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1.  Comparing Universal Lynch Syndrome Tumor Screening Programs to Evaluate Associations Between Implementation Strategies and Patient Follow-through 
Universal tumor screening (UTS) for all colorectal cancer (CRC) patients can improve the identification of Lynch syndrome, the most common cause of hereditary CRC. This multiple-case study explored how variability in UTS procedures influence patient follow-through (PF) with germline testing after a screen-positive result.
Data were obtained through web-based surveys and telephone interviews with institutional informants. Institutions were categorized as Low-PF (≤10% underwent germline testing), Medium-PF (11–40%), or High-PF (>40%). To identify implementation procedures (i.e., conditions) unique High-PF institutions, qualitative comparative analysis was performed.
Twenty-one informants from fifteen institutions completed surveys and/or interviews. Conditions present among all five High-PF institutions included: 1) disclosure of screen-positive results to patients by genetic counselors (GCs); and 2) GCs either facilitate physician referrals to genetics or eliminated the need for referrals. Although both of these High-PF conditions were present among two Medium-PF institutions, automatic reflex testing was lacking and difficulty contacting screen-positive patients was a barrier. The three remaining Medium-PF and five Low-PF institutions lacked High-PF conditions.
Methods for streamlining UTS procedures, incorporating a high level of involvement of GCs in results tracking and communication, and reducing barriers to patient contact are reviewed within a broader discussion on maximizing the effectiveness and public health impact of UTS.
PMCID: PMC4169758  PMID: 24651603
Qualitative comparative analysis; RE-AIM; hereditary colorectal cancer; effectiveness; Public Health Genomics; Lynch syndrome
2.  Use of Whole Genome Sequencing for Diagnosis and Discovery in the Cancer Genetics Clinic 
EBioMedicine  2015;2(1):74-81.
Despite the potential of whole-genome sequencing (WGS) to improve patient diagnosis and care, the empirical value of WGS in the cancer genetics clinic is unknown. We performed WGS on members of two cohorts of cancer genetics patients: those with BRCA1/2 mutations (n = 176) and those without (n = 82). Initial analysis of potentially pathogenic variants (PPVs, defined as nonsynonymous variants with allele frequency < 1% in ESP6500) in 163 clinically-relevant genes suggested that WGS will provide useful clinical results. This is despite the fact that a majority of PPVs were novel missense variants likely to be classified as variants of unknown significance (VUS). Furthermore, previously reported pathogenic missense variants did not always associate with their predicted diseases in our patients. This suggests that the clinical use of WGS will require large-scale efforts to consolidate WGS and patient data to improve accuracy of interpretation of rare variants. While loss-of-function (LoF) variants represented only a small fraction of PPVs, WGS identified additional cancer risk LoF PPVs in patients with known BRCA1/2 mutations and led to cancer risk diagnoses in 21% of non-BRCA cancer genetics patients after expanding our analysis to 3209 ClinVar genes. These data illustrate how WGS can be used to improve our ability to discover patients’ cancer genetic risks.
PMCID: PMC4444225  PMID: 26023681
Cancer genetics; BRCA1/2; Whole-genome sequence; ClinVar; Pathogenic variants; Single nucleotide variant
3.  How do we approach the goal of identifying everybody with Lynch Syndrome? 
Familial cancer  2013;12(2):313-317.
PMCID: PMC4337020  PMID: 23568035
4.  Mismatch Repair Analysis of Inherited MSH2 and/or MSH6 Variation Pairs Found in Cancer Patients 
Human mutation  2012;33(8):1294-1301.
Mismatch repair (MMR) malfunction causes the accumulation of mismatches in the genome leading to genomic instability and cancer. The inactivation of an MMR gene (MSH2, MSH6, MLH1, or PMS2) with an inherited mutation causes Lynch syndrome (LS), a dominant susceptibility to cancer. MMR gene variants of uncertain significance (VUS) may be pathogenic mutations, which cause LS, may result in moderately increased cancer risks, or may be harmless polymorphisms. Our study suggests that an inherited MMR VUS individually assessed as proficient may, however, in a pair with another MMR VUS found in the same colorectal cancer (CRC) patient have a concomitant contribution to the MMR deficiency. Here, eight pairs of MMR gene variants found in cancer patients were functionally analyzed in an in vitro MMR assay. Although the other pairs do not suggest a compound deficiency, the MSH2 VUS pair c.380A>G/c.982G>C (p.Asn127Ser/p.Ala328Pro), which nearly halves the repair capability of the wild-type MSH2 protein, is presumed to increase the cancer risk considerably. Moreover, two MSH6 variants, c.1304T>C (p.Leu435Pro) and c.1754T>C (p.Leu585Pro), were shown to be MMR deficient. The role of one of the most frequently reported MMR gene VUS, MSH2 c.380A>G (p.Asn127Ser), is especially interesting because its concomitant defect with another variant could finally explain its recurrent occurrence in CRC patients.
