Spring Holter

The PREMM1,2,6 Model Predicts Risk of MLH1, MSH2, and MSH6 Germline Mutations Based on Cancer History

BACKGROUND & AIMS We developed and validated a model to estimate the risks of mutations in the mismatch repair (MMR) genes MLH1, MSH2, and MSH6 based on personal and family history of cancer. METHODS Data were analyzed from 4539 probands tested for mutations in MLH1, MSH2, and MSH6. A multivariable polytomous logistic regression model (PREMM(1,2,6)) was developed to predict the overall risk of MMR gene mutations and the risk of mutation in each of the 3 genes. The discriminative ability of the model was validated in 1827 population-based colorectal cancer (CRC) cases. RESULTS Twelve percent of the original cohort carried pathogenic mutations (204 in MLH1, 250 in MSH2, and 71 in MSH6). The PREMM(1,2,6) model incorporated the following factors from the probands and first- and second-degree relatives (odds ratio; 95% confidence intervals [CIs]): male sex (1.9; 1.5-2.4), a CRC (4.3; 3.3-5.6), multiple CRCs (13.7; 8.5-22), endometrial cancer (6.1; 4.6-8.2), and extracolonic cancers (3.3; 2.4-4.6). The areas under the receiver operating characteristic curves were 0.86 (95% CI, 0.82-0.91) for MLH1 mutation carriers, 0.87 (95% CI, 0.83-0.92) for MSH2, and 0.81 (95% CI, 0.69-0.93) for MSH6; in validation, they were 0.88 for the overall cohort (95% CI, 0.86-0.90) and the population-based cases (95% CI, 0.83-0.92). CONCLUSIONS We developed the PREMM(1,2,6) model, which incorporates information on cancer history from probands and their relatives to estimate an individuals risk of mutations in the MMR genes MLH1, MSH2, and MSH6. This Web-based decision making tool can be used to assess risk of hereditary CRC and guide clinical management.

Analysis of the Gene Coding for the BRCA2-Interacting Protein PALB2 in Familial and Sporadic Pancreatic Cancer

Dear Sir: Current evidence indicates that about 5%–10% of pancreatic cancer has a familial component, although the vast majority of pancreatic cancer families remain unexplained. 1 PALB2 is a recently identified breast cancer susceptibility gene whose protein is closely associated with BRCA2, and is essential for BRCA2 anchorage to nuclear structures. This functional relationship made PALB2 a candidate gene for susceptibility to BRCA2-related cancers such as pancreas cancer. Recently, Jones et al2 screened 96 familial pancreatic cancer patients, 16 of whom had 1 first-degree relative with pancreatic cancer and 80 had ≥2 additional relatives with pancreatic cancer, ≥1 of which was first degree.2 Truncating PALB2 mutations were identified in 3 patients (3.1%) and there was no difference in average age of cancer onset between mutation-positive and -negative families. We sought to screen a larger cohort of pancreatic cancer cases, including both familial and sporadic types, to determine the wider contribution of PALB2 gene mutations in pancreatic cancer. We selected a total of 254 individuals with pancreas cancer (148 male, 106 female) at a median age of diagnosis of 61 years. Patients were identified between 1997 and 2007 via clinic-based recruitment in Toronto and Montreal and through either the Familial Gastrointestinal Cancer Registry at Mount Sinai Hospital in Toronto or the population-based Ontario Pancreas Cancer Study. In total, 203 patients were recruited in Toronto and 51 in Montreal. All probands were confirmed to have pancreatic adenocarcinoma by pathology report; 101 pro-bands had a family history of pancreatic cancer, of which 32 had 2 affected first-degree relatives. In these 101 cases, 74 had 1 affected relative, 18 had 2 affected relatives, 7 had 3 affected relatives, and 2 cases had >3 affected relatives (Table 1). Sixty probands had a family history of breast/ovarian cancer, including 21 cases with a family history of pancreatic cancer (included above). Because the cases were ascertained through pancreas cancer studies, the family history of breast cancer or ovarian cancer was not strong, with most families having a single relative affected, and no family having a BRCAPRO score >0.12 (ie, these were not primarily familial breast/ovarian cancer families with 1 or 2 additional cases of pancreas cancer). The median age at diagnosis of pancreatic cancer cases with no family history was 49 years (range, 31–85); 55% of these were <50 years old and 66% were <60 years old at diagnosis. Genomic DNA was obtained from blood, saliva, or buccal cells using standard extraction methods. Before analysis, 20 ng of total genomic DNA was used for whole genome amplification according to the manufacturer’s instructions using the Repli-g Mini Kit (Qiagen, Mississauga, Ontario, Canada). We screened the 13 coding exons of PALB2 by sequencing (n = 83) or high-resolution melt analysis (n = 171), which has similar sensitivity to sequencing.3 Samples with variants were reamplified by polymerase chain reaction (PCR) using the original, non–whole-genome-amplified DNA as template and the PCR product was sequenced in forward and reverse directions for confirmation. We performed multiplex ligation-dependent probe amplification assay (MLPA) for PALB2 on 228 samples where we had sufficient DNA of adequate quality, as previously described by Foulkes et al.4 Table 1 Pancreas Cancer Cases We identified a heterozygous, 6.7-kb germline deletion including exons 12 and 13 of PALB2 in a patient who was affected by breast and then pancreas cancer (ages 47 and 61, respectively) and whose mother died of pancreas cancer at age 83 (Figure 1). This result was confirmed by long range PCR (Takara Bio Inc., Madison, WI) using 2 different primer pairs, which determined the deleted region to span a region from the middle of intron 11 (2.7 kb from the beginning of exon 12), up to 1.8kb after exon 13. This deletion would disrupt the PALB2 WD40 motif, which is required for interaction with the BRCA2 protein. 5 Aside from the exonic deletion, 2 previously unreported missense variants (S285L and T911I) were identified, but neither were predicted to be pathogenic. Both these variants were seen in young-onset pancreas cancer cases (41 years and 48 years) with no family history. A number of previously reported variants were also identified (Table 2). Figure 1 (A) Pedigree of the proband (shown by arrow). PA, pancreatic cancer; BR, breast cancer. (B) MLPA showing deletion of exon 12 (161-bp fragment) and exon 3 (338-bp fragment)

