Diagnostic cancer genome sequencing and the contribution of germline variants.

Whole-genome sequencing (WGS) is revolutionizing medical research and has the potential to serve as a powerful and cost-effective diagnostic tool in the management of cancer. We review the progress to date in the use of WGS to reveal how germline variants and mutations may be associated with cancer. We use colorectal cancer as an example of how the current level of knowledge can be translated into predictions of predisposition. We also address challenges in the clinical implementation of the variants in germline DNA identified through cancer genome sequencing. We call for the international development of standards to facilitate the clinical use of germline information arising from diagnostic cancer genome sequencing.

miR-433 is aberrantly expressed in myeloproliferative neoplasms and suppresses hematopoietic cell growth and differentiation.

BCR-ABL-negative myeloproliferative neoplasms (MPNs) are most frequently characterized by the JAK2V617F gain-of-function mutation, but several studies showed that JAK2V617F may not be the initiating event in MPN development, and recent publications indicate that additional alterations such as chromatin modification and microRNA (miRNA) deregulation may have an important role in MPN pathogenesis. Here we report that 61 miRNAs were significantly deregulated in CD34+ cells from MPN patients compared with controls (P<0.01). Global miRNA analysis also revealed that polycythemia vera (JAKV617F) and essential thrombocythemia (JAK2 wild type) patients have significantly different miRNA expression profiles from each other. Among the deregulated miRNAs, expression of miR-134, -214 and -433 was not affected by changes in JAK2 activity, suggesting that additional signaling pathways are responsible for the deregulation of these miRNAs in MPN. Despite its upregulation in MPN CD34+ and during normal erythropoiesis, both overexpression and knockdown studies suggest that miR-433 negatively regulates CD34+ proliferation and differentiation ex vivo. Its novel target GBP2 is downregulated during normal erythropoiesis and regulates proliferation and erythroid differentiation in TF-1 cells, indicating that miR-433 negatively regulates hematopoietic cell proliferation and erythropoiesis by directly targeting GBP2.

Somatic MED12 mutations in uterine leiomyosarcoma and colorectal cancer.

BACKGROUND: Mediator complex participates in transcriptional regulation by connecting regulatory DNA sequences to the RNA polymerase II initiation complex. Recently, we discovered through exome sequencing that as many as 70% of uterine leiomyomas harbour specific mutations in exon 2 of mediator complex subunit 12 (MED12). In this work, we examined the role of MED12 exon 2 mutations in other tumour types. METHODS: The frequency of MED12 exon 2 mutations was analysed in altogether 1158 tumours by direct sequencing. The tumour spectrum included mesenchymal tumours (extrauterine leiomyomas, endometrial polyps, lipomas, uterine leiomyosarcomas, other sarcomas, gastro-intestinal stromal tumours), hormone-dependent tumours (breast and ovarian cancers), haematological malignancies (acute myeloid leukaemias, acute lymphoid leukaemias, myeloproliferative neoplasms), and tumours associated with abnormal Wnt-signalling (colorectal cancers (CRC)). RESULTS: Five somatic alterations were observed: three in uterine leiomyosarcomas (3/41, 7%; Gly44Ser, Ala38_Leu39ins7, Glu35_Leu36delinsVal), and two in CRC (2/392, 0.5%; Gly44Cys, Ala67Val). CONCLUSION: Somatic MED12 exon 2 mutations were observed in uterine leiomyosarcomas, suggesting that a subgroup of these malignant tumours may develop from a leiomyoma precursor. Mutations in CRC samples indicate that MED12 may, albeit rarely, contribute to CRC tumorigenesis.

IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic- or blast-phase essential thrombocythemia, polycythemia vera or myelofibrosis.

In a multi-institutional collaborative project, 1473 patients with myeloproliferative neoplasms (MPN) were screened for isocitrate dehydrogenase 1 (IDH1)/IDH2 mutations: 594 essential thrombocythemia (ET), 421 polycythemia vera (PV), 312 primary myelofibrosis (PMF), 95 post-PV/ET MF and 51 blast-phase MPN. A total of 38 IDH mutations (18 IDH1-R132, 19 IDH2-R140 and 1 IDH2-R172) were detected: 5 (0.8%) ET, 8 (1.9%) PV, 13 (4.2%) PMF, 1 (1%) post-PV/ET MF and 11 (21.6%) blast-phase MPN (P<0.01). Mutant IDH was documented in the presence or absence of JAK2, MPL and TET2 mutations, with similar mutational frequencies. However, IDH-mutated patients were more likely to be nullizygous for JAK2 46/1 haplotype, especially in PMF (P=0.04), and less likely to display complex karyotype, in blast-phase disease (P<0.01). In chronic-phase PMF, JAK2 46/1 haplotype nullizygosity (P<0.01; hazard ratio (HR) 2.9, 95% confidence interval (CI) 1.7-5.2), but not IDH mutational status (P=0.55; HR 1.3, 95% CI 0.5-3.4), had an adverse effect on survival. This was confirmed by multivariable analysis. In contrast, in both blast-phase PMF (P=0.04) and blast-phase MPN (P=0.01), the presence of an IDH mutation predicted worse survival. The current study clarifies disease- and stage-specific IDH mutation incidence and prognostic relevance in MPN and provides additional evidence for the biological effect of distinct JAK2 haplotypes.

