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1.  The Development of a Clinical Screening Instrument For Tumour Predisposition Syndromes In Childhood Cancer Patients 
Identification of tumour predisposition syndromes in patients who have cancer in childhood is paramount for optimal care. A screening instrument that can help to identify such patients will facilitate physicians caring for children with cancer. The complete screening instrument should consist of a standardized series of pictures and a screening form for manifestations not visible in the pictures. Here we describe the development of such a screening form based on an international two-stage Delphi process and an initial validation of the complete instrument.
Patients and Methods
We identified manifestations that may contribute to the diagnosis of a tumour predisposition syndrome through the Winter-Baraitser Dysmorphology Database and the textbook “Gorlin's Syndromes of the Head and Neck”. In a two-round Delphi process, eight international content-experts scored the contribution of each of these manifestations. We performed a clinical validation of the instrument in a selected cohort of ten paediatric cancer patients from another centre.
In total, 49 manifestations were found to contribute to the diagnosis of a tumour predisposition syndrome and were included in the screening form. The pilot validation study showed that patients suspect for having a tumour predisposition syndrome were recognized. Excellent correlation for indication for referral of a patient between the screening instrument and the reference standard (personal evaluation by an experienced clinical geneticist) was found.).
The Delphi process performed by international specialists with a function as opinion leaders in their field of expertise has led to a screening form and instrument with which those childhood cancer patients can be identified who may have a tumour predisposition syndrome and thus have an indication to be referred for further genetic analysis.
PMCID: PMC4277698  PMID: 23855994
Paediatric oncology; childhood cancer; screening instrument; tumour predisposition syndromes; Delphi process; questionnaires
2.  Pulmonary arterial hypertension in a patient with Cowden syndrome and the PTEN mutation 
Pulmonary Circulation  2014;4(4):728-731.
The pathogenesis of pulmonary arterial hypertension (PAH) exhibits many neoplastic-like features. Cowden syndrome is a difficult-to-recognize heritable cancer syndrome caused by a germline mutation in the phosphatase-and-tensin homolog deleted on the chromosome 10 (PTEN) gene. PTEN regulation has been implicated in cancer development and, more recently, PAH pathogenesis. Here we report a case of PAH in a patient with Cowden syndrome and the response to pulmonary vasodilators.
PMCID: PMC4278632  PMID: 25610608
pulmonary hypertension; PTEN mutation; Cowden syndrome
3.  The impact of pregnancy on breast cancer survival in women who carry a BRCA1 or BRCA2 mutation 
Physicians are often approached by young women with a BRCA mutation and a recent history of breast cancer who wish to have a baby. They wish to know if pregnancy impacts upon their future risks of cancer recurrence and survival. To date, there is little information on the survival experience of women who carry a mutation in one of the BRCA genes and who become pregnant. From an international multi-center cohort study of 12,084 women with a BRCA1 or BRCA2 mutation, we identified 128 case subjects who were diagnosed with breast cancer while pregnant or who became pregnant after a diagnosis of breast cancer. These women were age-matched to 269 mutation carriers with breast cancer who did not become pregnant (controls). Subjects were followed from the date of breast cancer diagnosis until the date of last follow-up or death from breast cancer. The Kaplan–Meier method was used to estimate 15-year survival rates. The hazard ratio for survival associated with pregnancy was calculated using a left-truncated Cox proportional hazard model, adjusting for other prognostic factors. Among women who were diagnosed with breast cancer when pregnant or who became pregnant thereafter, the 15-year survival rate was 91.5 %, compared to a survival of 88.6 % for women who did not become pregnant (adjusted hazard ratio = 0.76; 95 % CI 0.31–1.91; p = 0.56). Pregnancy concurrent with or after a diagnosis of breast cancer does not appear to adversely affect survival among BRCA1/2 mutation carriers.
PMCID: PMC3940343  PMID: 24136669
BRCA1; BRCA2; Breast cancer; Pregnancy; Survival
4.  A Novel Germline Mutation in BAP1 Predisposes to Familial Clear-Cell Renal Cell Carcinoma 
Molecular cancer research : MCR  2013;11(9):1061-1071.
Renal cell carcinoma (RCC) clusters in some families. Familial RCC arises from mutations in several genes, including VHL, which is also mutated in sporadic RCC. However, a significant percentage of familial RCC remains unexplained. Recently, we discovered that the BAP1 gene is mutated in sporadic RCC. BAP1, which encodes a nuclear deubiquitinase, is a two-hit tumor suppressor gene. Somatic BAP1 mutations are associated with high-grade ccRCC and poor patient outcomes. To determine whether BAP1 predisposes to familial RCC, we sequenced the BAP1 gene in 83 unrelated probands with unexplained familial RCC. We identified a novel variant (c.41T>A; p.L14H), which cosegregated with the RCC phenotype. The p.L14H variant disrupts a highly conserved residue in the catalytic domain, a domain frequently targeted by missense mutations. The family with the BAP1 variant was characterized by early-onset clear cell RCC, occasionally of high Fuhrman grade, and lacked other features that characterize von Hippel-Lindau syndrome. These findings suggest that BAP1 is a familial RCC predisposing gene.
PMCID: PMC4211292  PMID: 23709298
renal cell carcinoma; BAP1; cancer; inherited RCC; predisposition; tumor suppressor
5.  PTEN Germline Mutations in Patients Initially Tested for Other Hereditary Cancer Syndromes: Would Use of Risk Assessment Tools Reduce Genetic Testing? 
