Giovanni Parmigiani

An Integrated Genomic Analysis of Human Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. To identify the genetic alterations in GBMs, we sequenced 20,661 protein coding genes, determined the presence of amplifications and deletions using high-density oligonucleotide arrays, and performed gene expression analyses using next-generation sequencing technologies in 22 human tumor samples. This comprehensive analysis led to the discovery of a variety of genes that were not known to be altered in GBMs. Most notably, we found recurrent mutations in the active site of isocitrate dehydrogenase 1 (IDH1) in 12% of GBM patients. Mutations in IDH1 occurred in a large fraction of young patients and in most patients with secondary GBMs and were associated with an increase in overall survival. These studies demonstrate the value of unbiased genomic analyses in the characterization of human brain cancer and identify a potentially useful genetic alteration for the classification and targeted therapy of GBMs.

The genomic landscapes of human breast and colorectal cancers.

Human cancer is caused by the accumulation of mutations in oncogenes and tumor suppressor genes. To catalog the genetic changes that occur during tumorigenesis, we isolated DNA from 11 breast and 11 colorectal tumors and determined the sequences of the genes in the Reference Sequence database in these samples. Based on analysis of exons representing 20,857 transcripts from 18,191 genes, we conclude that the genomic landscapes of breast and colorectal cancers are composed of a handful of commonly mutated gene “mountains” and a much larger number of gene “hills” that are mutated at low frequency. We describe statistical and bioinformatic tools that may help identify mutations with a role in tumorigenesis. These results have implications for understanding the nature and heterogeneity of human cancers and for using personal genomics for tumor diagnosis and therapy.

Meta-analysis of BRCA1 and BRCA2 penetrance

PURPOSE Genetic counseling is now routinely offered to individuals at high risk of carrying a BRCA1 or BRCA2 mutation. Risk prediction provided by the counselor requires reliable estimates of the mutation penetrance. Such penetrance has been investigated by studies worldwide. The reported estimates vary. To facilitate clinical management and counseling of the at-risk population, we address this issue through a meta-analysis. METHODS We conducted a literature search on PubMed and selected studies that had nonoverlapping patient data, contained genotyping information, used statistical methods that account for the ascertainment, and reported risks in a useable format. We subsequently combined the published estimates using the DerSimonian and Laird random effects modeling approach. RESULTS Ten studies were eligible under the selection criteria. Between-study heterogeneity was observed. Study population, mutation type, design, and estimation methods did not seem to be systematic sources of heterogeneity. Meta-analytic mean cumulative cancer risks for mutation carriers at age 70 years were as follows: breast cancer risk of 57% (95% CI, 47% to 66%) for BRCA1 and 49% (95% CI, 40% to 57%) for BRCA2 mutation carriers; and ovarian cancer risk of 40% (95% CI, 35% to 46%) for BRCA1 and 18% (95% CI, 13% to 23%) for BRCA2 mutation carriers. We also report the prospective risks of developing cancer for currently asymptomatic carriers. CONCLUSION This article provides a set of risk estimates for BRCA1 and BRCA2 mutation carriers that can be used by counselors and clinicians who are interested in advising patients based on a comprehensive set of studies rather than one specific study.

Determining carrier probabilities for breast cancer-susceptibility genes BRCA1 and BRCA2.

Breast cancer-susceptibility genes BRCA1 and BRCA2 have recently been identified on the human genome. Women who carry a mutation of one of these genes have a greatly increased chance of developing breast and ovarian cancer, and they usually develop the disease at a much younger age, compared with normal individuals. Women can be tested to see whether they are carriers. A woman who undergoes genetic counseling before testing can be told the probabilities that she is a carrier, given her family history. In this paper we develop a model for evaluating the probabilities that a woman is a carrier of a mutation of BRCA1 and BRCA2, on the basis of her family history of breast and ovarian cancer in first- and second-degree relatives. Of special importance are the relationships of the family members with cancer, the ages at onset of the diseases, and the ages of family members who do not have the diseases. This information can be elicited during genetic counseling and prior to genetic testing. The carrier probabilities are obtained from Bayess rule, by use of family history as the evidence and by use of the mutation prevalences as the prior distribution. In addressing an individuals carrier probabilities, we incorporate uncertainty about some of the key inputs of the model, such as the age-specific incidence of diseases and the overall prevalence of mutations. There is some evidence that other, undiscovered genes may be important in explaining familial breast cancer. Users of the current version of the model should be aware of this limitation. The methodology that we describe can be extended to more than two genes, should data become available about other genes.

