Gordon Hutchins

Value of Mismatch Repair, KRAS, and BRAF Mutations in Predicting Recurrence and Benefits From Chemotherapy in Colorectal Cancer

PURPOSE It is uncertain whether modest benefits from adjuvant chemotherapy in stage II colorectal cancer justify the toxicity, cost, and inconvenience. We investigated the usefulness of defective mismatch repair (dMMR), BRAF, and KRAS mutations in predicting tumor recurrence and sensitivity to chemotherapy. PATIENTS AND METHODS Immunohistochemistry for dMMR and pyrosequencing for KRAS/BRAF were performed for 1,913 patients randomly assigned between fluorouracil and folinic acid chemotherapy and no chemotherapy in the Quick and Simple and Reliable (QUASAR) trial. RESULTS Twenty-six percent of 695 right-sided colon, 3% of 685 left-sided colon, and 1% of 407 rectal tumors were dMMR. Similarly, 17% of right colon, 2% of left colon, and 2% of rectal tumors were BRAF mutant. KRAS mutant tumors were more evenly distributed: 40% right colon, 28% left colon, and 36% rectal tumors. Recurrence rate for dMMR tumors was half that for MMR-proficient tumors (11% [25 of 218] v 26% [438 of 1,695] recurred; risk ratio [RR], 0.53; 95% CI, 0.40 to 0.70; P < .001). Risk of recurrence was also significantly higher for KRAS mutant than KRAS wild-type tumors (28% [150 of 542] v 21% [219 of 1,041]; RR, 1.40; 95% CI, 1.12 to 1.74; P = .002) but did not differ significantly between BRAF mutant and wild-type tumors (P = .36). No marker predicted benefit from chemotherapy with efficacy not differing significantly by MMR, KRAS, or BRAF status. The prognostic value of MMR and KRAS was similar in the presence and absence of chemotherapy. CONCLUSION MMR assays identify patients with a low risk of recurrence. KRAS mutational analysis provides useful additional risk stratification to guide use of chemotherapy.

The proportion of tumour cells is an independent predictor for survival in colorectal cancer patients

Background:The proportion of epithelial and stromal cells in tumours is thought to have an important role in the progression of epithelial malignancy. We aimed to determine whether the relative proportion of tumour (PoT) was related to survival in colorectal cancer.Methods:The PoT at the luminal surface was measured by point counting using virtual tissue sections in a series of 145 colorectal cancer cases. The relationship of PoT to clinicopathological parameters including cancer-specific survival was analysed. Modified receiver operating characteristic curves were used to determine the optimum cut off points to dichotomise the data for survival analyses.Results:Tumours with PoT-low (⩽47%) were associated with significantly lower cancer-specific survival when compared to PoT-high (hazard ratio (HR)=2.087, 95% CI=1.088–4.003, P=0.024). On sub-analysis, the prognostic effect remained significant in colonic tumours (HR=2.474, 95% CI=1.132–5.408, P=0.019) and tumour, node, metastasis stage III disease (HR=3.480, 95% CI=0.325–9.136, P=0.007). Multivariate Cox regression analysis demonstrated that PoT was an independent prognostic marker when adjusted for age, T stage, N stage and extramural vascular invasion (P=0.017).Conclusion:This study suggests that a low proportion of tumour cells in colorectal cancer is related to poor cancer-specific survival. A relatively quick, inexpensive and well-established method such as point counting on diagnostic tissue sections could be used to identify a subset of patients who may benefit from adjuvant therapy.

Comprehensive genomic meta-analysis identifies intra-tumoural stroma as a predictor of survival in patients with gastric cancer

Objective Gastric adenocarcinoma (gastric cancer, GC) is a major cause of global cancer mortality. Identifying molecular programmes contributing to GC patient survival may improve our understanding of GC pathogenesis, highlight new prognostic factors and reveal novel therapeutic targets. The authors aimed to produce a comprehensive inventory of gene expression programmes expressed in primary GCs, and to identify those expression programmes significantly associated with patient survival. Design Using a network-modelling approach, the authors performed a large-scale meta-analysis of GC transcriptome data integrating 940 gastric transcriptomes from multiple independent patient cohorts. The authors analysed a training set of 428 GCs and 163 non-malignant gastric samples, and a validation set of 288 GCs and 61 non-malignant gastric samples. Results The authors identified 178 gene expression programmes (‘modules’) expressed in primary GCs, which were associated with distinct biological processes, chromosomal location patterns, cis-regulatory motifs and clinicopathological parameters. Expression of a transforming growth factor β (TGF-β) signalling associated ‘super-module’ of stroma-related genes consistently predicted patient survival in multiple GC validation cohorts. The proportion of intra-tumoural stroma, quantified by morphometry in tissue sections from gastrectomy specimens, was also significantly associated with stromal super-module expression and GC patient survival. Conclusion Stromal gene expression predicts GC patient survival in multiple independent cohorts, and may be closely related to the intra-tumoural stroma proportion, a specific morphological GC phenotype. These findings suggest that therapeutic approaches targeting the GC stroma may merit evaluation.