The Oncologist  2013;18(10):1083-1090.
PTEN Hamartoma Tumor syndrome (PHTS) is a hereditary condition causing increased risk for neoplasia types seen in persons with Hereditary Breast and Ovarian Cancer, Lynch, and Adenomatous Polyposis syndromes. We reviewed our series of patients with PHTS to determine how often testing criteria for these syndromes were met and how often other-gene testing was ordered before testing PTEN.
Learning Objectives
Cite the risk assessment tools available for several hereditary cancer predisposition syndromes.Describe ways in which use of these risk assessment tools can lead to cost savings and decreased time to correct diagnosis.Explain the impact of correct genetic diagnosis on the patient's medical management and on predictive testing for family members.
PTEN Hamartoma Tumor syndrome (PHTS) includes patients with Cowden syndrome or other syndromes with germline mutation of the PTEN tumor suppressor gene. The risk for breast, colorectal, and endometrial cancer and polyposis is increased, creating clinical overlap with hereditary breast and ovarian cancer (HBOC), Lynch syndrome (LS), and adenomatous polyposis syndromes (APS). We reviewed our series of patients with PHTS to determine how often testing criteria for these syndromes were met and how often other-gene testing was ordered before testing PTEN.
Patients and Methods.
Patients were prospectively recruited by relaxed International Cowden Consortium criteria or presence of known germline PTEN mutation. Mutations were identified by mutation scanning/multiplex ligation-dependent probe amplification analysis and confirmed by sequencing/quantitative polymerase chain reaction. Patients were excluded if they were adopted, were <18 years of age, or if they were diagnosed with Cowden syndrome before 1998. Standard risk-assessment models were applied to determine whether patients met HBOC testing criteria, LS-relevant Amsterdam II/Bethesda 2004 criteria, or had adenomatous polyps. Prior probability of PTEN mutation was estimated with the Cleveland Clinic PTEN risk calculator.
Of 137 PTEN mutation-positive adult probands, 59 (43.1%) met testing criteria for HBOC or LS. Of these, 45 (32.8%) were first offered HBOC, LS, or APS testing. Of those who underwent APS testing, none of the six patients met criteria. Initial risk assessment by a genetics specialist was significantly associated with immediate PTEN testing in patients also meeting HBOC testing criteria. Using this PTEN risk assessment tool could have spared gene testing for 22 unlikely syndromes, at a total cost of $66,080.
PHTS is an important differential diagnosis for patients referred for HBOC, LS, or APS. Risk assessment tools may help focus genetic analysis and aid in the interpretation of multiplex testing.
PMCID: PMC3805149  PMID: 24037976
Risk assessment; Genetic testing; PTEN hamartoma tumor syndrome; Cowden syndrome; Hereditary cancer syndromes
6.  Cowden syndrome-associated germline SDHD variants alter PTEN nuclear translocation through SRC-induced PTEN oxidation 
Human Molecular Genetics  2014;24(1):142-153.
Germline mutations in the PTEN tumor-suppressor gene and germline variations in succinate dehydrogenase subunit D gene (SDHD-G12S, SDHD-H50R) are associated with a subset of Cowden syndrome and Cowden syndrome-like individuals (CS/CSL) and confer high risk of breast, thyroid and other cancers. However, very little is known about the underlying crosstalk between SDHD and PTEN in CS-associated thyroid cancer. Here, we show SDHD-G12S and SDHD-H50R lead to impaired PTEN function through alteration of its subcellular localization accompanied by resistance to apoptosis and induction of migration in both papillary and follicular thyroid carcinoma cell lines. Other studies have shown elevated proto-oncogene tyrosine kinase (SRC) activity in invasive thyroid cancer cells; so, we explore bosutinib, a specific inhibitor for SRC, to explore SRC as a mediator of SDH-PTEN crosstalk in this context. We show that SRC inhibition could rescue SDHD dysfunction-induced cellular phenotype and tumorigenesis only when wild-type PTEN is expressed, in thyroid cancer lines. Patient lymphoblast cells carrying either SDHD-G12S or SDHD-H50R also show increased nuclear PTEN and more oxidized PTEN after hydrogen peroxide treatment. Like in thyroid cells, bosutinib decreases oxidative PTEN in patient lymphoblast cells carrying SDHD variants, but not in patients carrying both SDHD variants and PTEN truncating mutations. In summary, our data suggest a novel mechanism whereby SDHD germline variants SDHD-G12S or SDHD-H50R induce thyroid tumorigenesis mediated by PTEN accumulation in the nucleus and may shed light on potential treatment with SRC inhibitors like bosutinib in PTEN-wild-type SDHD-variant/mutation positive CS/CSL patients and sporadic thyroid neoplasias.
PMCID: PMC4262496  PMID: 25149476
7.  Germline and somatic KLLN alterations in breast cancer dysregulate G2 arrest 
Human Molecular Genetics  2013;22(12):2451-2461.
