Stephen B. Gruber

Statins and cancer prevention

Randomized controlled trials for preventing cardiovascular disease indicated that statins had provocative and unexpected benefits for reducing colorectal cancer and melanoma. These findings have led to the intensive study of statins in cancer prevention, including recent, large population-based studies showing statin-associated reductions in overall, colorectal and prostate cancer. Understanding the complex cellular effects (for example, on angiogenesis and inflammation) and the underlying molecular mechanisms of statins (for example, 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase-dependent processes that involve geranylgeranylation of Rho proteins, and HMG-CoA-independent processes that involve lymphocyte-function-associated antigen 1) will advance the development of molecularly targeted agents for preventing cancer. This understanding might also help the development of drugs for other ageing-related diseases with interrelated molecular pathways.

Genome-wide association scan identifies a colorectal cancer susceptibility locus on 11q23 and replicates risk loci at 8q24 and 18q21.

In a genome-wide association study to identify loci associated with colorectal cancer (CRC) risk, we genotyped 555,510 SNPs in 1,012 early-onset Scottish CRC cases and 1,012 controls (phase 1). In phase 2, we genotyped the 15,008 highest-ranked SNPs in 2,057 Scottish cases and 2,111 controls. We then genotyped the five highest-ranked SNPs from the joint phase 1 and 2 analysis in 14,500 cases and 13,294 controls from seven populations, and identified a previously unreported association, rs3802842 on 11q23 (OR = 1.1; P = 5.8 × 10−10), showing population differences in risk. We also replicated and fine-mapped associations at 8q24 (rs7014346; OR = 1.19; P = 8.6 × 10−26) and 18q21 (rs4939827; OR = 1.2; P = 7.8 × 10−28). Risk was greater for rectal than for colon cancer for rs3802842 (P < 0.008) and rs4939827 (P < 0.009). Carrying all six possible risk alleles yielded OR = 2.6 (95% CI = 1.75–3.89) for CRC. These findings extend our understanding of the role of common genetic variation in CRC etiology.

Personalized Oncology Through Integrative High-Throughput Sequencing: A Pilot Study

The mutations present in advanced cancers can be identified by integrative high-throughput sequencing to enable biomarker-driven clinical trials and, ultimately, treatment. First Steps to Personalized Cancer Treatment In an optimistic vision of personalized medicine, each cancer patient is treated with drugs tailored for their particular tumor. This sounds appealing, but is it even possible? Roychowdhury and his colleagues tested this approach by extensively characterizing cancers in several patients and then convening a Sequencing Tumor Board of experts to determine the appropriate treatment. With a combination of whole genome and exome sequencing plus sequencing of transcribed RNA, the authors were able to find informative mutations within 3 to 4 weeks, a short enough time to be useful clinically. To verify that their sequencing strategy would work before testing it on actual patients, they assessed two xenografts established from patients with metastatic prostate cancer. They found that one of these carried the common prostate cancer–specific gene fusion of TMPRSS2 and ERG and another, previously undescribed, gene fusion. Also, the androgen receptor gene was amplified and two tumor suppressors were inactivated. The Board concluded that this pattern of mutations could in theory be treated by combined block of the PI3K and androgen receptor signaling pathways. The authors then turned to an actual patient, a 46 year old with colorectal cancer, who had been unsuccessfully treated. Characterization of his metastatic tumor showed mutations in the oncogene NRAS, the tumor suppressor TP53, aurora kinase A, a myosin heavy chain and the FAS death receptor, plus amplification of CDK8. Of these, the Sequencing Tumor Board concluded that the NRAS and CDK8 aberrations could potentially be matched to clinical trials, although none were available at the time. Similar analysis of another patient with metastatic melanoma revealed a structural rearrangement in CDKN2C and HRas. Although the HRAS mutation has not been described before in melanoma, the Sequencing Tumor Board suggested that combined treatment with PI3K and MEK inhibitors would be suitable for this patient. The good news resulting from these studies was that the patients’ tumors were analyzed with in 24 days for ~

Frequency and Spectrum of Cancers in the Peutz-Jeghers Syndrome

3600, well within the cost of routine clinical tests. But aspects need improvement: Additional testing for epigenetic and small RNA variants will allow more informative characterization. Sequencing at higher depth or enrichment methods will be needed for tumors of lower purity. And perhaps most important, we need a broader array of clinical trials, as highlighted by the fact that none was available for these two patients. Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology.