Prevalence of Germline Mutations in Cancer Predisposition Genes in Patients With Pancreatic Cancer

BACKGROUND & AIMS We investigated the prevalence of germline mutations in APC, ATM, BRCA1, BRCA2, CDKN2A, MLH1, MSH2, MSH6, PALB2, PMS2, PRSS1, STK11, and TP53 in patients with pancreatic cancer. METHODS The Ontario Pancreas Cancer Study enrolls consenting participants with pancreatic cancer from a province-wide electronic pathology database; 708 probands were enrolled from April 2003 through August 2012. To improve the precision of BRCA2 prevalence estimates, 290 probands were selected from 3 strata, based on family history of breast and/or ovarian cancer, pancreatic cancer, or neither. Germline DNA was analyzed by next-generation sequencing using a custom multiple-gene panel. Mutation prevalence estimates were calculated from the sample for the entire cohort. RESULTS Eleven pathogenic mutations were identified: 3 in ATM, 1 in BRCA1, 2 in BRCA2, 1 in MLH1, 2 in MSH2, 1 in MSH6, and 1 in TP53. The prevalence of mutations in all 13 genes was 3.8% (95% confidence interval, 2.1%-5.6%). Carrier status was associated significantly with breast cancer in the proband or first-degree relative (P < .01), and with colorectal cancer in the proband or first-degree relative (P < .01), but not family history of pancreatic cancer, age at diagnosis, or stage at diagnosis. Of patients with a personal or family history of breast and colorectal cancer, 10.7% (95% confidence interval, 4.4%-17.0%) and 11.1% (95% confidence interval, 3.0%-19.1%) carried pathogenic mutations, respectively. CONCLUSIONS A small but clinically important proportion of pancreatic cancer is associated with mutations in known predisposition genes. The heterogeneity of mutations identified in this study shows the value of using a multiple-gene panel in pancreatic cancer.

Identification of Individuals at Risk for Lynch Syndrome Using Targeted Evaluations and Genetic Testing: National Society of Genetic Counselors and the Collaborative Group of the Americas on Inherited Colorectal Cancer Joint Practice Guideline

Identifying individuals who have Lynch syndrome (LS) involves a complex diagnostic work up that includes taking a detailed family history and a combination of various genetic and immunohistochemical tests. The National Society of Genetic Counselors (NSGC) and the Collaborative Group of the Americas on Inherited Colorectal Cancer (CGA-ICC) have come together to publish this clinical practice testing guideline for the evaluation of LS. The purpose of this practice guideline is to provide guidance and a testing algorithm for LS as well as recommendations on when to offer testing. This guideline does not replace a consultation with a genetics professional. This guideline includes explanations in support of this and a summary of background data. While this guideline is not intended to serve as a review of LS, it includes a discussion of background information on LS, and cites a number of key publications which should be reviewed for a more in-depth understanding of LS. These guidelines are intended for genetic counselors, geneticists, gastroenterologists, surgeons, medical oncologists, obstetricians and gynecologists, nurses and other healthcare providers who evaluate patients for LS.