PTEN is a well-described predisposition gene for Cowden syndrome (CS), a familial cancer syndrome characterized by a high risk of breast and other cancers. KLLN, which shares a bidirectional promoter with PTEN, causes cell cycle arrest and apoptosis. We previously identified germline hypermethylation of the KLLN promoter in 37% of PTEN mutation-negative CS/CS-like (CSL) patients. Patients with germline KLLN hypermethylation have an increased prevalence of breast and renal cancers when compared with PTEN mutation carriers. We have consequently sought to identify and characterize germline KLLN variants/mutations in CS/CSL and in apparently sporadic breast cancer patients. KLLN variants in CS/CSL patients are rare (1 of 136, 0.007%). Interestingly, among 438 breast cancer patients, 13 (3%) have germline KLLN variants when compared with none in 128 controls (P = 0.049). Patients with KLLN variants have a family history of breast cancer when compared with those without (P = 0.02). We demonstrate that germline KLLN variants dysregulate the cell cycle at G2. Of 24 breast carcinomas analyzed, 3 (13%) have somatic KLLN hemizygous deletions, with somatic loss of the wild-type allele in a patient with germline KLLN p.Leu119Leu. Of 452 breast carcinomas in The Cancer Genome Atlas project, 93 (21%) have KLLN hemizygous or homozygous deletions. This is the first study to associate germline KLLN variants with sporadic breast cancer and to recognize somatic KLLN deletions in breast carcinomas. Our observations suggest that KLLN may be a low penetrance susceptibility factor for apparently sporadic breast cancer.
PMCID: PMC3858115  PMID: 23446638
8.  Prevalence of Germline PTEN, BMPR1A, SMAD4, STK11, and ENG Mutations in Patients with Moderate-Load Colorectal Polyps 
Gastroenterology  2013;144(7):1402-1409.e5.
Gastrointestinal polyposis is a common clinical problem, yet there is no consensus on how to best manage patients with moderate-load polyposis. Identifying genetic features of this disorder could improve management, and especially surveillance, of these patients. We sought to determine the prevalence of hamartomatous polyposis associated mutations in the susceptibility genes PTEN, BMPR1A, SMAD4, ENG, and STK11 in individuals with 5 or more gastrointestinal polyps, including at least 1 hamartomatous or hyperplastic/serrated polyp.
We performed a prospective, referral-based study of 603 patients (median age 51 y; range, 2–89 y), enrolled from June 2006 through January 2012. Genomic DNA was extracted from peripheral lymphocytes and analyzed for specific mutations and large rearrangements in PTEN, BMPR1A, SMAD4, and STK11, as well as mutations in ENG. Recursive partitioning analysis was used to determine cutoffs for continuous variables. The prevalence of mutations was compared using Fischer’s exact test. Logistic regression analyses were used to determine univariate and multivariate risk factors.
Of 603 patients, 119 (20%) had a personal history of colorectal cancer and most (461; 76%) had fewer than 30 polyps. Seventy-seven patients (13%) were found to have polyposis-associated mutations, comprising 11 in ENG (1.8%), 13 in PTEN (2.2%), 13 in STK11 (2.2%), 20 in BMPR1A (3.3%), and 21 in SMAD4 (3.5%). Univariate clinical predictors for risk of having these mutations included age at presentation less than 40 years (19% vs 10%; P=.008), a polyp burden of 30 or more (19% vs 11%; P=0.014), and male sex (16% vs 10%; P=.03). Patients who had 1 or more ganglioneuromas (29% vs 2%; P<.001) or presented with polyps of 3 or more histologic types (20% vs 2%; P=.003) were more likely to have germline mutations in PTEN.
Age less than 40 years, male sex, and specific polyp histologies are significantly associated with risk of germline mutations in hamartomatous-polyposis associated genes. These associations could guide clinical decision making and further investigations.
PMCID: PMC3969031  PMID: 23399955
Hamartomatous polyposis; Juvenile Polyposis Syndrome; Peutz-Jeghers syndrome; Cowden syndrome
9.  Formative Evaluation of Clinician Experience with Integrating Family History-Based Clinical Decision Support into Clinical Practice 
Journal of Personalized Medicine  2014;4(2):115-136.
Family health history is a leading predictor of disease risk. Nonetheless, it is underutilized to guide care and, therefore, is ripe for health information technology intervention. To fill the family health history practice gap, Cleveland Clinic has developed a family health history collection and clinical decision support tool, MyFamily. This report describes the impact and process of implementing MyFamily into primary care, cancer survivorship and cancer genetics clinics. Ten providers participated in semi-structured interviews that were analyzed to identify opportunities for process improvement. Participants universally noted positive effects on patient care, including increases in quality, personalization of care and patient engagement. The impact on clinical workflow varied by practice setting, with differences observed in the ease of integration and the use of specific report elements. Tension between the length of the report and desired detail was appreciated. Barriers and facilitators to the process of implementation were noted, dominated by the theme of increased integration with the electronic medical record. These results fed real-time improvement cycles to reinforce clinician use. This model will be applied in future institutional efforts to integrate clinical genomic applications into practice and may be useful for other institutions considering the implementation of tools for personalizing medical management.
PMCID: PMC4263968  PMID: 25563219
family health history; clinical decision support; implementation; risk stratification; barriers; facilitators
10.  Specific kinesin expression profiles associated with taxane resistance in breast cancer 
Breast cancer is a genetically heterogenous disease with subtypes differing in prognosis and chemosensitivity. The basal-like breast cancer (BLBC) molecular subtype is associated with poorer outcomes, but is more responsive to taxane-based chemotherapy, which is used in multiple settings of breast cancer. Kinesins are intracellular transport proteins that interact with microtubules, which are also the mechanistic target for taxanes. We investigated the relationship between taxane resistance in BLBC and kinesins using both expression and functional studies.