The genetic landscape of the childhood cancer medulloblastoma

Genomic analysis of a childhood cancer reveals markedly fewer mutations than what is typically seen in adult cancers. Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.

Comparative lesion sequencing provides insights into tumor evolution

We show that the times separating the birth of benign, invasive, and metastatic tumor cells can be determined by analysis of the mutations they have in common. When combined with prior clinical observations, these analyses suggest the following general conclusions about colorectal tumorigenesis: (i) It takes ≈17 years for a large benign tumor to evolve into an advanced cancer but <2 years for cells within that cancer to acquire the ability to metastasize; (ii) it requires few, if any, selective events to transform a highly invasive cancer cell into one with the capacity to metastasize; (iii) the process of cell culture ex vivo does not introduce new clonal mutations into colorectal tumor cell populations; and (iv) the rates at which point mutations develop in advanced cancers are similar to those of normal cells. These results have important implications for understanding human tumor pathogenesis, particularly those associated with metastasis.

Exomic Sequencing Identifies PALB2 as a Pancreatic Cancer Susceptibility Gene

Through complete sequencing of the protein-coding genes in a patient with familial pancreatic cancer, we identified a germline, truncating mutation in PALB2 that appeared responsible for this patients predisposition to the disease. Analysis of 96 additional patients with familial pancreatic cancer revealed three distinct protein-truncating mutations, thereby validating the role of PALB2 as a susceptibility gene for pancreatic cancer. PALB2 mutations have been previously reported in patients with familial breast cancer, and the PALB2 protein is a binding partner for BRCA2. These results illustrate that complete, unbiased sequencing of protein-coding genes can lead to the identification of a gene responsible for a hereditary disease.

BRCAPRO Validation, Sensitivity of Genetic Testing of BRCA1/BRCA2, and Prevalence of Other Breast Cancer Susceptibility Genes

PURPOSE To compare genetic test results for deleterious mutations of BRCA1 and BRCA2 with estimated probabilities of carrying such mutations; to assess sensitivity of genetic testing; and to assess the relevance of other susceptibility genes in familial breast and ovarian cancer. PATIENTS AND METHODS Data analyzed were from six high-risk genetic counseling clinics and concern individuals from families for which at least one member was tested for mutations at BRCA1 and BRCA2. Predictions of genetic predisposition to breast and ovarian cancer for 301 individuals were made using BRCAPRO, a statistical model and software using Mendelian genetics and Bayesian updating. Model predictions were compared with the results of genetic testing. RESULTS Among the test individuals, 126 were Ashkenazi Jewish, three were male subjects, 243 had breast cancer, 49 had ovarian cancer, 34 were unaffected, and 139 tested positive for BRCA1 mutations and 29 for BRCA2 mutations. BRCAPRO performed well: for the 150 probands with the smallest BRCAPRO carrier probabilities (average, 29.0%), the proportion testing positive was 32.7%; for the 151 probands with the largest carrier probabilities (average, 95.2%), 78.8% tested positive. Genetic testing sensitivity was estimated to be at least 85%, with false-negatives including mutations of susceptibility genes heretofore unknown. CONCLUSION BRCAPRO is an accurate counseling tool for determining the probability of carrying mutations of BRCA1 and BRCA2. Genetic testing for BRCA1 and BRCA2 is highly sensitive, missing an estimated 15% of mutations. In the populations studied, breast cancer susceptibility genes other than BRCA1 and BRCA2 either do not exist, are rare, or are associated with low disease penetrance.

Analysis of the human protein interactome and comparison with yeast, worm and fly interaction datasets.

