Pharmacogenomics and metastatic colorectal cancer:current knowledge and perspectives

The pharmacogenomics field is crucial for optimizing the selection of which chemotherapy regimen to use according to the patient's genomic profile. Indeed, the individual's inherited genome accounts for a large proportion of the variation in his or her response to chemotherapeutic agents both in terms of efficiency and toxicity. Patients with metastatic disease are more likely to receive different lines of chemotherapy with variable efficacy and experience some related complications. It is therefore critical to tailor the best therapeutic arsenal to improve the efficacy and avoid as much as possible related complications that are susceptible to interrupt the treatment. The pharmacogenomics approach investigates for each drug the implicated metabolic pathway and the potential personal variations in gene function. The aim of this review is to present a clear overview of the most accurate polymorphisms that have been identified as related to drug response in patients with mCRC.

Common cancer stem cell gene variants predict colon cancer recurrence

PURPOSE: Recent evidence suggests that cancer stem cells (CSC) are responsible for key elements of colon cancer progression and recurrence. Germline variants in CSC genes may result in altered gene function and/or activity, thereby causing interindividual differences in a patient's tumor recurrence capacity and chemoresistance. We investigated germline polymorphisms in a comprehensive panel of CSC genes to predict time to tumor recurrence (TTR) in patients with stage III and high-risk stage II colon cancer.

EXPERIMENTAL DESIGN: A total of 234 patients treated with 5-fluorouracil-based chemotherapy at the University of Southern California were included in this study. Whole blood samples were analyzed for germline polymorphisms in genes that have been previously associated with colon CSC (CD44, Prominin-1, DPP4, EpCAM, ALCAM, Msi-1, ITGB1, CD24, LGR5, and ALDH1A1) by PCR-RFLP or direct DNA-sequencing.

RESULTS: The minor alleles of CD44 rs8193 C>T, ALCAM rs1157 G>A, and LGR5 rs17109924 T>C were significantly associated with increased TTR (9.4 vs. 5.4 years; HR, 0.51; 95% CI: 0.35-0.93; P = 0.022; 11.3 vs. 5.7 years; HR, 0.56; 95% CI: 0.33-0.94; P = 0.024, and 10.7 vs. 5.7 years; HR, 0.33; 95% CI: 0.12-0.90; P = 0.023, respectively) and remained significant in the multivariate analysis stratified by ethnicity. In recursive partitioning, a specific gene variant profile including LGR5 rs17109924, CD44 rs8193, and ALDH1A1 rs1342024 represented a high-risk subgroup with a median TTR of 1.7 years (HR, 6.71, 95% CI: 2.71-16.63, P < 0.001).

CONCLUSION: This is the first study identifying common germline variants in colon CSC genes as independent prognostic markers for stage III and high-risk stage II colon cancer patients.

A review of excision repair cross-complementation group 1 in colorectal cancer

Oxaliplatin-based chemotherapy is the standard of care in patients with high-risk stage II and stage III colorectal cancer as well as in patients with advanced disease. Unfortunately, a large proportion of patients offered oxaliplatin fail to benefit from it. In the era of personalized treatment, there are strong efforts to identify biomarkers that will predict efficacy to oxaliplatin-based treatments. Excision repair cross-complementation group 1 (ERCC1) is a key element in the nucleotide excision repair (NER) pathway, which is responsible for repairing DNA adducts induced by platinum compounds. ERCC1 has recently been shown to be closely associated with outcome in patients with non-small-cell lung cancer (NSCLC): both high ERCC1 protein and gene expression are associated with resistance to cisplatin-based chemotherapy and better outcome without treatment. Therefore, ERCC1 has the potential to be used as a strong candidate biomarker, both predictive and prognostic, for colorectal cancer. This review will focus on the preclinical and clinical evidences supporting ERCC1 as a major molecule in oxaliplatin resistance. In addition, the important technologies used to assess ERCC1 gene and protein expression will be highlighted.

Predictive molecular classifiers in colorectal cancer

The introduction of predictive molecular markers has radically enhanced the identification of which patients may benefit from a given treatment. Despite recent controversies, KRAS mutation is currently the most recognized molecular predictive marker in colorectal cancer (CRC), predicting efficacy of anti-epidermal growth factor receptor (anti-EGFR) antibodies. However, other relevant markers have been reported and claimed to identify patients that will benefit from anti-EGFR therapies. This group of markers includes BRAF mutations, PI3KCA mutations, and loss of PTEN expression. Similarly, molecular markers for cytotoxic agents' efficacy also may predict outcome in patients with CRC. This review aims to summarize the most important predictive molecular classifiers in patients with CRC and further discuss any inconsistent or conflicting findings for these molecular classifiers.