Kinesin (KIF) expression was evaluated in three settings in relation to taxane resistance: (i) the NCI-60 cancer cell lines, (ii) pre-treatment samples from four BLBC patient cohorts receiving neoadjuvant chemotherapy regimens with and without taxanes, and (iii) post-treatment samples from residual breast cancer following neoadjuvant taxane-containing chemotherapy. We used a novel functional approach to gene modification, validation-based insertional mutagenesis, to select kinesin-overexpressing clones of BLBC cells for evaluation of related mechanisms of taxane resistance.
In the NCI-60 cell line dataset, overexpression of the kinesin KIFC3 is significantly correlated with resistance to both docetaxel (p<0.001) and paclitaxel (p<0.001), but not to platinum-based chemotherapy, including carboplatin (p=0.49) and cisplatin (p=0.10). Overexpression of KIFC3 and KIF5A in pre-chemotherapy samples similarly predicted resistance to paclitaxel in the MDACC cohorts (p=0.01); no KIF predicted resistance to fluorouracil-epirubicin-cyclophosphamide or cisplatin in BLBC patient cohorts treated without taxanes. KIF12 is the most overexpressed KIF gene in post-chemotherapy taxane-resistant residual breast cancers (2.8 fold-change). Functional studies established that overexpression of KIFC3, KIF5A and KIF12 were specific in mediating resistance to docetaxel and not vincristine or doxorubicin. We demonstrated that mutation of the ATP-binding domain of a kinesin abolishes its ability to mediate docetaxel resistance.
We show that kinesin overexpression correlates with specific taxane resistance in BLBC cell lines and tissue. Our results suggest a novel approach to overcoming taxane resistance in breast cancer through concurrent or sequential use of kinesin inhibitors, highlighting the ATP-binding domain as a drug development target.
PMCID: PMC4038085  PMID: 21479552
11.  Cowden syndrome-related mutations in PTEN associate with enhanced proteasome activity 
Cancer research  2013;73(10):3029-3040.
Germline mutations in PTEN have been described in a spectrum of syndromes that are collectively known as PTEN hamartoma tumor syndrome (PHTS). In addition to being mutated in the germline in PHTS, somatic loss-of-function PTEN mutations are seen in a wide range of sporadic human tumors. Here, we show evidence of upregulated proteasome activity in PHTS-derived lymphoblasts, Pten knock-in mice and cell lines expressing missense and nonsense PTEN mutations. Notably, elevated nuclear proteasome activity occurred in cells expressing the nuclear mislocalized PTEN-K62R mutant, whereas elevated cytosolic proteasome activity was observed in cells expressing the cytosolic-predominant mutant PTEN (M3M4 and C136R). Treatment with proteasome inhibitor MG-132 was able to restore both nonsense and missense mutant PTEN protein levels in vitro. PHTS patients with destabilizing PTEN mutations and proteasome hyperactivity are more susceptible to develop neurological symptoms such as mental retardation and autism than mutation-positive patients with normal proteasome activity. A detailed molecular and functional analysis shows that PTEN mutants most likely cause proteasome hyperactivity via two different mechanisms, namely, induction of proteotoxic stress and loss of protein phosphatase activity. These results provide novel insights into the cellular functions of PTEN and reveal molecular mechanisms whereby PTEN mutations increase proteasome activity and lead to neurological phenotypes.
PMCID: PMC3655114  PMID: 23475934
Cowden syndrome; PHTS; PTEN instability; localization; germline mutation
12.  Elevated Plasma Succinate Among PTEN, SDHB and SDHD Mutation Positive Individuals 
Cowden Syndrome (CS) results from germline mutations in phosphatase and tensin homologue deleted on chromosome ten (PTEN) and from variants in succinate dehydrogenase (SDH) B and D subunits. We hypothesized that succinate accumulation may be common among individuals with SDH variants/mutations and those with PTEN mutations.
Urine and blood were collected from individuals meeting full or partial CS diagnostic criteria, those with paraganglioma or a known susceptibility PGL-associated gene mutation and succinate was measured. PTEN, SDHB, SDHC, and SDHD-genes were sequenced from genomic DNA.
Elevated plasma succinate was observed in 13/21 (62%) individuals with germline PTEN, SDHB or SDHD mutations compared to 5/32 (16%) controls (p<0.001); 10/15 (67%) individuals with pathogenic PTEN mutations, but in <20% of mutation negative individuals meeting identical criteria; and among individuals with mutations in SDHB (1/1, 100%) and SDHD (2/5, 40%).
Our data suggest that mutations in PTEN, SDHB and SDHD reduce catalytic activity of SDH and result in succinate accumulation and identify a common biochemical alteration between these two patient populations. Plasma organic acid analysis may provide an effective and inexpensive screening method to determine when more expensive gene sequencing of PTEN and SDH-genes is warranted.
PMCID: PMC4019996  PMID: 22261759
PTEN; SDH; Succinate; Cowden Syndrome
13.  Hypomethylation of Noncoding DNA Regions and Overexpression of the Long Noncoding RNA, AFAP1-AS1, in Barrett’s Esophagus and Esophageal Adenocarcinoma 
Gastroenterology  2013;144(5):956-966.e4.