JAK2 and MPL mutations in myeloproliferative neoplasms: discovery and science.

Although it has long been known that the myeloproliferative neoplasms (MPN) polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are clonal hematopoietic stem-cell disorders, for many years the genetic basis for these disorders was elusive. A new era in MPN biology began in 2005 with the discovery of a somatic point mutation in JAK2 tyrosine kinase (JAK2V617F), which was identified in a significant proportion of patients with PV, ET and PMF. Based on the hypothesis that JAK-STAT signaling is central to the pathogenesis of JAK2V617F-negative MPN, genomic studies have identified JAK2 exon 12 mutations in JAK2V617F-negative PV and activating mutations in MPL in patients with JAK2V617F-negative ET and PMF. In this review, we will discuss the role of these mutant alleles in the pathogenesis of PV, ET and PMF, the potential therapeutic implications of these discoveries, and the implications of these discoveries for genomic studies of hematopoietic malignancies.

The DNA damage signalling kinase ATM is aberrantly reduced or lost in BRCA1/BRCA2-deficient and ER/PR/ERBB2-triple-negative breast cancer.

The ataxia-telangiectasia-mutated (ATM) kinase is a key transducer of DNA damage signals within the genome maintenance machinery and a tumour suppressor whose germline mutations predispose to familial breast cancer. ATM signalling is constitutively activated in early stages of diverse types of human malignancies and cell culture models in response to oncogene-induced DNA damage providing a barrier against tumour progression. As BRCA1 and BRCA2 are also components of the genome maintenance network and their mutations predispose to breast cancer, we have examined the ATM expression in human breast carcinomas of BRCA1/2 mutation carriers, sporadic cases and familial non-BRCA1/2 patients. Our results show that ATM protein expression is aberrantly reduced more frequently among BRCA1 (33%; P=0.0003) and BRCA2 (30%; P=0.0009) tumours than in non-BRCA1/2 tumours (10.7%). Furthermore, the non-BRCA1/2 tumours with reduced ATM expression were more often estrogen receptor (ER) negative (P=0.0002), progesterone receptor (PR) negative (P=0.004) and were of higher grade (P=0.0004). In our series of 1013 non-BRCA1/2 cases, ATM was more commonly deficient (20%; P=0.0006) and p53 was overabundant (47%; P<0.0000000001) among the difficult-to-treat ER/PR/ERBB2-triple-negative subset of tumours compared with cases that expressed at least one of these receptors (10 and 16% of aberrant ATM and p53, respectively). We propose a model of 'conditional haploinsufficiency' for BRCA1/2 under conditions of enhanced DNA damage in precancerous lesions resulting in more robust activation and hence increased selection for inactivation or loss of ATM in tumours of BRCA1/2 mutation carriers, with implications for genomic instability and curability of diverse subsets of human breast cancer.

CHEK2 I157T associates with familial and sporadic colorectal cancer.

BACKGROUND: Recently, a functionally defective CHEK2 variant I157T has been proposed to associate with an increased risk of several types of cancer. We investigated the CHEK2 I157T variant for colorectal cancer (CRC) predisposition in a large population based study including a significant number of familial CRC cases. METHODS: We screened the CHEK2 I157T variant in a population based series of 1042 Finnish CRC patients using restriction fragment length polymorphism. Mutation status was studied for correlation with clinical characteristics and family history of CRC and other cancers. RESULTS: The frequency of CHEK2 I157T was significantly higher in CRC patients (7.8%, 76/972) than in healthy population controls (5.3%, 100/1885) (OR = 1.5, 95% CI 1.1 to 2.1, p = 0.008). The significant association of CHEK2 I157T with CRC was observed among patients with (10.4%, 14/135) and without (7.4%, 62/837) a family history of CRC (OR = 2.1, 95% CI 1.1 to 3.7, p = 0.01; OR = 1.4, 95% CI 1.0 to 2.0, p = 0.03; respectively). A trend towards higher variant frequency was also noted among patients with multiple primary tumours and a family history of any cancer. CONCLUSIONS: CHEK2 I157T associates with an increased risk of CRC: the association was observed both among familial and sporadic CRC patients. Furthermore, the higher frequency of I157T among patients with multiple primary tumours as well as those with a family history of any cancer supports a role for CHEK2 I157T as a susceptibility allele for multiple cancer types.