PMCID: PMC4273566  PMID: 22581703
functional analysis; Lynch syndrome; mismatch repair; MSH2; MSH6; VUS
5.  Use of Whole Genome Sequencing for Diagnosis and Discovery in the Cancer Genetics Clinic 
EBioMedicine  2014;2(1):74-81.
Despite the potential of whole-genome sequencing (WGS) to improve patient diagnosis and care, the empirical value of WGS in the cancer genetics clinic is unknown. We performed WGS on members of two cohorts of cancer genetics patients: those with BRCA1/2 mutations (n = 176) and those without (n = 82). Initial analysis of potentially pathogenic variants (PPVs, defined as nonsynonymous variants with allele frequency < 1% in ESP6500) in 163 clinically-relevant genes suggested that WGS will provide useful clinical results. This is despite the fact that a majority of PPVs were novel missense variants likely to be classified as variants of unknown significance (VUS). Furthermore, previously reported pathogenic missense variants did not always associate with their predicted diseases in our patients. This suggests that the clinical use of WGS will require large-scale efforts to consolidate WGS and patient data to improve accuracy of interpretation of rare variants. While loss-of-function (LoF) variants represented only a small fraction of PPVs, WGS identified additional cancer risk LoF PPVs in patients with known BRCA1/2 mutations and led to cancer risk diagnoses in 21% of non-BRCA cancer genetics patients after expanding our analysis to 3209 ClinVar genes. These data illustrate how WGS can be used to improve our ability to discover patients' cancer genetic risks.
•We used WGS to analyze the germline variations of 258 cancer genetics patients.•To interpret variants, BRCA mutation carrier genomes were used as controls for patients that did not have BRCA mutations.•The numbers of genetic diagnoses were increased when we expanded our focus to all genes annotated in the ClinVar database.•This study investigates the pitfalls and the potential for diagnosis and discovery using whole-genome germline sequencing.
PMCID: PMC4444225  PMID: 26023681
Cancer genetics; BRCA1/2; Whole-genome sequence; ClinVar; Pathogenic variants; Single nucleotide variant
6.  Immunodepletion Plasma Proteomics by TripleTOF 5600 and Orbitrap Elite/LTQ-Orbitrap Velos/Q Exactive Mass Spectrometers 
Journal of proteome research  2013;12(10):10.1021/pr400307u.
Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions. 480 LC-MS/MS runs delivered >250 GB of data in two months. Several analysis algorithms were compared. At 1 % false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.
PMCID: PMC3817719  PMID: 24004147
immunodepletion; Seppro; IgY14; iTRAQ; EMMOL normalization; TripleTOF; Orbitrap; Q Exactive
7.  Identification of Lynch Syndrome Among Patients With Colorectal Cancer 
JAMA : the journal of the American Medical Association  2012;308(15):10.1001/jama.2012.13088.
Lynch syndrome is the most common form of hereditary colorectal cancer (CRC) and is caused by germline mutations in DNA mismatch repair (MMR) genes. Identification of gene carriers currently relies on germline analysis in patients with MMR-deficient tumors, but criteria to select individuals in whom tumor MMR testing should be performed are unclear.
To establish a highly sensitive and efficient strategy for the identification of MMR gene mutation carriers among CRC probands.
Design, Setting, and Patients
Pooled-data analysis of 4 large cohorts of newly diagnosed CRC probands recruited between 1994 and 2010 (n = 10 206) from the Colon Cancer Family Registry, the EPICOLON project, the Ohio State University, and the University of Helsinki examining personal, tumor-related, and family characteristics, as well as microsatellite instability, tumor MMR immunostaining, and germline MMR mutational status data.
