over 1% were found only in patients older than 6 yr. The prevalence of dystrophin-positive fibers is about the same in patients with detectable and with undetectable deletions. The occurrence of positive fibers in small clusters supports the hypothesis of their clonal origin, suggesting that they may result from genetic reversion. No clinical differences were noticed in DMD patients of similar age with respect to the occurrence of dystrophin-positive fibres in their muscle biopsies.

Cytotoxicity of trichothecene mycotoxins isolated from Fusarium sporotrichioides (MC-72083) and Fusarium sambucinum in baby hamster kidney (BHK-21) cells.

Twenty-six trichothecene mycotoxins produced by Fusarium sporotrichioides (MC-72083) and Fusarium sambucinum were screened for relative cytotoxicity in cultured baby hamster kidney (BHK-21) cells. The relative cytotoxicity was measured as LC100. The most cytotoxic trichothecenes were T-2 toxin (5 ng/ml) and the recently isolated 4-propanoyl HT-2 (5 ng/ml) and 3'-hydroxy T-2 toxin (5 ng/ml). T-2 tetraol (1 x 10(4) ng/ml), 8-beta-hydroxytrichothecene (1 x 10(4) ng/ml), sporotrichiol (2 x 10(4) ng/ml), 8-oxodiacetoxyscirpenol (6 x 10(4) ng/ml) and 8-acetyl T-2 tetraol (1 x 10(5) ng/ml) were the least toxic of the regular trichothecenes. None of the modified trichothecenes or the apotrichothecene were very cytotoxic: 8-beta-hydroxysambucoin (2 x 10(3) ng/ml), FS-1 (5 x 10(3) ng/ml), 8-alpha-hydroxysambucoin (8 x 10(4) ng/ml) and trichotriol (1 x 10(5) ng/ml). The modified trichothecenes, FS-2 and FS-3, were not toxic even at 1 x 10(5) ng/ml. The baby hamster kidney cell bioassay proved to be a very sensitive and reproducible means of screening new trichothecene mycotoxins for relative cytotoxicity.