Pharmacogenetic angiogenesis profiling for first-line Bevacizumab plus oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer

PURPOSE: There is substantial germline genetic variability within angiogenesis pathway genes, thereby causing interindividual differences in angiogenic capacity and resistance to antiangiogenesis therapy. We investigated germline polymorphisms in genes involved in VEGF-dependent and -independent angiogenesis pathways to predict clinical outcome and tumor response in metastatic colorectal cancer (mCRC) patients treated with bevacizumab and oxaliplatin-based chemotherapy.

EXPERIMENTAL DESIGN: A total of 132 patients treated with first-line bevacizumab and FOLFOX or XELOX were included in this study. Genomic DNA was isolated from whole-blood samples by PCR-RFLP or direct DNA sequencing. The endpoints of the study were progression-free survival (PFS), overall survival (OS), and response rate (RR).

RESULTS: The minor alleles of EGF rs444903 A>G and IGF-1 rs6220 A>G were associated with increased OS and remained significant in multivariate Cox regression analysis (HR: 0.52; 95% CI: 0.31-0.87; adjusted P = 0.012 and HR: 0.60; 95% CI: 0.36-0.99; adjusted P = 0.046, respectively). The minor allele of HIF1α rs11549465 C>T was significantly associated with increased PFS but lost its significance in multivariate analysis. CXCR1 rs2234671 G>C, CXCR2 rs2230054 T>C, EGFR rs2227983 G>A, and VEGFR-2 rs2305948 C>T predicted tumor response, with CXCR1 rs2234671 G>C remaining significant in multiple testing (P(act) = 0.003).

CONCLUSION: In this study, we identified common germline variants in VEGF-dependent and -independent angiogenesis genes predicting clinical outcome and tumor response in patients with mCRC receiving first-line bevacizumab and oxaliplatin-based chemotherapy.

EGF61 polymorphism predicts complete pathologic response to cetuximab-based chemoradiation independent of KRAS status in locally advanced rectal cancer patients

BACKGROUND: Cetuximab has shown significant clinical activity in metastatic colon cancer. However, cetuximab-containing neoadjuvant chemoradiation has not been shown to improve tumor response in locally advanced rectal cancer patients in recent phase I/II trials. We evaluated functional germline polymorphisms of genes involved in epidermal growth factor receptor pathway, angiogenesis, antibody-dependent cell-mediated cytotoxicity, DNA repair, and drug metabolism, for their potential role as molecular predictors for clinical outcome in locally advanced rectal cancer patients treated with preoperative cetuximab-based chemoradiation.

METHODS: 130 patients (74 men and 56 women) with locally advanced rectal cancer (4 with stage II, 109 with stage III, and 15 with stage IV, 2 unknown) who were enrolled in phase I/II clinical trials treated with cetuximab-based chemoradiation in European cancer centers were included. Genomic DNA was extracted from formalin-fixed paraffin-embedded tumor samples and genotyping was done by using PCR-RFLP assays. Fisher's exact test was used to examine associations between polymorphisms and complete pathologic response (pCR) that was determined by a modified Dworak classification system (grade III vs. grade IV: complete response).

RESULTS: Patients with the epidermal growth factor (EGF) 61 G/G genotype had pCR of 45% (5/11), compared with 21% (11/53) in patients heterozygous, and 2% (1/54) in patients homozygous for the A/A allele (P < 0.001). In addition, this association between EGF 61 G allele and pCR remained significant (P = 0.019) in the 59 patients with wild-type KRAS.

CONCLUSION: This study suggested EGF A+61G polymorphism to be a predictive marker for pCR, independent of KRAS mutation status, to cetuximab-based neoadjuvant chemoradiation of patients with locally advanced rectal cancer.

Gender-specific genomic profiling in metastatic colorectal cancer patients treated with 5-fluorouracil and oxaliplatin

AIMS: Survival and response rates in metastatic colorectal cancer remain poor, despite advances in drug development. There is increasing evidence to suggest that gender-specific differences may contribute to poor clinical outcome. We tested the hypothesis that genomic profiling of metastatic colorectal cancer is dependent on gender.

MATERIALS & METHODS: A total of 152 patients with metastatic colorectal cancer who were treated with oxaliplatin and continuous infusion 5-fluorouracil were genotyped for 21 polymorphisms in 13 cancer-related genes by PCR. Classification and regression tree analysis tested for gender-related association of polymorphisms with overall survival, progression-free survival and tumor response.

