Germline polymorphisms as biomarkers of tumor response in colorectal cancer patients treated with anti-EGFR monoclonal antibodies: a systematic review and meta-analysis.

Studies of germline polymorphisms as predictors of tumor response to anti-epidermal growth factor receptor (EGFR) monoclonal antibody agents in metastatic colorectal cancer have reported inconsistent results. We performed a systematic review of studies from 1990 to September 2015, followed by random-effects meta-analyses for polymorphisms examined in at least three studies. Of 87 studies, 40 passed the criteria for systematic review and 23 for meta-analysis. The polymorphisms suitable for meta-analysis were CCND1 (rs17852153), COX2 (rs20417), EGF (rs4444903), EGFR (rs712829, rs11543848, 3'UTR CA repeat), FCGR2A (rs1801274), FCGR3A (rs396991), IL8 (rs4073), KRAS (rs61764370) and VEGFA (rs3025039). Meta-analysis yielded nominal significance (at α=0.05) for rs4444903 and rs11543848, but showed no significant results after multiple testing correction; this was unchanged by sensitivity analyses to address subgroups, funnel-plot asymmetries, and study quality. This highlights a tendency for lack of replication in the face of initial positive results, and possibly the unsuitability of relying on tumor response as a surrogate marker in this setting.

Associations between the IASLC/ATS/ERS lung adenocarcinoma classification and EGFR and KRAS mutations.

We sought to investigate the frequency of mutations in epidermal growth factor receptor (EGFR) and Kirsten-RAS (KRAS) by each pathological subtype for patients with resected pulmonary adenocarcinoma as defined by the IASLC/ATS/ERS classification. Histological examination determined the predominant subtype according to the IASLC/ATS/ERS classification. EGFR and KRAS mutations were determined by high-resolution melting and Sanger sequencing. Clinical data were collected from medical records and clinicians. The 178 consecutive patients consisted of 48% males, median age 68 years (range 20-87) and smoking history 78%. The tumour stage was I in 62%, II in 18% and III in 20%. The mutation rates were: EGFR 30%; KRAS 28%. The rate of EGFR mutations in the acinar predominant reference group (n=76), was 37%. The solid predominant subtype showed significantly fewer EGFR mutations [3/33 (9%), odds ratio 0.17 (0.05-0.61), p=0.007]. No differences in mutation rate were observed in other subtypes. No association was found between KRAS mutations and predominant histological subtype. Advanced stage and solid predominant subtype were negative prognostic factors. EGFR mutations can be present in adenocarcinoma of any predominant subtype, however rarely in solid predominant tumours. No association was found between KRAS mutation and the predominant histological subtype.

Assessing the clinical value of targeted massively parallel sequencing in a longitudinal, prospective population-based study of cancer patients.

INTRODUCTION: Recent discoveries in cancer research have revealed a plethora of clinically actionable mutations that provide therapeutic, prognostic and predictive benefit to patients. The feasibility of screening mutations as part of the routine clinical care of patients remains relatively unexplored as the demonstration of massively parallel sequencing (MPS) of tumours in the general population is required to assess its value towards the health-care system. METHODS: Cancer 2015 study is a large-scale, prospective, multisite cohort of newly diagnosed cancer patients from Victoria, Australia with 1094 patients recruited. MPS was performed using the Illumina TruSeq Amplicon Cancer Panel. RESULTS: Overall, 854 patients were successfully sequenced for 48 common cancer genes. Accurate determination of clinically relevant mutations was possible including in less characterised cancer types; however, technical limitations including formalin-induced sequencing artefacts were uncovered. Applying strict filtering criteria, clinically relevant mutations were identified in 63% of patients, with 26% of patients displaying a mutation with therapeutic implications. A subset of patients was validated for canonical mutations using the Agena Bioscience MassARRAY system with 100% concordance. Whereas the prevalence of mutations was consistent with other institutionally based series for some tumour streams (breast carcinoma and colorectal adenocarcinoma), others were different (lung adenocarcinoma and head and neck squamous cell carcinoma), which has significant implications for health economic modelling of particular targeted agents. Actionable mutations in tumours not usually thought to harbour such genetic changes were also identified. CONCLUSIONS: Reliable delivery of a diagnostic assay able to screen for a range of actionable mutations in this cohort was achieved, opening unexpected avenues for investigation and treatment of cancer patients.

The role of BRAF mutations in primary melanoma growth rate and survival.