Main Outcome Measures
Performance characteristics of selected strategies (Bethesda guidelines, Jerusalem recommendations, and those derived from a bivariate/multivariate analysis of variables associated with Lynch syndrome) were compared with tumor MMR testing of all CRC patients (universal screening).
Of 10 206 informative, unrelated CRC probands, 312 (3.1%) were MMR gene mutation carriers. In the population-based cohorts (n=3671 probands), the universal screening approach (sensitivity, 100%;95% CI, 99.3%–100%; specificity, 93.0%; 95% CI, 92.0%–93.7%; diagnostic yield, 2.2%; 95% CI, 1.7%–2.7%) was superior to the use of Bethesda guidelines (sensitivity, 87.8%; 95% CI, 78.9%–93.2%; specificity, 97.5%; 95% CI, 96.9%–98.0%; diagnostic yield, 2.0%; 95% CI, 1.5%–2.4%; P <.001), Jerusalem recommendations (sensitivity, 85.4%; 95% CI, 77.1%–93.6%; specificity, 96.7%; 95% CI, 96.0%–97.2%; diagnostic yield, 1.9%; 95% CI, 1.4%–2.3%; P < .001), and a selective strategy based on tumor MMR testing of cases with CRC diagnosed at age 70 years or younger and in older patients fulfilling the Bethesda guidelines (sensitivity, 95.1%; 95% CI, 89.8%–99.0%; specificity, 95.5%; 95% CI, 94.7%–96.1%; diagnostic yield, 2.1%; 95% CI, 1.6%–2.6%; P <.001). This selective strategy missed 4.9% of Lynch syndrome cases but resulted in 34.8% fewer cases requiring tumor MMR testing and 28.6% fewer cases undergoing germline mutational analysis than the universal approach.
Universal tumor MMR testing among CRC probands had a greater sensitivity for the identification of Lynch syndrome compared with multiple alternative strategies, although the increase in the diagnostic yield was modest.
PMCID: PMC3873721  PMID: 23073952
8.  Analysis of Induced Pluripotent Stem Cells from a BRCA1 Mutant Family 
Stem Cell Reports  2013;1(4):336-349.
Understanding BRCA1 mutant cancers is hampered by difficulties in obtaining primary cells from patients. We therefore generated and characterized 24 induced pluripotent stem cell (iPSC) lines from fibroblasts of eight individuals from a BRCA1 5382insC mutant family. All BRCA1 5382insC heterozygous fibroblasts, iPSCs, and teratomas maintained equivalent expression of both wild-type and mutant BRCA1 transcripts. Although no difference in differentiation capacity was observed between BRCA1 wild-type and mutant iPSCs, there was elevated protein kinase C-theta (PKC-theta) in BRCA1 mutant iPSCs. Cancer cell lines with BRCA1 mutations and hormone-receptor-negative breast cancers also displayed elevated PKC-theta. Genome sequencing of the 24 iPSC lines showed a similar frequency of reprogramming-associated de novo mutations in BRCA1 mutant and wild-type iPSCs. These data indicate that iPSC lines can be derived from BRCA1 mutant fibroblasts to study the effects of the mutation on gene expression and genome stability.
•Induced pluripotent stem cell (iPSC) lines from a BRCA1 mutant family were made•BRCA1 was elevated in iPSC lines compared with progenitor fibroblast lines•Protein kinase C-theta was elevated in mutant iPSCs and ER-negative breast cancer•Increased de novo mutations were found in only one BRCA1 mutant iPSC line
Twenty-four induced pluripotent stem cell (iPSC) lines were generated from a BRCA1 mutant family. Protein kinase C-theta (PKC-theta) was elevated in BRCA1 mutant compared to wild-type iPSC lines. PKC-theta was also increased in ER-negative breast cancers. A bioinformatic filter using the genome sequence of lymphoblasts, fibroblasts, and iPSCs from each patient identified reprogramming-associated de novo mutations. Increased mutation numbers were found in one of the BRCA1 mutant iPSC lines and none of the wild-type iPSC lines.