Alterations in methylation of protein-coding genes are associated with Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC). Dys-regulation of noncoding RNAs occurs during carcinogen-esis but has never been studied in BE or EAC. We applied high-resolution methylome analysis to identify changes at genomic regions that encode noncoding RNAs in BE and EAC.
We analyzed methylation of 1.8 million CpG sites using massively parallel sequencing-based HELP tagging in matched EAC, BE, and normal esophageal tissues. We also analyzed human EAC (OE33, SKGT4, and FLO-1) and normal (HEEpic) esophageal cells.
BE and EAC exhibited genome-wide hypomethylation, significantly affecting intragenic and repetitive genomic elements as well as noncoding regions. These methylation changes targeted small and long noncoding regions, discriminating normal from matched BE or EAC tissues. One long noncoding RNA, AFAP1-AS1, was extremely hypomethylated and overexpressed in BE and EAC tissues and EAC cells. Its silencing by small interfering RNA inhibited proliferation and colony-forming ability, induced apoptosis, and reduced EAC cell migration and invasion without altering the expression of its protein-coding counterpart, AFAP1.
BE and EAC exhibit reduced methylation that includes noncoding regions. Methylation of the long noncoding RNA AFAP1-AS1 is reduced in BE and EAC, and its expression inhibits cancer-related biologic functions of EAC cells.
PMCID: PMC3739703  PMID: 23333711
Esophageal Cancer Progression; Tumor Development; Gene Regulation; Noncoding RNA
14.  The Cognitive Characteristics of PTEN Hamartoma Tumor Syndromes 
To characterize cognition in individuals with germline PTEN mutations (N=23) as well as in PTEN mutation-negative individuals with classic Cowden Syndrome or Bannayan-Riley-Ruvalcaba Syndrome (N=2).
Twenty-five individuals completed a comprehensive neuropsychological evaluation. One sample t-tests and effect sizes were used to examine differences in participant test scores compared with normal controls. Composite scores were created for each patient within each of the cognitive domains assessed and classified as above average, average, or below average according to normative standards. Chi-square analyses compared these classifications to expected proportions in normal control samples.
The mean IQ was in the average range, and there was a wide range of intellectual functioning (extremely low to very superior). However, scores were lower than expected on measures of motor functioning, executive functioning, and memory recall suggesting a pattern of frontal lobe dysfunction in a large subset of participants.
This is the first study to characterize cognition in individuals with PTEN mutations and associated syndromes using a comprehensive neuropsychological battery. Contrary to previous association with intellectual disability, mean IQ was average, and there was a broad range of intellectual abilities. Specific evidence of frontal lobe dysfunction may have implications for treatment compliance and cancer surveillance and warrants further investigation.
PMCID: PMC3956109  PMID: 23470840
PTEN; PTEN Hamartoma Tumor Syndromes; Cowden Syndrome; Bannayan-Riley-Ruvalcaba Syndrome; Cognition; Neuropsychology
15.  Evil lurks in the heart of man: cardiac paraganglioma presenting as recurrent dyspnoea and chronic cough 
BMJ Case Reports  2011;2011:bcr1120115170.
A man in his 60s presents with chronic dyspnoea and cough for 3 years. EKG and nuclear stress test were not diagnostic. An echocardiogram revealed moderate pericardial effusion. His symptoms improved with ibuprofen temporarily and a repeat echocardiogram showed resolution of the effusion. However, when his symptoms recurred, re-imaging showed a large intracardiac tumour causing right ventricular outflow obstruction. Subsequent histological examination revealed metastatic paraganglioma. He was found to carry a germline mutation in the SDHB gene which is associated with higher malignant risk. Knowledge of his underlying mutation allowed the patient and his family to receive appropriate gene-specific counselling and surveillance.
PMCID: PMC3246175  PMID: 22670000
16.  Vitamin E Protects against Lipid Peroxidation and Rescues Tumorigenic Phenotypes in Cowden/Cowden-like Patient-derived Lymphoblast Cells with Germline SDHx Variants 
Cowden syndrome (CS), a Mendelian autosomal-dominant disorder, predisposes to breast, thyroid, and other cancers. Germline variations in succinate dehydrogenase genes (SDHx) occur in ~10% PTEN mutation-negative CS and CS-like (CSL) individuals (SDHvar+). We previously showed that SDHx variants result in elevated reactive oxygen species (ROS), disruption of nicotinamide adenine dinucleotide (NAD) equilibrium, and destabilization of p53 hence apoptosis resistance in CS/CSL patient-derived lymphoblastoid cells. In the present study, we sought to address the tumorigenic impacts of increased ROS and the potential of protecting SDHvar+ cells with antioxidants.
Experimental Design
We measured the lipid peroxidation levels in patient-derived SDHvar+ lymphoblastoid cells and sequenced 74 controls or SDHvar+ germline DNA samples for mitochondrial hypervariable region II (HVRII) polymorphisms. SDHvar+ lymphoblastoid cells were treated with various antioxidants to check p53 expression and SubG1 cell population with cell cycle analysis.
We demonstrated that elevated ROS results in higher lipid peroxidation in SDHvar+ cells. Accumulation of polymorphisms in mitochondrial HVRII were observed in SDHvar+ samples. Interestingly, α-tocopherol (vitamin E) treatment, but not other antioxidants, rescued SDHvar+ cells from apoptosis resistance and protected SDHvar+ cells from oxidative damage such as decreased lipid peroxidation as well as partially recovered p53 expression and NAD/NADH levels.