RESULTS: Classification and regression tree analysis of all polymorphisms, age and race resulted in gender-specific predictors of overall survival, progression-free survival and tumor response. Polymorphisms in the following genes were associated with gender-specific clinical outcome: estrogen receptor β, EGF receptor, xeroderma pigmentosum group D, voltage-gated sodium channel and phospholipase A2.

CONCLUSION: Genetic profiling to predict the clinical outcome of patients with metastatic colorectal cancer may depend on gender.

Learning from the QUEST multicentre feasibility randomization trials in breast reconstruction after mastectomy

BACKGROUND: Breast reconstruction aims to improve health-related quality of life after mastectomy. However, evidence guiding patients and surgeons in shared decision-making concerning the optimal type or timing of surgery is lacking.

METHODS: QUEST comprised two parallel feasibility phase III randomized multicentre trials to assess the impact of the type and timing of latissimus dorsi breast reconstruction on health-related quality of life when postmastectomy radiotherapy is unlikely (QUEST A) or highly probable (QUEST B). The primary endpoint for the feasibility phase was the proportion of women who accepted randomization, and it would be considered feasible if patient acceptability rates exceeded 25 per cent of women approached. A companion QUEST Perspectives Study (QPS) of patients (both accepting and declining trial participation) and healthcare professionals assessed trial acceptability.

RESULTS: The QUEST trials opened in 15 UK centres. After 18 months of recruitment, 17 patients were randomized to QUEST A and eight to QUEST B, with overall acceptance rates of 19 per cent (17 of 88) and 22 per cent (8 of 36) respectively. The QPS recruited 56 patients and 51 healthcare professionals. Patient preference was the predominant reason for declining trial entry, given by 47 (53 per cent) of the 88 patients approached for QUEST A and 22 (61 per cent) of the 36 approached for QUEST B. Both trials closed to recruitment in December 2012, acknowledging the challenges of achieving satisfactory patient accrual.

CONCLUSION: Despite extensive efforts to overcome recruitment barriers, it was not feasible to reach timely recruitment targets within a feasibility study. Patient preferences for breast reconstruction types and timings were common, rendering patients unwilling to enter the trial.

Expression of proteins associated with hypoxia and Wnt pathway activation is of prognostic significance in hepatocellular carcinoma

In the development and progression of hepatocellular carcinoma, tumor hypoxia plays an important role, as does activation of the Wnt pathway. The aim of this study was to characterize the expression and interrelationship between hypoxia and Wnt-pathway-associated proteins as prognostic factors for hepatocellular carcinoma. Expression of HIF-1α, CA-IX, E-cadherin, β-catenin, and Ki-67 was assessed by immunohistochemistry in 179 primary hepatocellular carcinoma cases. Univariate and multivariate analyses were performed to assess the relationship between the clinicopathological factors, protein expression, overall survival (OS), and recurrence-free survival (RFS). By univariate analysis, tumor stage, size, satellitosis, and vascular invasion were confirmed as prognostic factors for worse OS and RFS. High expression of HIF-1α, CA-IX, β-catenin, Ki-67, and E-cadherin was observed in 60, 15, 64, 8, and 64 % of tumors, respectively, and this was significantly associated with poor OS. CA-IX, HIF-1α, and E-cadherin were independent predictors of poor prognosis. We stratified 169 patients into four groups according to the expression level of hypoxia and Wnt pathway markers. The group with high expression of both hypoxia and Wnt-pathway-associated proteins showed worst OS. The poor survival of this group was also significant in patients with early stage disease and tumor size of less than 5 cm (p < 0.05). We identified a subgroup of hepatocellular carcinoma patients with high expression of both hypoxia and Wnt pathway proteins and found this predictive of poor survival. The therapeutic options for this group might need to be revisited.

Guidance for laboratories performing molecular pathology for cancer patients

Molecular testing is becoming an important part of the diagnosis of any patient with cancer. The challenge to laboratories is to meet this need, using reliable methods and processes to ensure that patients receive a timely and accurate report on which their treatment will be based. The aim of this paper is to provide minimum requirements for the management of molecular pathology laboratories. This general guidance should be augmented by the specific guidance available for different tumour types and tests. Preanalytical considerations are important, and careful consideration of the way in which specimens are obtained and reach the laboratory is necessary. Sample receipt and handling follow standard operating procedures, but some alterations may be necessary if molecular testing is to be performed, for instance to control tissue fixation. DNA and RNA extraction can be standardised and should be checked for quality and quantity of output on a regular basis. The choice of analytical method(s) depends on clinical requirements, desired turnaround time, and expertise available. Internal quality control, regular internal audit of the whole testing process, laboratory accreditation, and continual participation in external quality assessment schemes are prerequisites for delivery of a reliable service. A molecular pathology report should accurately convey the information the clinician needs to treat the patient with sufficient information to allow for correct interpretation of the result. Molecular pathology is developing rapidly, and further detailed evidence-based recommendations are required for many of the topics covered here.