BACKGROUND: The clinical behaviour and prognosis of primary melanomas harbouring BRAF mutations is not fully understood. OBJECTIVES: To investigate the effect of mutation status on primary melanoma growth rate and melanoma-specific survival (MSS). METHODS: A prospective cohort of 196 patients with stage I-III primary cutaneous melanoma were followed for a median of 92 months, pre-dating the institution of BRAF inhibitor therapy. Clinicopathological variables were correlated with mutation status and hazard ratios (HRs) estimated for MSS. RESULTS: Of 196 tumours, 77 (39.2%) were BRAF V600E, 10 (5.1%) BRAF V600K and 33 (16.8%) were NRAS mutant. BRAF V600E mutant melanomas were associated with favourable clinical characteristics and tended to be slower growing compared with BRAF V600K, NRAS mutant or BRAF/NRAS wild-type tumours (0.12 mm per month, 0.61 mm per month, 0.36 mm per month and 0.23 mm per month, respectively; P = 0.05). There were 39 melanoma deaths, and BRAF mutant melanomas were associated with poorer MSS in stage I-III disease [HR 2.60, 95% confidence interval (CI) 1.20-5.63; P = 0.02] and stage I-II disease (HR 3.39, 95% CI 1.12-10.22; P = 0.03) after adjusting for other prognostic variables. Considered separately, BRAF V600E mutant melanomas were strongly associated with MSS independently of thickness and nodal status (HR 3.89, 95% CI 1.67-9.09; P < 0.01) but BRAF V600K mutant tumours were not (HR 1.19, 95% CI 0.36-3.92; P = 0.77). CONCLUSIONS: The presence of a BRAF mutation does not necessarily 'drive' more rapid tumour growth but is associated with poorer MSS in patients with early-stage disease.

Mutational profiling of familial male breast cancers reveals similarities with luminal A female breast cancer with rare TP53 mutations.

BACKGROUND: Male breast cancer (MBC) is still poorly understood with a large proportion arising in families with a history of breast cancer. Genomic studies have focused on germline determinants of MBC risk, with minimal knowledge of somatic changes in these cancers. METHODS: Using a TruSeq amplicon cancer panel, this study evaluated 48 familial MBCs (3 BRCA1 germline mutant, 17 BRCA2 germline mutant and 28 BRCAX) for hotspot somatic mutations and copy number changes in 48 common cancer genes. RESULTS: Twelve missense mutations included nine PIK3CA mutations (seven in BRCAX patients), two TP53 mutations (both in BRCA2 patients) and one PTEN mutation. Common gains were seen in GNAS (34.1%) and losses were seen in GNAQ (36.4%), ABL1 (47.7%) and ATM (34.1%). Gains of HRAS (37.5% vs 3%, P=0.006), STK11 (25.0% vs 0%, P=0.01) and SMARCB1 (18.8% vs 0%, P=0.04) and the loss of RB1 (43.8% vs 13%, P=0.03) were specific to BRCA2 tumours. CONCLUSIONS: This study is the first to perform high-throughput somatic sequencing on familial MBCs. Overall, PIK3CA mutations are most commonly seen, with fewer TP53 and PTEN mutations, similar to the profile seen in luminal A female breast cancers. Differences in mutation profiles and patterns of gene gains/losses are seen between BRCA2 (associated with TP53/PTEN mutations, loss of RB1 and gain of HRAS, STK11 and SMARCB1) and BRCAX (associated with PIK3CA mutations) tumours, suggesting that BRCA2 and BRCAX MBCs may be distinct and arise from different tumour pathways. This has implications on potential therapies, depending on the BRCA status of MBC patients.

Docetaxel plus cetuximab as second-line treatment for docetaxel-refractory oesophagogastric cancer: the AGITG ATTAX2 trial.

BACKGROUND: Cetuximab can reverse chemotherapy resistance in colorectal cancer. This study evaluated the efficacy and safety of the combination of docetaxel and cetuximab as a second-line treatment in docetaxel-refractory oesophagogastric cancer. METHODS: Patients received docetaxel 30 mg m(-2) on days 1 and 8, every 3 weeks and cetuximab 400 mg m(-2) on day 1, then 250 mg m(-2) weekly. Biomarker mutation analysis was performed. RESULTS: A total of 38 patients were enrolled. Response rates were PR 6% (95% CI 2-19%), s.d. 43% (95% CI 28-59%). Main grade 3/4 toxicities were febrile neutropenia, anorexia, nausea, diarrhoea, stomatitis, and acneiform rash. Median progression-free and overall survival were 2.1 and 5.4 months, respectively. A landmark analysis showed a trend to improved survival times with increased grade of acneiform rash. No KRAS, BRAF or PIK3CA mutations were observed. CONCLUSION: Cetuximab and docetaxel achieve modest responses rates, but maintain comparable survival times to other salvage regimens with low rates of toxicity.

Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer.

MicroRNAs (miRNAs) are small non-coding RNAs of ∼20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located ∼2 kb upstream of the 5' stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.

Using tumour pathology to identify people at high genetic risk of breast and colorectal cancers.

Genes have been identified for which germline mutations are associated with high lifetime risks of breast, colorectal and other cancers. Identification of mutation carriers through genetic testing is important as it could help lower cancer incidence and mortality. The translation of genetic information into better health outcomes is expensive because of the costs of genetic counselling as well as laboratory testing. Approaches to triage for mutation screening of known genes which rely on cancer family history are not necessarily sensitive and specific or the most cost-effective. Recent population-based research has shown that the cancers and precancerous lesions arising in mutation carriers have specific molecular and morphological characteristics. People with colorectal cancer, especially those diagnosed at a young age, whose tumours exhibit microsatellite instability and some specific pathology and immunohistochemically-defined features are more likely to carry a germline mutation in one of four mismatch repair genes. Some morphological and immunohistochemically-defined features are associated with breast cancers arising in women who carry BRCA1 or BRCA2 germline mutations, especially if at a young age. Screening paradigms based on molecular and morphological features that predict mutation status, especially if focused on early-onset disease, have the potential to identify mutation carriers with greater sensitivity and specificity, and in a more cost-effective way, than those based on family history alone. Genetic testing results could help inform treatment if those affected are tested soon after diagnosis using pathology-led selection strategies to identify cases most likely to carry germline mutations. We propose how this new approach could be undertaken by having genetic testing and counselling prioritised to those with the greatest probability of carrying a germline mutation in these known cancer predisposition genes.

Clinical responses observed with imatinib or sorafenib in melanoma patients expressing mutations in KIT.

BACKGROUND: Mutations in KIT are more frequent in specific melanoma subtypes, and response to KIT inhibition is likely to depend on the identified mutation. METHODS: A total of 32 patients with metastatic acral or mucosal melanoma were screened for mutations in KIT exons 11, 13 and 17. RESULTS: KIT mutations were found in 38% of mucosal and in 6% of acral melanomas. Three patients were treated with imatinib and one with sorafenib. All four patients responded to treatment, but three have since progressed within the brain. CONCLUSION: The observed clinical responses support further investigation of KIT inhibitors in metastatic melanoma, selected according to KIT mutation status.

In vitro sensitivity testing of minimally passaged and uncultured gliomas with TRAIL and/or chemotherapy drugs.

TRAIL/Apo-2L has shown promise as an anti-glioma drug, based on investigations of TRAIL sensitivity in established glioma cell lines, but it is not known how accurately TRAIL signalling pathways of glioma cells in vivo are reproduced in these cell lines in vitro. To replicate as closely as possible the in vivo behaviour of malignant glioma cells, 17 early passage glioma cell lines and 5 freshly resected gliomas were exposed to TRAIL-based agents and/or chemotherapeutic drugs. Normal human hepatocytes and astrocytes and established glioma cell lines were also tested. Cross-linked TRAIL, but not soluble TRAIL, killed both normal cell types and cells from three tumours. Cells from only one glioma were killed by soluble TRAIL, although only inefficiently. High concentrations of cisplatin were lethal to glioma cells, hepatocytes and astrocytes. Isolated combinations of TRAIL and chemotherapy drugs were more toxic to particular gliomas than normal cells, but no combination was generally selective for glioma cells. This study highlights the widespread resistance of glioma cells to TRAIL-based agents, but suggests that a minority of high-grade glioma patients may benefit from particular combinations of TRAIL and chemotherapy drugs. In vitro sensitivity assays may help identify effective drug combinations for individual glioma patients.

Incorporation of somatic BRAF mutation testing into an algorithm for the investigation of hereditary non-polyposis colorectal cancer.