PMCID: PMC3849250  PMID: 24319668
9.  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
10.  Implementing screening for Lynch syndrome among patients with newly diagnosed colorectal cancer: summary of a public health/clinical collaborative meeting 
Lynch syndrome is the most common cause of inherited colorectal cancer, accounting for approximately 3% of all colorectal cancer cases in the United States. In 2009, an evidence-based review process conducted by the independent Evaluation of Genomic Applications in Practice and Prevention Working Group resulted in a recommendation to offer genetic testing for Lynch syndrome to all individuals with newly diagnosed colorectal cancer, with the intent of reducing morbidity and mortality in family members. To explore issues surrounding implementation of this recommendation, the Centers for Disease Control and Prevention convened a multidisciplinary working group meeting in September 2010. This article reviews background information regarding screening for Lynch syndrome and summarizes existing clinical paradigms, potential implementation strategies, and conclusions which emerged from the meeting. It was recognized that widespread implementation will present substantial challenges, and additional data from pilot studies will be needed. However, evidence of feasibility and population health benefits and the advantages of considering a public health approach were acknowledged. Lynch syndrome can potentially serve as a model to facilitate the development and implementation of population-level programs for evidence-based genomic medicine applications involving follow-up testing of at-risk relatives. Such endeavors will require multilevel and multidisciplinary approaches building on collaborative public health and clinical partnerships.
PMCID: PMC3762677  PMID: 22237445
colorectal cancer; genetic screening; genetic testing; HNPCC; Lynch syndrome
11.  An American Founder Mutation in MLH1 
Mutations in the mismatch repair genes cause Lynch syndrome (LS), conferring high risk of colorectal, endometrial and some other cancers. After the same splice site mutation in the MLH1 gene (c.589-2A>G) had been observed in 4 ostensibly unrelated American families with typical LS cancers, its occurrence in comprehensive series of LS cases (Mayo Clinic, Germany and Italy) was determined. It occurred in 10 out of 995 LS mutation carriers (1.0%) diagnosed in the Mayo Clinic diagnostic laboratory. It did not occur among 1803 cases tested for MLH1 mutations by the German HNPCC consortium, while it occurred in 3 probands and an additional 5 family members diagnosed in Italy. In the U.S., the splice site mutation occurs on a large (~4.8 Mb) shared haplotype that also harbors the variant c.2146G>A which predicts a missense change in codon 716 referred to here as V716M. In Italy, it occurs on a different, shorter shared haplotype (~2.2 Mb) that does not carry V716M. The V716M variant was found to be present by itself in the U.S., German and Italian populations with individuals sharing a common haplotype of 280 kb, allowing us to calculate that the variant arose around 5600 years ago (225 generations; 95% confidence interval 183–272). The splice site mutation in America arose or was introduced some 450 years ago (18 generations; 95% confidence interval 14–23); it accounts for 1.0% all LS in the Unites States and can be readily screened for.
PMCID: PMC3266960  PMID: 21671475
12.  Reflex Immunohistochemistry and Microsatellite Instability Testing of Colorectal Tumors for Lynch Syndrome Among US Cancer Programs and Follow-Up of Abnormal Results 
Journal of Clinical Oncology  2012;30(10):1058-1063.
Immunohistochemistry (IHC) for MLH1, MSH2, MSH6, and PMS2 protein expression and microsatellite instability (MSI) are well-established tools to screen for Lynch syndrome (LS). Although many cancer centers have adopted these tools as reflex LS screening after a colorectal cancer diagnosis, the standard of care has not been established, and no formal studies have described this practice in the United States. The purpose of this study was to describe prevalent practices regarding IHC/MSI reflex testing for LS in the United States and the subsequent follow-up of abnormal results.
Materials and Methods
A 12-item survey was developed after interdisciplinary expert input. A letter of invitation, survey, and online-survey option were sent to a contact at each cancer program. A modified Dillman strategy was used to maximize the response rate. The sample included 39 National Cancer Institute–designated Comprehensive Cancer Centers (NCI-CCCs), 50 randomly selected American College of Surgeons–accredited Community Hospital Comprehensive Cancer Programs (COMPs), and 50 Community Hospital Cancer Programs (CHCPs).
The overall response rate was 50%. Seventy-one percent of NCI-CCCs, 36% of COMPs, and 15% of CHCPs were conducting reflex IHC/MSI for LS; 48% of the programs used IHC, 14% of the programs used MSI, and 38% of the programs used both IHC and MSI. One program used a presurgical information packet, four programs offered an opt-out option, and none of the programs required written consent.