Endoscopic tri-modal imaging improves detection of gastric intestinal metaplasia among a high-risk patient population in Singapore

BACKGROUND: Detection of pre-neoplastic gastric mucosal changes and early gastric cancer (EGC) by white-light endoscopy (WLE) is often difficult. In this study we investigated whether combined autofluorescence imaging (AFI) and narrow band imaging (NBI) can improve detection of pre-neoplastic lesions and early gastric cancer in high-risk patients.

PATIENTS AND METHODS: Chinese patients who were 50-years-old or above with dyspepsia were examined by both high-resolution WLE and combined AFI followed by NBI (AFI-NBI), consecutively in a prospective randomized cross-over setting, by two experienced endoscopists. The primary outcome was diagnostic ability of the two methods for patients with pre-neoplastic lesions such as intestinal metaplasia (IM) and mucosal atrophy.

RESULTS: Sixty-five patients were recruited. One patient with large advanced gastric cancer was found and excluded from the analysis. Among the remaining 64 patients, 38 (59%) had IM; of these, 26 (68%) were correctly identified by AFI-NBI (sensitivity 68%, specificity 23%) and only 13 (34%) by WLE (sensitivity 34%, specificity 65%). AFI-NBI detected more patients with IM than did WLE (p=0.011). Thirty-one patients (48%) had mucosal atrophy. Ten patients (32%) were identified by AFI-NBI (sensitivity 32%, specificity 79%) and four patients (13%) by WLE (sensitivity 13%, specificity 88%) (p=0.100). No dysplasia or EGC was found.

CONCLUSION: AFI-NBI identified significantly more patients with IM than did WLE. Our result warrants further studies to define the role of combined AFI-NBI endoscopy for detection of precancerous conditions.

POU2F1 activity regulates HOXD10 and HOXD11 promoting a proliferative and invasive phenotype in head and neck cancer

HOX genes are master regulators of organ morphogenesis and cell differentiation during embryonic development, and continue to be expressed throughout post-natal life. To test the hypothesis that HOX genes are dysregulated in head and neck squamous cell carcinoma (HNSCC) we defined their expression profile, and investigated the function, transcriptional regulation and clinical relevance of a subset of highly expressed HOXD genes. Two HOXD genes, D10 and D11, showed strikingly high levels in HNSCC cell lines, patient tumor samples and publicly available datasets. Knockdown of HOXD10 in HNSCC cells caused decreased proliferation and invasion, whereas knockdown of HOXD11 reduced only invasion. POU2F1 consensus sequences were identified in the 5' DNA of HOXD10 and D11. Knockdown of POU2F1 significantly reduced expression of HOXD10 and D11 and inhibited HNSCC proliferation. Luciferase reporter constructs of the HOXD10 and D11 promoters confirmed that POU2F1 consensus binding sites are required for optimal promoter activity. Utilizing patient tumor samples a significant association was found between immunohistochemical staining of HOXD10 and both the overall and the disease-specific survival, adding further support that HOXD10 is dysregulated in head and neck cancer. Additional studies are now warranted to fully evaluate HOXD10 as a prognostic tool in head and neck cancers.

LNK mutations and myeloproliferative disorders

The lymphocyte adaptor protein (LNK) is one of a family of adaptor proteins involved cell signalling and control of B cell populations. It has a critical role in regulation of signalling in hematopoiesis. Lnk negatively regulates cytokine initiated cell signalling and it functions as a negative regulator of the mutant protein in myeloproliferative neoplasms JAK2V617F. A number of mutations in LNK have been described in a variety of myeloproliferative neoplasms some of which have been demonstrated to cause increased cellular proliferation. The majority of mutations occur in exon 2. In a small number of cases idiopathic erythrocytosis with subnormal erythropoietin levels LNK mutations have been found which may account for the clinical phenotype. Thus investigation for LNK mutations should be considered in the investigation of idiopathic erythrocytosis and perhaps other myeloproliferative neoplasms.