Patients suspected on clinical grounds to have hereditary non-polyposis colorectal cancer (HNPCC) may be offered laboratory testing in order to confirm the diagnosis and to facilitate screening of pre-symptomatic family members. Tumours from an affected family member are usually pre-screened for microsatellite instability (MSI) and/or loss of immunohistochemical expression of mismatch repair (MMR) genes prior to germline MMR gene mutation testing. The efficiency of this triage process is compromised by the more frequent occurrence of sporadic colorectal cancer (CRC) showing high levels of MSI (MSI-H) due to epigenetic loss of MLH1 expression. Somatic BRAF mutations, most frequently V600E, have been described in a significant proportion of sporadic MSI-H CRC but not in HNPCC-associated cancers. BRAF mutation testing has therefore been proposed as a means to more definitively identify and exclude sporadic MSI-H CRC cases from germline MMR gene testing. However, the clinical validity and utility of this approach have not been previously evaluated in a familial cancer clinic setting. Testing for the V600E mutation was performed on MSI-H CRC samples from 68 individuals referred for laboratory investigation of suspected HNPCC. The V600E mutation was identified in 17 of 40 (42%) tumours showing loss of MLH1 protein expression by immunohistochemistry but in none of the 28 tumours that exhibited loss of MSH2 expression (P < 0.001). The assay was negative in all patients with an identified germline MMR gene mutation. Although biased by the fact that germline testing was not pursued beyond direct sequencing in many cases lacking a high clinical index of suspicion of HNPCC, BRAF V600E detection was therefore considered to be 100% specific and 48% sensitive in detecting sporadic MSI-H CRC amongst those cases showing loss of MLH1 protein expression, in a population of patients with MSI-H CRC and clinical features suggestive of HNPCC. Accordingly, we recommend the incorporation of BRAF V600E mutation testing into the laboratory algorithm for pre-screening patients with suspected HNPCC, whose CRCs show loss of expression of MLH1. In such tumours, the presence of a BRAF V600E mutation indicates the tumour is not related to HNPCC and that germline testing of MLH1 in that individual is not warranted. We also recommend that in families where the clinical suspicion of HNPCC is high, germline testing should not be performed on an individual whose CRC harbours a somatic BRAF mutation, as this may compromise identification of the familial mutation.

KIT immunohistochemistry and mutation status in gastrointestinal stromal tumours (GISTs) evaluated for treatment with imatinib.

AIMS: With the availability of effective but expensive treatment in the form of imatinib, accurate diagnosis of gastrointestinal stromal tumour (GIST) is extremely important. The aims of this study were: to describe the clinicopathological, immunohistochemical and molecular features of cases referred to a cancer centre with a possible diagnosis of GIST; to identify pitfalls in the performance and interpretation of KIT immunohistochemistry; to define the role of KIT mutation testing in making a diagnosis of GIST. METHODS AND RESULTS: Morphological review, KIT immunohistochemistry and mutation testing were performed on all cases referred with a diagnosis of GIST or where the diagnosis was under serious consideration on the basis of KIT immunopositivity with a view to treating with imatinib. Thirty-seven cases met the inclusion criteria. Of these, 26 were classified as GIST and 11 as non-GIST. Most GISTs showed strong diffuse membranous, cytoplasmic or paranuclear KIT immunopositivity. Some non-GISTs demonstrated patchy cytoplasmic KIT immunopositivity related to the immunohistochemical protocol used in the external laboratory, which led to erroneous diagnoses of GIST in nine (24%) cases. KIT mutations involving exons 11 or 9 were identified in 22 (88%) GISTs tested and none of the non-GISTs. CONCLUSIONS: An accurate diagnosis of GIST can be made on clinicopathological and immunohistochemical criteria without the need for mutational analysis in most cases, provided proper attention is paid to the immunohistochemical protocol used and, most importantly, control material. False-positive diagnoses of GIST potentially leading to inappropriate treatment with imatinib are more common than missed diagnoses.

Fusion of the NUP98 gene with the LEDGF/p52 gene defines a recurrent acute myeloid leukemia translocation.

BACKGROUND: The NUP98 gene is involved in multiple rearrangements in haematological malignancy. The leukemic cells in an acute myeloid leukemia (AML) patient with a t(9;11)(p22;p15) were recently shown to have a fusion between the NUP98 gene and the LEDGF gene but it was not demonstrated that this fusion was recurrent in other leukaemia patients with the same translocation. RESULTS: We used RT-PCR to analyse the leukemic cells from an AML patient who presented with a cytogenetically identical translocation as the sole chromosomal abnormality. A NUP98-LEDGF fusion transcript was observed and confirmed by sequencing. The reciprocal transcript was also observed. The fusion transcript was not detectable during remission and recurred at relapse. The breakpoints in the NUP98 and LEDGF genes were different to those previously reported. The NUP98 breakpoint occurs in the intron between exons 8 and 9. It is the most 5' breakpoint reported in a translocation involving the NUP98 gene. All of the LEDGF gene is included in the fusion except for exon 1 which codes for the first 24 amino terminal amino acids. CONCLUSIONS: Our results show that fusion of the NUP98 and LEDGF genes is a new recurrent translocation in AML.

Loss of red cell A, B, and H antigens is frequent in myeloid malignancies.