Although most NCI-CCCs use reflex IHC/MSI to screen for LS, this practice is not well-adopted by community hospitals. These findings may indicate an emerging standard of care and diffusion from NCI-CCC to community cancer programs. Our findings also described an important trend away from requiring written patient consent for screening.
PMCID: PMC3341150  PMID: 22355048
13.  Variants in the Netrin-1 Receptor UNC5C Prevent Apoptosis and Increase Risk for Familial Colorectal Cancer 
Gastroenterology  2011;141(6):2039-2046.
Background & Aims
Expression of the netrin-1 dependence receptor UNC5C is reduced in many colorectal tumors; mice with the UNC5C mutations have increased progression of intestinal tumors. We investigated whether specific variants in UNC5C increase risk for colorectal cancer (CRC).
We analyzed the sequence of UNC5C in blood samples from 1801 patients with CRC and 4152 controls from 3 cohorts (France, USA, and Finland). Almost all cases from France and the USA had familial CRC; of the Finnish cases, 92/984 were familial. We analyzed whether CRC segregates with the UNC5C variant A628K in 3 families with histories of CRC. We also performed haplotype analysis, to determine the origin of this variant.
Of 817 patients with familial CRC, 14 had 1 of 4 different, unreported missense variants in UNC5C. The variants p.Asp353Asn (encodes D353N), p.Arg603Cys (encodes R603C), and p.Gln630Glu (encodes Q630E) did not occur significantly more often in cases than controls. The variant p.Ala628Lys (A628K) was detected in 3 families in the French cohort (odds ratio [OR], 8.8; Wald’s 95% confidence interval [CI], 1.47–52.93; P=.03) and in 2 families the US cohort (OR, 1.9; P=.6), but was not detected in the Finnish cohort; UNC5C A628K segregated with CRC in families. Three families with A628K had a 109 kb identical haplotype that spanned most of UNC5C, indicating recent origin of this variant in Caucasians (14 generations; 95% CI, 6–36 generations). Transfection of HEK293T cells with UNC5C-A628K significantly reduced apoptosis compared to wild-type UNC5C, measured in an assay of active caspase-3.
Inherited mutations in UNC5C prevent apoptosis and increase risk for CRC.
PMCID: PMC3221775  PMID: 21893118
Colon cancer; tumor suppression; tumorigenesis; neoplasm; UNC5H3
14.  Performance of PREMM1,2,6, MMRpredict, and MMRpro in detecting Lynch syndrome among endometrial cancer cases 
Genetics in Medicine  2012;14(7):670-680.
Lynch syndrome accounts for 2–5% of endometrial cancer cases. Lynch syndrome prediction models have not been evaluated among endometrial cancer cases.
Area under the receiver operating curve (AUC), sensitivity and specificity of PREMM1,2,6, MMRpredict, and MMRpro scores were assessed among 563 population-based and 129 clinic-based endometrial cancer cases.
A total of 14 (3%) population-based and 80 (62%) clinic-based subjects had pathogenic mutations. PREMM1,2,6, MMRpredict, and MMRpro were able to distinguish mutation carriers from noncarriers (AUC of 0.77, 0.76, and 0.77, respectively), among population-based cases. All three models had lower discrimination for the clinic-based cohort, with AUCs of 0.67, 0.64, and 0.54, respectively. Using a 5% cutoff, sensitivity and specificity were as follows: PREMM1,2,6, 93% and 5% among population-based cases and 99% and 2% among clinic-based cases; MMRpredict, 71% and 64% for the population-based cohort and 91% and 0% for the clinic-based cohort; and MMRpro, 57% and 85% among population-based cases and 95% and 10% among clinic-based cases.
Currently available prediction models have limited clinical utility in determining which patients with endometrial cancer should undergo genetic testing for Lynch syndrome. Immunohistochemical analysis and microsatellite instability testing may be the best currently available tools to screen for Lynch syndrome in endometrial cancer patients.
PMCID: PMC3396560  PMID: 22402756
endometrial cancer; genetic screening; genetic testing; Lynch syndrome; prediction models
15.  Evaluation of Allele-Specific Somatic Changes of Genome-Wide Association Study Susceptibility Alleles in Human Colorectal Cancers 
PLoS ONE  2012;7(5):e37672.