Comparison of clinical schemas and morphologic features in predicting Lynch syndrome in mutation-positive patients with endometrial cancer encountered in the context of familial gastrointestinal cancer registries.

Endometrial cancer (EC) is the most common extraintestinal malignancy in Lynch syndrome (LS) and often is the sentinel malignancy, yet there is no consensus regarding LS‐EC detection algorithms. In this study, the authors determined the efficacy of family/personal history and tumor morphology in predicting LS in a cohort of patients with EC who had mutation‐proven LS.

The Gastrointestinal Phenotype of Germline Biallelic Mismatch Repair Gene Mutations

OBJECTIVES:A novel cancer syndrome associated with biallelic mismatch repair (MMR) mutations has been described recently. Patients presenting with childhood-onset gastrointestinal (GI) cancers may carry biallelic MMR mutations and have a distinct phenotype from classic Lynch syndrome. The aim of this study was to characterize patients with GI small bowel and/or colorectal cancers (CRCs) who have germline biallelic MMR mutations.METHODS:A search of a Canadian GI cancer registry and literature review to identify patients with biallelic MMR was conducted.RESULTS:The database identified 237 patients with intestinal cancer diagnosed before the age of 35 years. Five (2.1%) patients had biallelic MMR mutations. Overall, 32 individuals, from 29 families, with biallelic MMR gene mutations and GI cancers were identified by the registry and literature review. Among the 29 patients with CRCs, the mean age of first cancer diagnosis was 16.4 years (range: 5–28). More than one-third of patients had multiple colorectal adenomas (>10 polyps). Six individuals with biallelic MMR gene mutations have been reported with small bowel adenocarcinoma (mean age 20 years (range: 11–41)). Café-au-lait (CAL) macules were reported in 72% and, based on mutation analysis, consanguinity was suspected in 52% of kindred. Of the 29 kindred, 19 (66%) had PMS2 mutations, 6 (21%) had MSH6 mutations, 3 (10%) had MLH1 mutations, and 1 (3%) had MSH2 mutation.CONCLUSIONS:Biallelic MMR mutations are an underrecognized cause of small bowel and colonic cancers in children and young adults. This distinct phenotype includes multiple adenomatous polyps and CAL skin lesions. It is important to identify such patients, so that families can be referred for genetic testing and counseling.

The histomorphology of Lynch syndrome-associated ovarian carcinomas: toward a subtype-specific screening strategy.

Women with Lynch syndrome (LS) are at increased risk for the development of epithelial ovarian cancer (OC). Analogous to previous studies on BRCA1/2 mutation carriers, there is evidence to suggest a histotype-specific association in LS-associated OCs (LS-OC). Whereas the diagnosis of high-grade serous carcinoma is an indication for BRCA1/2 germline testing, in contrast, there are no screening guidelines in place for triaging OC patients for LS testing based on histotype. We performed a centralized pathology review of tumor subtype on 20 germline mutation-confirmed LS-OCs, on the basis of morphologic assessment of hematoxylin and eosin–stained slides, with confirmation by immunohistochemistry when necessary. Results from mismatch-repair immunohistochemistry (MMR-IHC) and microsatellite instability (MSI) phenotype status were documented, and detailed pedigrees were analyzed to determine whether previously proposed clinical criteria would have selected these patients for genetic testing. Review of pathology revealed all LS-OCs to be either pure endometrioid carcinoma (14 cases), mixed carcinoma with an endometrioid component (4 cases), or clear cell carcinoma (2 cases). No high-grade or low-grade serous carcinomas or mucinous carcinomas of intestinal type were identified. Tumor-infiltrating lymphocytes were prominent (≥40 per 10 high-powered fields) in 2 cases only. With the exception of 1 case, all tumors tested for MMR-IHC or MSI had an MMR-deficient phenotype. Within this cohort, 50%, 55%, 65%, and 85% of patients would have been selected for genetic workup by Amsterdam II, revised Bethesda Guidelines, SGO 10% to 25%, and SGO 5% to 10% criteria, respectively, with <60% of index or sentinel cases detected by any of these schemas. To further support a subtype-driven screening strategy, MMR-IHC reflex testing was performed on all consecutive non-serous OCs diagnosed at 1 academic hospital over a 2-year period; MMR deficiency was identified in 10/48 (21%) cases, all with endometrioid or clear cell histology. We conclude that there is a strong association between endometrioid and clear cell ovarian carcinomas and hereditary predisposition due to MMR gene mutation. These findings have implications for the role of tumor subtype in screening patients with OC for further genetic testing and support reflex MMR-IHC and/or MSI testing for newly diagnosed cases of endometrioid or clear cell ovarian carcinoma.