Loss of A, B, and H antigens from the surface of red blood cells has been a recurrent observation in patients with hematologic malignancy, particularly those malignancies in which the myeloid lineage is involved. To better understand this phenomenon, a 2-color flow cytometric method was developed to determine quantitative and qualitative alterations of A, B, and H antigens in patients with myeloid malignancies. Characteristic patterns, dependent on the genotype, were seen for healthy individuals from each of the blood groups. Fifty-five percent (16/29) of patients of blood group A, B, or AB had a proportion of red cells with decreased expression of A or B antigens compared with no changes in 127 healthy A, B, and AB individuals. In most cases, the changes were not detected by routine serologic typing. The loss of A or B antigens was the primary change in 28% (8/29) of patients. In 17% (5/29) of patients, loss of A or B antigens was an indirect consequence of loss of the precursor H antigen. Alterations involving both the H and the A or B antigens were seen in 10% (3/29) of patients. Loss of H was also detected in 21% (6/28) of group O patients whereas none of 51 healthy O individuals showed changes. Alterations of ABO antigens can now be considered a common event in myeloid malignancy. (Blood. 2001;97:3633-3639)

Molecular detection of blood-borne epithelial cells in colorectal cancer patients and in patients with benign bowel disease.

In colorectal cancer (CRC), a proportion of patients with early stage disease still die of metastatic or recurrent disease within 5 years of "curative" resection. Detection of carcinoma cells in the peripheral circulation at presentation may identify a subgroup of patients with micro-metastatic disease who may benefit from adjuvant chemotherapy or radiotherapy. Our aim was to determine the presence and clinical significance of colon carcinoma cells in peripheral blood at the time of surgery. Preoperative peripheral blood samples were collected from 94 patients with CRC and 64 patients undergoing bowel resection for benign conditions (adenoma, diverticular disease or Crohn's colitis). Blood was also obtained from 20 normal donors not undergoing bowel surgery. Immunomagnetic beads were used to isolate epithelial cells followed by reverse transcription-polymerase chain reaction (RT-PCR) analysis of expression of cytokeratin (CK) 19, CK 20, mucin (MUC) 1 and MUC 2. Nineteen of 94 (20%) CRC patients were positive for epithelial cells in preoperative blood, including 6 with early stage disease. Kaplan-Meier survival analysis showed that detection of epithelial cells in preoperative blood was associated with reduced disease-free and overall survival (log-rank test, p = 0.0001). Surprisingly, circulating epithelial cells were detected in 3/30 (10%) patients resected for adenoma, and in 4/34 (12%) patients resected for benign inflammatory conditions, suggesting that cells from nonmalignant colonic epithelium may also gain entry into the bloodstream in the presence of bowel pathology. All 20 normal control bloods were negative for epithelial cells.

Tumour-specific distribution of BRCA1 promoter region methylation supports a pathogenetic role in breast and ovarian cancer.

The role of BRCA1 in sporadic breast and ovarian cancers remains elusive. Direct involvement of BRCA1 in the development of breast and ovarian cancer is suggested by the finding that the BRCA1 promoter region CpG island is methylated in a proportion of breast and ovarian cancers. The aim of this study was to compare the incidence of BRCA1 promoter region methylation in tumours in which loss of BRCA1 has been shown to play a role in pathogenesis (breast and ovarian carcinomas) with the incidence in tumours in which BRCA1 is unlikely to play a role in pathogenesis. Promoter region hypermethylation was significantly more common (P < 0.008) in breast and ovarian cancer (6/38 tumours methylated) than in colon cancer (0/35 tumours methylated) or in leukaemias (0/19 samples methylated). The restriction of BRCA1 promoter region hypermethylation to breast and ovarian cancer is consistent with a pathogenetic role of BRCA1 promoter methylation in these tumours. We suggest that the rarity of observed BRCA1 mutations in sporadic breast and ovarian cancer is due to the greater likelihood of BRCA1 inactivation by non-mutational mechanisms such as methylation.

Promoter region methylation does not account for the frequent loss of expression of the Fas gene in colorectal carcinoma.

Expression of the apoptosis-promoting Fas gene is frequently reduced or lost during the development of colorectal carcinoma. However, loss of heterozygosity at the Fas locus or Fas gene rearrangements do not account for the loss of expression of Fas, raising the possibility that methylation of the Fas promoter may inhibit gene expression in colorectal carcinomas. We have examined the Fas promoter region CpG island for evidence of hypermethylation in colorectal tumours. Forty-seven specimens of colorectal adenoma and carcinoma, as well as six samples of normal colonic mucosa, were examined by Southern blotting for methylation at HpaII and Cfol sites in this region. No methylation was detected in any of the specimens, suggesting that hypermethylation is not primarily responsible for the loss of expression of the Fas gene during colorectal tumorigenesis.