Tumors frequently exhibit loss of tumor suppressor genes or allelic gains of activated oncogenes. A significant proportion of cancer susceptibility loci in the mouse show somatic losses or gains consistent with the presence of a tumor susceptibility or resistance allele. Thus, allele-specific somatic gains or losses at loci may demarcate the presence of resistance or susceptibility alleles. The goal of this study was to determine if previously mapped susceptibility loci for colorectal cancer show evidence of allele-specific somatic events in colon tumors.
We performed quantitative genotyping of 16 single nucleotide polymorphisms (SNPs) showing statistically significant association with colorectal cancer in published genome-wide association studies (GWAS). We genotyped 194 paired normal and colorectal tumor DNA samples and 296 paired validation samples to investigate these SNPs for allele-specific somatic gains and losses. We combined analysis of our data with published data for seven of these SNPs.
No statistically significant evidence for allele-specific somatic selection was observed for the tested polymorphisms in the discovery set. The rs6983267 variant, which has shown preferential loss of the non-risk T allele and relative gain of the risk G allele in previous studies, favored relative gain of the G allele in the combined discovery and validation samples (corrected p-value = 0.03). When we combined our data with published allele-specific imbalance data for this SNP, the G allele of rs6983267 showed statistically significant evidence of relative retention (p-value = 2.06×10−4).
Our results suggest that the majority of variants identified as colon cancer susceptibility alleles through GWAS do not exhibit somatic allele-specific imbalance in colon tumors. Our data confirm previously published results showing allele-specific imbalance for rs6983267. These results indicate that allele-specific imbalance of cancer susceptibility alleles may not be a common phenomenon in colon cancer.
PMCID: PMC3357346  PMID: 22629442
16.  The Search for Unaffected Individuals with Lynch Syndrome: Do the Ends Justify the Means? 
Lynch syndrome is the most common cause of inherited colorectal and endometrial cancers yet it is under-recognized in clinical practice. The relative merits of screening for Lynch syndrome among healthy adults without cancer versus among adults with colorectal or endometrial cancer are discussed in this Perspectives article. Newly diagnosed colorectal cancer patients are a much easier target population for screening and leads to more informative genetic test results, at a lower cost in most cases.
PMCID: PMC3076593  PMID: 21205737
Lynch syndrome; population screening; genetic testing; mismatch repair genes
17.  Clinical Relevance of Microsatellite Instability in Colorectal Cancer 
Journal of Clinical Oncology  2010;28(20):3380-3387.
Microsatellite instability (MSI) is a clonal change in the number of repeated DNA nucleotide units in microsatellites. It arises in tumors with deficient mismatch repair due to the inactivation of one of the four mismatch repair genes: MSH2, MLH1, MSH6, and PMS2. In order to determine the MSI status of a tumor, microdissection and polymerase chain reaction–based detection strategies are required. For practical purposes, MSI is equivalent to the loss of staining by immunohistochemistry (IHC) of one of the mismatch repair genes since both signify an abnormality in mismatch repair. Of all colorectal cancers (CRCs), 15% to 20% display MSI or abnormal IHC (often referred to as microsatellite instability [MIN] pathway). The remaining 80% to 85% of CRCs are microsatellite stable but most are characterized by chromosomal instability (CIN pathway). Almost all Lynch syndrome tumors have MSI or abnormal IHC and they account for up to one third of all MIN CRCs (3% to 5% of all CRCs). The remaining MIN tumors are sporadic as a result of somatic inactivation of the MLH1 gene caused by methylation of its promoter. Thus, the presence of a MSI/IHC abnormality prompts further investigations to diagnose Lynch syndrome, whereas its absence excludes Lynch syndrome. We recommend screening all CRC tumors for IHC or MSI. MIN tumors have a more favorable outcome than CIN tumors, and fluorouracil-based adjuvant chemotherapy does not improve the outcome of stage II or stage III MIN tumors. More data are needed to determine how best to treat patients with stage II and stage III MIN CRCs.
PMCID: PMC2903331  PMID: 20516444
19.  Risks of Lynch Syndrome Cancers for MSH6 Mutation Carriers 
Germline mutations in MSH6 account for 10%–20% of Lynch syndrome colorectal cancers caused by hereditary DNA mismatch repair gene mutations. Because there have been only a few studies of mutation carriers, their cancer risks are uncertain.