Rhabdomyosarcoma in patients with constitutional mismatch-repair-deficiency syndrome

Background: Biallelic germline mutations in the mismatch repair genes MLH1, MSH2, MSH6 or PMS2 cause a recessive childhood cancer syndrome characterised by early-onset malignancies and signs reminiscent of neurofibromatosis type 1 (NF1). Alluding to the underlying genetic defect, we refer to this syndrome as constitutional mismatch repair-deficiency (CMMR-D) syndrome. The tumour spectrum of CMMR-D syndrome includes haematological neoplasias, brain tumours and Lynch syndrome-associated tumours. Other tumours, such as neuroblastoma, Wilm tumour, ovarian neuroectodermal tumour or infantile myofibromatosis, have so far been found only in individual cases. Results: We analysed two consanguineous families that had members with suspected CMMR-D syndrome who developed rhabdomyosarcoma among other neoplasias. In the first family, we identified a pathogenic PMS2 mutation for which the affected patient was homozygous. In family 2, immunohistochemistry analysis showed isolated loss of PMS2 expression in all tumours in the affected patients, including rhabdomyosarcoma itself and the surrounding normal tissue. Together with the family history and microsatellite instability observed in one tumour this strongly suggests an underlying PMS2 alteration in family 2 also. Conclusion: Together, these two new cases show that rhabdomyosarcoma and possibly other embryonic tumours, such as neuroblastoma and Wilm tumour, belong to the tumour spectrum of CMMR-D syndrome. Given the clinical overlap of CMMR-D syndrome with NF1, we suggest careful examination of the family history in patients with embryonic tumours and signs of NF1 as well as analysis of the tumours for loss of one of the mismatch repair genes and microsatellite instability. Subsequent mutation analysis will lead to a definitive diagnosis of the underlying disorder.

Patients with Lynch syndrome mismatch repair gene mutations are at higher risk for not only upper tract urothelial cancer but also bladder cancer.

BACKGROUND Lynch syndrome (LS), or hereditary nonpolyposis colorectal cancer, is caused by mutations in mismatch repair (MMR) genes. An increased risk for upper tract urothelial carcinoma (UTUC) has been described in this population; however, data regarding the risk for bladder cancer (BCa) are sparse. OBJECTIVE To assess the risk of BCa in MMR mutation carriers and suggest screening and management recommendations. DESIGN, SETTING, AND PARTICIPANTS Cancer data from 1980 to 2007 were obtained from the Familial Gastrointestinal Cancer Registry in Toronto for 321 persons with known MMR mutations: mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) (MLH1); mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli) (MSH2); mutS homolog 6 (E. coli) (MSH6); and PMS2 postmeiotic segregation increased 2 (S. cerevisiae) (PMS2). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Standardized incidence ratios from the Ontario Cancer Registry, using the Surveillance Epidemiology and End Results public database, were used to compare cancer risk in patients with MMR mutations with the Canadian population. Microsatellite instability analysis and immunohistochemistry (IHC) of the MMR proteins were also performed and the results compared with matched sporadic bladder tumors. RESULTS AND LIMITATIONS Eleven of 177 patients with MSH2 mutations (6.21%, p<0.001 compared with the Canadian population) were found to have BCa, compared with 3 of 129 patients with MLH1 mutations (2.32%, p>0.05). Of these 11 tumors, 81.8% lacked expression of MSH2 on IHC, compared with the matched sporadic cases, which all displayed normal expression of MSH2 and MLH1. The incidence of UTUC among MSH2 carriers was 3.95% (p<0.001), and all tumors were found to be deficient in MSH2 expression on IHC. Mutations in the intron 5 splice site and exon 7 of the MSH2 gene increased the risk of urothelial cancer. Limitations include possible inflated risk estimates due to ascertainment bias. CONCLUSIONS LS patients with MSH2 mutations are at an increased risk for not only UTUC but also BCa and could be offered appropriate screening.

Café-au-lait macules and pediatric malignancy caused by biallelic mutations in the DNA mismatch repair (MMR) gene PMS2.

A 14‐year‐old male presented with a T4 sigmoid adenocarcinoma, <10 colonic adenomas and multiple café‐au‐lait macules. Family history was not suggestive of a dominant hereditary form of colorectal cancer. Evaluation of the tumor revealed abnormal immunohistochemical staining of the PMS2 protein and high frequency microsatellite instability. Germline analysis identified biallelic PMS2 missense mutations. A new cancer syndrome caused by biallelic mutations in the mismatch repair genes, including PMS2, is now emerging and is characterized by café‐au‐lait macules, colonic polyps and a distinctive tumor spectrum. Pediatr Blood Cancer 2008;50:1268–1270.