We conclude that disruption of complex II due to SDHx variants leads to increased ROS generation, specifically accompanied by lipid peroxidation. The lipid soluble antioxidant α-tocopherol can selectively protect SDHxvar+ cells from oxidative damage, apoptosis resistance, and rebalance redox metabolites NAD/NADH.
PMCID: PMC3445717  PMID: 22829200
SDH; CS/CSL; α-tocopherol; ROS; mitochondria
17.  Characterization of the colorectal cancer–associated enhancer MYC-335 at 8q24: the role of rs67491583 
Cancer genetics  2012;205(0):25-33.
Recent genome-wide association studies have identified multiple regions at 8q24 that confer susceptibility to many cancers. In our previous work, we showed that the colorectal cancer (CRC) risk variant rs6983267 at 8q24 resides within a TCF4 binding site at the MYC-335 enhancer, with the risk allele G having a stronger binding capacity and Wnt responsiveness. Here, we searched for other potential functional variants within MYC-335. Genetic variation within MYC-335 was determined in samples from individuals of European, African, and Asian descent, with emphasis on variants in putative transcription factor binding sites. A 2-bp GA deletion rs67491583 was found to affect a growth factor independent (GFI) binding site and was present only in individuals with African ancestry. Chromatin immunoprecipitation performed in heterozygous cells showed that the GA deletion had an ability to reduce binding of the transcriptional repressors GFI1 and GFI1b. Screening of 1,027 African American colorectal cancer cases and 1,773 healthy controls did not reveal evidence for association (odds ratio: 1.17, 95% confidence interval: 0.97–1.41, P = 0.095). In this study, rs67491583 was identified as another functional variant in the CRC-associated enhancer MYC-335, but further studies are needed to establish the role of rs67491583 in the colorectal cancer predisposition of African Americans.
PMCID: PMC3770308  PMID: 22429595
Enhancer; transcription factor; susceptibility variant; colorectal cancer; association
18.  Estimate of de novo mutation frequency in probands with PTEN Hamartoma Tumor syndrome 
PTEN Hamartoma Tumor Syndrome (PHTS) is an autosomal dominant disorder with increased risks of neoplasias, macrocephaly, and developmental disabilities. While both familial and sporadic cases exist, actual de novo mutation frequency remains unknown. We sought to estimate this within our PTEN-mutation positive patient series.
Patients were prospectively accrued if they had known pathogenic germline PTEN mutations or phenotypic features suspicious for PHTS. Only families with pathogenic PTEN mutations were included. Likelihood for de novo mutation was graded from 1 (confirmed inherited) to 5 (confirmed de-novo) based on family history and mutation-status. Fisher’s 2-tailed exact and unpaired t-tests were used to compare between groups.
187 pathogenic PTEN-mutation positive families were eligible for this study. De novo (grade 5) status was confirmed in 20 (10.7%) probands, and in 36 (19.3%) was suspected based on family history. Demographics, mutations, and phenotypes were similar for probands graded 1 versus 5 (all p>0.06). In grade 1 probands, mutations were inherited equally from maternal and paternal lineages (p=0.55).
The frequency of de novo PTEN mutation is minimally 10.7% and maximally 47.6%. Absence of PHTS features within a family history should not preclude consideration of this diagnosis for patients with relevant personal history.
PMCID: PMC3651836  PMID: 22595938
Germline PTEN mutation; Cowden syndrome; Bannayan-Riley-Ruvalcaba syndrome; inherited cancer syndrome; de novo mutation
19.  Oophorectomy after Menopause and the Risk of Breast Cancer in BRCA1 and BRCA2 Mutation Carriers 
To evaluate the effect of the cumulative number of ovulatory cycles and its contributing components on the risk of breast cancer among BRCA mutation carriers.
We conducted a matched case-control study on 2,854 pairs of women with a BRCA1 or BRCA2 mutation. Conditional logistic regression was used to estimate the association between the number of ovulatory cycles and various exposures and the risk of breast cancer. Information from a subset of these women enrolled in a prospective cohort study was used to calculate age-specific breast cancer rates.
The annual risk of breast cancer decreased with the number of ovulatory cycles experienced (ρ = −0.69; P = 0.03). Age at menarche and duration of breastfeeding were inversely related with risk of breast cancer among BRCA1 (P-trend < 0.0001) but not among BRCA2 (P-trend ≥ 0.28) mutation carriers. The reduction in breast cancer risk associated with surgical menopause (OR = 0.52; 95%CI 0.40–0.66; P-trend < 0.0001) was greater than that associated with natural menopause (OR = 0.81; 95%CI 0.62–1.07; P-trend = 0.14). There was a highly significant reduction in breast cancer risk among women who had an oophorectomy after natural menopause (OR = 0.13; 95%CI 0.02–0.54; P = 0.006).
These data challenge the hypothesis that breast cancer risk can be predicted by the lifetime number of ovulatory cycles in women with a BRCA mutation. Both pre- and post-menopausal oophorectomy protect against breast cancer.
Understanding the basis for the protective effect of oophorectomy has important implications for chemoprevention.