We identified 113 families of MSH6 mutation carriers from five countries that we ascertained through family cancer clinics and population-based cancer registries. Mutation status, sex, age, and histories of cancer, polypectomy, and hysterectomy were sought from 3104 of their relatives. Age-specific cumulative risks for carriers and hazard ratios (HRs) for cancer risks of carriers, compared with those of the general population of the same country, were estimated by use of a modified segregation analysis with appropriate conditioning depending on ascertainment.
For MSH6 mutation carriers, the estimated cumulative risks to ages 70 and 80 years, respectively, were as follows: for colorectal cancer, 22% (95% confidence interval [CI] = 14% to 32%) and 44% (95% CI = 28% to 62%) for men and 10% (95% CI = 5% to 17%) and 20% (95% CI = 11% to 35%) for women; for endometrial cancer, 26% (95% CI = 18% to 36%) and 44% (95% CI = 30% to 58%); and for any cancer associated with Lynch syndrome, 24% (95% CI = 16% to 37%) and 47% (95% CI = 32% to 66%) for men and 40% (95% CI = 32% to 52%) and 65% (95% CI = 53% to 78%) for women. Compared with incidence for the general population, MSH6 mutation carriers had an eightfold increased incidence of colorectal cancer (HR = 7.6, 95% CI = 5.4 to 10.8), which was independent of sex and age. Women who were MSH6 mutation carriers had a 26-fold increased incidence of endometrial cancer (HR = 25.5, 95% CI = 16.8 to 38.7) and a sixfold increased incidence of other cancers associated with Lynch syndrome (HR = 6.0, 95% CI = 3.4 to 10.7).
We have obtained precise and accurate estimates of both absolute and relative cancer risks for MSH6 mutation carriers.
PMCID: PMC2815724  PMID: 20028993
20.  Genetic Testing for Hereditary Colorectal Cancer 
PMCID: PMC2821160  PMID: 19793575
Genetic; testing; Lynch syndrome; polyposis syndromes
21.  Identifying Lynch Syndrome 
PMCID: PMC2771416  PMID: 19536819
22.  Allele-specific expression of TGFBR1 in colon cancer patients 
Carcinogenesis  2010;31(10):1800-1804.
The genetic component of colorectal cancer (CRC) predisposition has been only partially explained. We recently suggested that a subtle decrease in the expression of one allele of the TGFBR1 gene was a heritable quantitative trait predisposing to CRC. Here, we refined the measurements of allele-specific expression (ASE) of TGFBR1 in a population-based series of CRC patients and controls. Five single-nucleotide polymorphisms (SNPs) in the 3′-untranslated region of the gene were genotyped and used for ASE determination by pyrosequencing. After eliminating non-informative samples and samples with RNA of insufficient quality 109 cases and 125 controls were studied. Allelic ratios ranged between 0.74 and 1.69 without evidence of bimodality or cutoff points for ‘ASE’ versus ‘non-ASE’. Treating ASE as a continuous variable, cases had non-significantly different values than controls (P = 0.081 when comparing means by permutation test). However, cases had significantly higher ASE values when comparing medians by permutation test (P = 0.0027) and when using Wilcoxon test (P = 0.0094). We conclude that with the present-day technology, ASE differences between individuals and between cases and controls are too subtle to be used to assess CRC risk. More advanced technology is expected to resolve this issue as well as the low informativity caused by the limited heterozygosity of transcribed SNPs.
PMCID: PMC2950937  PMID: 20705955
23.  Current and emerging trends in Lynch syndrome identification in women with endometrial cancer 
Gynecologic oncology  2009;114(1):128-134.
Lynch syndrome is a heritable, cancer susceptibility syndrome. This study aims to review current and emerging trends in the identification of Lynch syndrome in the endometrial cancer patient population.
We performed a comprehensive review of past and present screening algorithms for Lynch syndrome, including a review of the utility of both the Amsterdam criteria and Bethesda guidelines. Because non-colon cancers have historically not been the focus of Lynch syndrome research, current literature is ripe with questions regarding screening among this patient population. Low BMI, age less than 50, positive family history and pathologic features have all been identified as risk factors in endometrial cancer patients who might benefit from Lynch screening. Additionally, based on experience at our own institution we offer a feasible screening algorithm for these patients.