Microsatellite instability in colorectal cancer-the stable evidence.

Background: Although an increased cancer risk in Peutz-Jeghers syndrome is established, data on the spectrum of tumors associated with the disease and the influence of germ-line STK11/LKB1 (serine/threonine kinase) mutation status are limited. Experimental Design: We analyzed the incidence of cancer in 419 individuals with Peutz-Jeghers syndrome, and 297 had documented STK11/LKB1 mutations. Results: Ninety-six cancers were found among individuals with Peutz-Jeghers syndrome. The risk for developing cancer at ages 20, 30, 40, 50, 60, and 70 years was 2%, 5%, 17%, 31%, 60%, and 85%, respectively. The most common cancers represented in this analysis were gastrointestinal in origin, gastroesophageal, small bowel, colorectal, and pancreatic, and the risk for these cancers at ages 30, 40, 50, and 60 years was 1%, 9%, 15%, and 33%, respectively. In women with Peutz-Jeghers syndrome, the risk of breast cancer was substantially increased, being 8% and 31% at ages 40 and 60 years, respectively. Kaplan-Meier analysis showed that cancer risks were similar in Peutz-Jeghers syndrome patients with identified STK11/LKB1 mutations and those with no detectable mutation (log-rank test of difference χ2 = 0.62; 1 df; P = 0.43). Furthermore, the type or site of STK11/LKB1 mutation did not significantly influence cancer risk. Conclusions: The results from our study provide quantitative information on the spectrum of cancers and risks of specific cancer types associated with Peutz-Jeghers syndrome.

Small changes in expression affect predisposition to tumorigenesis

Microsatellite instability (MSI) is the molecular fingerprint of a deficient mismatch repair system. Approximately 15% of colorectal cancers (CRC) display MSI owing either to epigenetic silencing of MLH1 or a germline mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2. Methods to detect MSI are well established and routinely incorporated into clinical practice. A clinical and molecular profile of MSI tumors has been described, leading to the concept of an MSI phenotype in CRC. Studies have confirmed that MSI tumors have a better prognosis than microsatellite stable CRC, but MSI cancers do not necessarily have the same response to the chemotherapeutic strategies used to treat microsatellite stable tumors. Specifically, stage II MSI tumors might not benefit from 5-fluorouracil-based adjuvant chemotherapy regimens. New data suggest possible advantages of irinotecan-based regimens, but these findings require further clarification. Characterization of the molecular basis of MSI in CRC is underway and initial results show that mutations in genes encoding kinases and candidate genes with microsatellite tracts are over-represented in MSI tumors. Transcriptome expression profiles of MSI tumors and systems biology approaches are providing the opportunity to develop targeted therapeutics for MSI CRC.

Calculation of Risk of Colorectal and Endometrial Cancer Among Patients With Lynch Syndrome

We have used quantitative measures of gene expression to show that constitutional 50% decreases in expression of one adenomatous polyposis coli tumor suppressor gene (APC) allele can lead to the development of familial adenomatous polyposis.

Risk of pancreatic cancer in families with Lynch syndrome

BACKGROUND & AIMS Lynch syndrome is the most common hereditary colorectal cancer (CRC) syndrome. Some previous estimates of lifetime risk for CRC and endometrial cancer (EC) did not control for ascertainment and were susceptible to bias toward overestimated risk. METHODS We studied 147 families with mismatch repair gene mutations (55 MLH1, 81 MSH2, and 11 MSH6) identified at 2 US cancer genetics clinics. Age-specific cumulative risks (penetrance) and hazard ratio (HR) estimates of CRC and EC risks were calculated and compared with the general population using modified segregation analysis. The likelihood for each pedigree was conditioned on the proband and first-degree relatives affected with CRC to reduce ascertainment bias and overestimation of penetrance. RESULTS We analyzed 628 cases of CRC, diagnosed at the median ages of 42 and 47 years for men and women, respectively. The cumulative risk of CRC was 66.08% (95% confidence interval [CI], 59.47%-76.17%) for men and 42.71% (95% CI, 36.57%-52.83%) for women, with overall HRs of 148.4 and 51.1, respectively. CRC risk was highest for males with mutations in MLH1. There were 155 cases of EC, diagnosed at a median age of 47.5 years. The cumulative risk of EC was 39.39% (95% CI, 30.78%-46.94%) with an overall HR of 39.0 (95% CI, 30.4-50.2). For women, the cumulative risk of CRC or EC was 73.42% (95% CI, 63.76%-80.54%). CONCLUSIONS Lifetime risks of CRC and EC in mismatch repair gene mutation carriers are high even after adjusting for ascertainment. These estimates are valuable for patients and providers; specialized cancer surveillance is necessary.