PMCID: PMC3593267  PMID: 22564871
BRCA1; ovulatory cycles; breast cancer; oophorectomy
20.  Hunting for cancer in the microbial jungle 
Genome Medicine  2013;5(5):42.
PMCID: PMC3707053  PMID: 23731547
21.  Germline Epigenetic Regulation of KILLIN in Cowden and Cowden-Like Syndromes 
Germline loss-of-function PTEN mutations cause 80% of Cowden syndrome (CS), an autosomal dominant disorder characterized by high risks of breast, thyroid, and other cancers. A large heterogenous group of CS-like (CSL) individuals, who have various combinations of CS features but who do not meet CS diagnostic criteria, have PTEN mutations <10% of the time, making molecular-diagnosis, prediction, genetic-counseling and risk-management challenging. Other mechanisms of loss-of-function such as hypermethylation, which should result in under-expression of PTEN, or of KILLIN, a novel tumor suppressor transcribed in the opposite direction, may account for the remainder of CS/CSL individuals. Screening for such promoter methylation, may improve the sensitivity of molecular diagnosis.
To determine whether germline methylation is found in CS/CSL individuals lacking germline PTEN mutations.
Nucleic-acids from prospective nested series of 123 CS/CSL patients and 50 unaffected individuals without PTEN germline variants analyzed for germline methylation and expression of PTEN and KILLIN (Cleveland Clinic, 2008/8–2010/6). Prevalence of component cancers between groups was compared with Fisher’s exact-test.
Main outcome measures
Frequency of germline methylation in PTEN mutation negative CS/CS-like individuals. Prevalence of component cancers in methylation-positive and PTEN mutation-positive patients.
Of 123 CS/CS-like patients, 45 (37%, 95%CI 29–45%) showed hypermethylation upstream of PTEN but no transcriptional repression. The germline methylation was found to transcriptionally downregulate KILLIN 250-fold (95%CI 45–14286, P=0.007); and exclusively disrupted p53-activation of KILLIN by 30% (95%CI 7–45% (P=0.008). Demethylation treatment increased only KILLIN expression 4.88-fold (95%CI 1.4–18.1, P<0.05). Individuals with KILLIN-promoter methylation had 3-fold increased prevalence of breast cancer (35/42 vs 24/64, P<0.0001) and a >2-fold increase of kidney cancer (4/45 vs 6/155, P=0.004) over those with germline PTEN mutations.
Germline KILLIN- methylation is common among CS/CSL patients, and associated with increased risks of breast and renal cancer over PTEN mutation-positive patients. These observations need to be replicated.
PMCID: PMC3655326  PMID: 21177507
Cowden syndrome; PTEN; DNA methylation; p53; bidirectional promoter
22.  Frequent gastrointestinal polyps and colorectal adenocarcinomas in prospective series of PTEN mutation carriers 
Gastroenterology  2010;139(6):1927-1933.
Germline PTEN mutations cause Cowden syndrome (CS), associated with breast and thyroid cancers. Case reports found 35–85% of CS patients had gastrointestinal (GI) hamartomas. The association of benign and malignant GI neoplasias with CS remains debatable. Our goal is to describe the GI phenotype in a prospective series of PTEN mutation carriers.
Patients who met relaxed International Cowden Consortium criteria (N=2548) or with ≥5 GI polyps, ≥1 of which was hyperplastic or hamartomatous (N=397) were prospectively recruited. Germline PTEN mutation/deletion analysis was performed. Of the 2945, 127 patients having clear pathogenic PTEN mutations (123/2548+4/397) were eligible for this study. EGD and colonoscopy were performed and pathology reports reviewed. Fisher’s 2-tailed exact test, unpaired t-tests, and age- and gender-adjusted SIR were calculated.
Of 127 PTEN mutation carriers, 67 underwent ≥1 endoscopy with 62 (95%) having polyps, making GI polyps the second most common feature, after macrocephaly (74.8%). Of the 65, half had hyperplastic polyps and ¼ each with hamartomatous, ganglioneuromatous or adenomatous polyps. There were one to “innumerable” polyps in the colorectum, ileum, duodenum, stomach and/or esophagus, with 24 subjects having both upper and lower GI polyps. Nine (13%) subjects had colorectal cancer, all under the age of 50. The adjusted SIR was 224.1 (95%CI 109.3–411.3, p<0.0001). Cancers were commonly associated with adenomatous and/or hyperplastic polyps. One had gastric signet ring cell carcinoma.
PTEN-associated CS should be considered a mixed polyp syndrome, with hyperplastic polyps most prevalent, and a risk of early-onset colorectal cancer. Routine colonoscopy should be considered in PTEN-associated CS especially in the context of hyperplastic and/or adenomatous polyps.