A comprehensive review of the data demonstrated that immunohistochemistry is becoming an efficient, inexpensive way to screen tumors at risk for mismatch repair deficiency. The sensitivity and specificity of immunohistochemistry for predicting Lynch syndrome approaches 100%. Ideally, prospective screening of all endometrial cancer patients with IHC is a feasible, cost-efficient way to detect Lynch in this patient population given the limitations of using personal/family history of malignancy as well as pathologic risk factors.
It is imperative that clinicians be mindful of the risk of Lynch syndrome in women with endometrial cancer. Given the opportunity for colon cancer screening and prevention strategies to be initiated, the identification of probands with endometrial cancer as a result of Lynch syndrome will lead to a reduction in morbidity and mortality for these patients and their families.
PMCID: PMC2841434  PMID: 19375789
Lynch syndrome; Endometrial cancer; Screening
24.  Phenotypic diversity in patients with multiple serrated polyps: a genetics clinic study 
Hyperplastic polyposis is a colonic polyposis condition of unknown aetiology. The purpose of this study was to examine the spectrum of phenotypic variation in patients with multiple serrated polyps as a basis for gene discovery.
One hundred and twenty-six patients with multiple (≥5) serrated polyps were recruited to the study. Polyp counts were extracted from histology and colonoscopy reports. Ethnicity was self-reported. Family history of cancer data were derived from pedigrees. Ascertainment status was classified as either index case or identified by screening.
The average reported polyp count was 39. Patients with highest polyp numbers were more likely to be male (P  = 0.02). Colorectal cancer (CRC) was identified in 49 of 119 patients (41%) and 28% of these patients had multiple CRC. Young onset patients had higher polyp numbers (P  = 0.03) and were more likely to have their CRC in the distal colon (P  = 0.02). CRC was significantly associated with the presence of adenomas (P  = 0.03). Patients were divided into moderate polyposis (5-79 serrated polyps) and dense polyposis (80 or more) categories. The dense polyposis category was associated with a lack of family history for CRC (P  = 0.034) and male gender (P  = 0.014), independent of ascertainment status and recruitment site.
Multiple serrated polyps were associated with an increased personal risk of CRC. A subset of patients with the highest polyp numbers was more likely to be male and to have no family history of CRC. This result suggests heterogeneous modes of inheritance and has implications for studies investigating the genetic basis of multiple serrated polyps.
PMCID: PMC2862176  PMID: 20213458
Hyperplastic polyposis; Serrated neoplasia; Smoking; Family history; Ethnicity
25.  Feasibility of Screening for Lynch Syndrome Among Patients With Colorectal Cancer 
Journal of Clinical Oncology  2008;26(35):5783-5788.
Identifying individuals with Lynch syndrome (LS) is highly beneficial. However, it is unclear whether microsatellite instability (MSI) or immunohistochemistry (IHC) should be used as the screening test and whether screening should target all patients with colorectal cancer (CRC) or those in high-risk subgroups.
Patients and Methods
MSI testing and IHC for the four mismatch repair proteins was performed on 500 tumors from unselected patients with CRC. If either MSI or IHC was abnormal, complete mutation analysis for the mismatch repair genes was performed.
Among the 500 patients, 18 patients (3.6%) had LS. All 18 patients detected with LS (100%) had MSI-high tumors; 17 (94%) of 18 patients with LS were correctly predicted by IHC. Of the 18 probands, only eight patients (44%) were diagnosed at age younger than 50 years, and only 13 patients (72%) met the revised Bethesda guidelines. When these results were added to data on 1,066 previously studied patients, the entire study cohort (N = 1,566) showed an overall prevalence of 44 of 1,566 patients (2.8%; 95% CI, 2.1% to 3.8%) for LS. For each proband, on average, three additional family members carried MMR mutations.
One of every 35 patients with CRC has LS, and each has at least three relatives with LS; all of whom can benefit from increased cancer surveillance. For screening, IHC is almost equally sensitive as MSI, but IHC is more readily available and helps to direct gene testing. Limiting tumor analysis to patients who fulfill Bethesda criteria would fail to identify 28% (or one in four) cases of LS.
PMCID: PMC2645108  PMID: 18809606

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