Anti-oncogenic role of the endoplasmic reticulum differentially activated by mutations in the MAPK pathway

CONTEXT Lynch syndrome is an inherited cause of colorectal cancer caused by mutations of DNA mismatch repair (MMR) genes. A number of extracolonic tumors have been associated with the disorder, including pancreatic cancer; however, the risk of pancreatic cancer in Lynch syndrome is uncertain and not quantified. OBJECTIVE To estimate pancreatic cancer risk in families with germline MMR gene mutations. DESIGN, SETTING, AND PATIENTS Cancer histories of probands and their relatives were evaluated in MMR gene mutation carriers in the familial cancer registries of the Dana-Farber Cancer Institute (n = 80), Boston, Massachusetts, and University of Michigan Comprehensive Cancer Center (n = 67), Ann Arbor, Michigan. Families enrolled before the study start date (June 2008) were eligible. Age-specific cumulative risks and hazard ratio estimates of pancreatic cancer risk were calculated and compared with the general population using modified segregation analysis, with correction for ascertainment. MAIN OUTCOME MEASURES Age-specific cumulative risks and hazard ratio estimates of pancreatic cancer risk. RESULTS Data on 6342 individuals from 147 families with MMR gene mutations were analyzed. Thirty-one families (21.1%) reported at least 1 case of pancreatic cancer. Forty-seven pancreatic cancers were reported (21 men and 26 women), with no sex-related difference in age of diagnosis (51.5 vs 56.5 years for men and women, respectively). The cumulative risk of pancreatic cancer in these families with gene mutations was 1.31% (95% confidence interval [CI], 0.31%-2.32%) up to age 50 years and 3.68% (95% CI, 1.45%-5.88%) up to age 70 years, which represents an 8.6-fold increase (95% CI, 4.7-15.7) compared with the general population. CONCLUSIONS Among 147 families with germline MMR gene mutations, the risk of pancreatic cancer was increased compared with the US population. Individuals with MMR gene mutations and a family history of pancreatic cancer are appropriate to include in studies to further define the risk of premalignant and malignant pancreatic neoplasms and potential benefits and limitations of surveillance.

Organ-Specific Molecular Classification of Primary Lung, Colon, and Ovarian Adenocarcinomas Using Gene Expression Profiles

Dysfunction of the endoplasmic reticulum (ER) has been reported in a variety of human pathologies, including cancer. However, the contribution of the ER to the early stages of normal cell transformation is largely unknown. Using primary human melanocytes and biopsies of human naevi (moles), we show that the extent of ER stress induced by cellular oncogenes may define the mechanism of activation of premature senescence. Specifically, we found that oncogenic forms of HRAS (HRASG12V) but not its downstream target BRAF (BRAFV600E), engaged a rapid cell-cycle arrest that was associated with massive vacuolization and expansion of the ER. However, neither p53, p16INK4a nor classical senescence markers – such as foci of heterochromatin or DNA damage – were able to account for the specific response of melanocytes to HRASG12V. Instead, HRASG12V-driven senescence was mediated by the ER-associated unfolded protein response (UPR). The impact of HRAS on the UPR was selective, as it was poorly induced by activated NRAS (more frequently mutated in melanoma than HRAS). These results argue against premature senescence as a converging mechanism of response to activating oncogenes and support a direct role of the ER as a gatekeeper of tumour control.