PMCID: PMC3652614  PMID: 20600018
colorectal cancer; Cowden syndrome; hamartomatous polyposis; PTEN
23.  Prospective comparison of family medical history with personal genome screening for risk assessment of common cancers 
Family history-based risk assessment (FHRA) is a genetic tool for identifying those at risk of disease. Genome-wide association studies have shown that single nucleotide polymorphisms (SNP) are statistically associated with low- to moderate-level risks of diseases. There has been limited study of complementarity for these two assessment methods. We sought to compare cancer risk categorizations from FHRA and from Navigenics Personal Genome Screening (PGS). We compared FHRA with PGS for breast (22 females), prostate (22 males), and colon cancer (44 males and females) assessed by kappa (κ) statistic. We also assessed each participant's hereditary risk based on clinical criteria and/or gene-test results. Both FHRA and PGS placed 59%, 68% and 44% of participants into the same risk categories for breast, prostate, and colon cancer, respectively. Overall, however, there was little concordance in FHRA versus PGS for all three cancer risks (κ<0.2). FHRA assigned 22 with hereditary risk compared with PGS, which identified one as high risk (P<0.0001). We assessed nine with hereditary colorectal cancer risk, five with germline mutations, but none were classified as PGS high risk (P=0.0001). FHRA and PGS may be complementary tools for cancer risk assessment. However, evaluation of family history remains the standard to evaluate an individual's cancer risk until further research.
PMCID: PMC3330209  PMID: 22215421
breast cancer; colon cancer; Navigenics; prostate cancer; risk assessment
24.  Papillary Renal Cell Carcinoma is Associated with PTEN Hamartoma Tumor Syndrome 
Urology  2012;79(5):1187.e1-1187.e7.
To formally study the prevalence and histological classification of renal cell carcinoma (RCC) in a series of patients with PTEN Hamartoma Tumor syndrome (PHTS).
We evaluated prevalence of RCC within a prospectively-accrued series of 219 patients found to have pathogenic germline PTEN mutations. Clinical data including pathology reports were requested for all participants. Slides and tumor blocks were requested for central pathology re-review and immunohistochemistry (IHC) analysis.
Nine patients were identified with RCC. Based on SEER data 0.28 RCC cases were expected for the group, giving an overall age-adjusted Standardized Incidence Ratio (SIR) of 31.7 (95% CI 15.4–58.1, p<0.001) with a higher sex-adjusted SIR for females (46.7 vs. 21.6 for males). Reported histology of each mutation positive patient’s RCC was variable. However, on central pathology re-review of 8 patients, six examined lesions were determined to be of papillary subhistology (pRCC), with the other two patients’ tumors consistent with the initial report of chromophobe RCC (chRCC). IHC demonstrated complete loss of PTEN protein in all PTEN mutation positive patients’ pRCCs and patchy positivity in one chRCC.
PHTS is a hereditary syndrome newly associated with pRCC, and PTEN IHC may be a helpful screening tool to identify pRCC patients with PHTS. Physicians caring for PHTS patients should note the >31-fold increased risk for RCC and have a low threshold for investigating possible RCC in patients with relevant complaints. Renal ultrasound is not sensitive for detecting pRCC and so PHTS patients should have alternate renal imaging (CT or MRI).
PMCID: PMC3341468  PMID: 22381246
PTEN germline mutations; Cowden syndrome; Bannayan-Riley-Ruvalcaba syndrome; Carcinoma, Kidney
25.  PTEN Hamartoma of Soft Tissue: A Distinctive Lesion in PTEN Syndromes 
PTEN hamartoma tumor syndrome (PHTS) presents in a spectrum that encompasses the epononymous disorders, Cowden’s and Bannayan-Riley-Ruvalcaba. Herein, we delineate the distinctive histopathology of a predominantly intramuscular lesion in PHTS, often called “arteriovenous malformation” because of certain imaging and histopathologic features. Cases were identified by review of lesions resected from patients with PHTS registered in our Vascular Anomalies Center and of unusual intramuscular vascular anomalies in our pathology database from 1985 to 2008. Thirty-four patients with this lesion were identified: 20 had a clinical diagnosis of, or were suspected to have, PHTS (genetically confirmed in 16). In 4 patients without clinical manifestations of PHTS, 2 had PTEN mutations, 1 did not, and in another the mutation was intronic. In the remaining 10, there was insufficient clinical information to fully assess whether they had manifestations of PHTS. Lesions manifested by 15 years of age, normally with pain and swelling, most often located in the lower extremity. The major mass was usually intramuscular, but often there were fascial and subcutaneous components and not infrequently a cutaneous vascular stain. MRI generally showed an infiltrative soft tissue lesion involving muscle, fascia and subcutis with frequent enlarged, serpiginous vessels and a prominent adipocytic component. Some lesions involved contiguous muscles and 20% were multifocal. Resected specimens ranged in size from 3–25 cm; in one patient, amputation was necessary.
Histopathologically, these unencapsulated masses, oftentimes with a nodular appearance at scanning magnification, consisted of: 1) variable admixture of mature adipocytic and dense and/or myxoid fibrous tissues (50–90% of surface area); 2) vascular component (10–50% of surface area) with: a) clusters of venous channels, some with excessively and irregularly muscularized complex walls and lumens, and others with thin walls resembling pulmonary alveoli, b) tortuous, thick-walled arteries with concentric muscular hyperplasia and relatively small lumens, c) numerous small vessels (arteries, veins and indeterminate channels), and d) occasional arteriovenous communications; 3) lymphoid follicles (50%); 4) foci of bone (20%); 5) hypertrophic nerves with “onion bulb” proliferation of periaxonal spindled cells (9%).
We designate this disorganized overgrowth of essentially mesenchymal elements, PTEN hamartoma of soft tissue (PHOST). It differs from other vascular and connective tissue lesions that occur in patients with PHTS. PHOST is histopathologically distinctive and its identification should prompt a thorough investigation for PHTS.
PMCID: PMC3367317  PMID: 22446940

Results 1-25 (65)