We present the first analysis of the human proteome with regard to interactions between proteins. We also compare the human interactome with the available interaction datasets from yeast (Saccharomyces cerevisiae), worm (Caenorhabditis elegans) and fly (Drosophila melanogaster). Of >70,000 binary interactions, only 42 were common to human, worm and fly, and only 16 were common to all four datasets. An additional 36 interactions were common to fly and worm but were not observed in humans, although a coimmunoprecipitation assay showed that 9 of the interactions do occur in humans. A re-examination of the connectivity of essential genes in yeast and humans indicated that the available data do not support the presumption that the number of interaction partners can accurately predict whether a gene is essential. Finally, we found that proteins encoded by genes mutated in inherited genetic disorders are likely to interact with proteins known to cause similar disorders, suggesting the existence of disease subnetworks. The human interaction map constructed from our analysis should facilitate an integrative systems biology approach to elucidating the cellular networks that contribute to health and disease states.

Detection of Chromosomal Alterations in the Circulation of Cancer Patients with Whole-Genome Sequencing

Massively parallel sequencing directly detects tumor-derived chromosomal alterations in plasma DNA from cancer patients. Getting Harder to Hide It might be challenging, but game players can usually answer the question: “Where’s Waldo?” After all, we’ve met the traveler before and can comb the baroque illustrations for his characteristic striped ensemble and walking stick. But if we didn’t know what Waldo looked like, it would take a powerful detective and at least a clue or two to find him in a crowd. Now, Leary et al. use a well-characterized clue—the universal nature of chromosomal alterations in human cancer—along with powerful DNA sequencing technology to pinpoint tumor-specific chromosomal aberrations in the circulation of patients without knowing, in advance, precisely what the edited DNA looks like. The authors compared circulating cell-free DNA from 10 late-stage colorectal and breast cancer patients and 10 healthy individuals using massively parallel whole-genome sequencing (WGS) and detected chromosomal aberrations—copy number changes and rearrangements—present only in plasma DNA from patients. Two known cancer driver genes were amplified in the patients: ERBB2, which encodes HER2/Neu, the protein target of the anticancer drug trastuzumab, and a cell-cycle regulatory gene, CDK6. For three colorectal cancer cases where both tumor and blood samples were analyzed by WGS, the copy number patterns observed in blood samples resembled those of the resected tumor. The authors quantified the ability of their approach to discriminate between cancer patients and healthy subjects by analyzing simulated mixtures of varying concentrations of tumor and control DNA. Under certain defined conditions, tumor DNA concentrations of ≥0.75% could be detected in the circulation of breast and colorectal cancer patients with a sensitivity >90% and a specificity >99%. Leary et al. outline several current limitations of their method. For example, the patients studied were all in the late stages of cancer progression, and the sensitivity and specificity parameters were dependent on the amount of sequence data obtained. As the cost of WGS falls, this new approach may provide a powerful way to detect cancers in a noninvasive and unbiased manner even without prior knowledge of disease. Clinical management of cancer patients could be improved through the development of noninvasive approaches for the detection of incipient, residual, and recurrent tumors. We describe an approach to directly identify tumor-derived chromosomal alterations through analysis of circulating cell-free DNA from cancer patients. Whole-genome analyses of DNA from the plasma of 10 colorectal and breast cancer patients and 10 healthy individuals with massively parallel sequencing identified, in all patients, structural alterations that were not present in plasma DNA from healthy subjects. Detected alterations comprised chromosomal copy number changes and rearrangements, including amplification of cancer driver genes such as ERBB2 and CDK6. The level of circulating tumor DNA in the cancer patients ranged from 1.4 to 47.9%. The sensitivity and specificity of this approach are dependent on the amount of sequence data obtained and are derived from the fact that most cancers harbor multiple chromosomal alterations, each of which is unlikely to be present in normal cells. Given that chromosomal abnormalities are present in nearly all human cancers, this approach represents a useful method for the noninvasive detection of human tumors that is not dependent on the availability of tumor biopsies.