In situ validation of an intestinal stem cell signature in colorectal cancer

Objective Wnt/Tcf, Lgr5, Ascl2 and/or Bmi1 signalling is believed to define the mouse intestinal stem cell niche(s) from which adenomas arise. The aim of this study was to determine the relevance of these putative intestinal stem cell markers to human colorectal cancer. Design 19 putative intestinal stem cell markers, including Ascl2 and Lgr5, were identified from published data and an evaluation of a human colorectal gene expression database. Associations between these genes were assessed by isotopic in situ hybridisation (ISH) in 57 colorectal adenocarcinomas. Multiplex fluorescent ISH and chromogenic non-isotopic ISH were performed to confirm expression patterns. The prognostic significance of Lgr5 was assessed in 891 colorectal adenocarcinomas. Results Ascl2 and Lgr5 were expressed in 85% and 74% of cancers respectively, and expression was positively correlated (p=0.003). Expression of Bmi1 was observed in 47% of cancers but was very weak in 98% of cases with expression. Both Ascl2 and/or Lgr5 were positively correlated with the majority of genes in the signature but neither was correlated with Cdk6, Gpx2, Olfm4 or Tnfrsf19. Lgr5 did not have prognostic significance. Conclusion These data suggest that 74–85% of colorectal cancers express a Lgr5/Ascl2 associated signature and support the hypothesis that they derive from Lgr5+/Ascl2+ crypt stem cells, not Bmi1+ stem cells. However, Olfm4 was not found to be a useful marker of Lgr5+ cells in normal colon or tumours. In this large series, Lgr5 expression is not associated with increased tumour aggressiveness, as might be expected from a cancer stem cell marker.

HER2 overexpression and amplification as a potential therapeutic target in colorectal cancer: analysis of 3256 patients enrolled in the QUASAR, FOCUS and PICCOLO colorectal cancer trials

HER2 overexpression/amplification is linked to trastuzumab response in breast/gastric cancers. One suggested anti‐EGFR resistance mechanism in colorectal cancer (CRC) is aberrant MEK–AKT pathway activation through HER2 up‐regulation. We assessed HER2‐amplification/overexpression in stage II–III and IV CRC patients, assessing relationships to KRAS/BRAF and outcome. Pathological material was obtained from 1914 patients in the QUASAR stage II–III trial and 1342 patients in stage IV trials (FOCUS and PICCOLO). Tissue microarrays were created for HER2 immunohistochemistry. HER2‐amplification was assessed using FISH and copy number variation. KRAS/BRAF mutation status was assessed by pyrosequencing. Progression‐free survival (PFS) and overall survival (OS) data were obtained for FOCUS/PICCOLO and recurrence and mortality for QUASAR; 29/1342 (2.2%) stage IV and 25/1914 (1.3%) stage II–III tumours showed HER2 protein overexpression. Of the HER2‐overexpressing cases, 27/28 (96.4%) stage IV tumours and 20/24 (83.3%) stage II–III tumours demonstrated HER2 amplification by FISH; 41/47 (87.2%) also showed copy number gains. HER2‐overexpression was associated with KRAS/BRAF wild‐type (WT) status at all stages: in 5.2% WT versus 1.0% mutated tumours (p < 0.0001) in stage IV and 2.1% versus 0.2% in stage II–III tumours (p = 0.01), respectively. HER2 was not associated with OS or PFS. At stage II–III, there was no significant correlation between HER2 overexpression and 5FU/FA response. A higher proportion of HER2‐overexpressing cases experienced recurrence, but the difference was not significant. HER2‐amplification/overexpression is identifiable by immunohistochemistry, occurring infrequently in stage II–III CRC, rising in stage IV and further in KRAS/BRAF WT tumours. The value of HER2‐targeted therapy in patients with HER2‐amplified CRC must be tested in a clinical trial.

Systemic neutrophil-to-lymphocyte ratio in colorectal cancer: the relationship to patient survival, tumour biology and local lymphocytic response to tumour.

Background:Colorectal cancer (CRC) is a major cause of mortality and morbidity. The impact of inflammatory biomarkers (C-reactive protein etc.) on CRC is increasingly studied including systemic neutrophil-to-lymphocyte ratio (NLR) as they seem to predict outcome.Methods:All patients who underwent curative resection for CRC from 2000 to 2004 at Leeds Teaching Hospitals NHS Trust had pre-operative NLR calculated. Demographic, histopathological and survival data were collected. Tissue microarrays were created and stained to determine the mismatch repair (MMR) protein status of each tumour. Local lymphocytic response to the tumour was assessed and graded.Results:About 358 patients were eligible. Of these 88 had an NLR ⩾5, which predicted lower overall survival and greater disease recurrence. A high NLR is associated with higher pT- and pN-stage and a greater incidence of extramural venous invasion. MMR protein status was not associated with NLR. A pronounced lymphocytic reaction at the invasive margin (IM) indicated a better prognosis and was associated with a lower NLR.Conclusion:Neutrophil-to-lymphocyte ratio predicts disease-free and overall survival and is associated with a more aggressive tumour phenotype. The lymphocytic response to tumour at the IM is associated with NLR however dMMR is not. Neutrophil-to-lymphocyte ratio is a cheap, easy-to-access test that predicts outcome in CRC.

Histopathological Work-Up of Resection Specimens, Local Excisions and Biopsies in Colorectal Cancer

The pathological examination of material removed from patients with colorectal neoplasia is important. It provides a wide range of information on, for example, the quality and completeness of excision, the stage and biological aggressiveness, the need for further therapy, and response to therapy. Molecular testing adds valuable information on genetic risk and is required before treatment with anti-EGF-r antibodies. This article highlights the value derived from macroscopic inspection of surgical specimens, careful microscopy and excellent reporting according to national guidelines. Increasing use of a number of preoperative therapies and combinations in rectal cancer change the pathological features found and a standardised approach to the diagnosis of complete response is required. It touches upon the issues with frequent changes in TNM staging and the difficulties these changes are causing. The widespread introduction of bowel cancer screening is changing the stage of presentation of colorectal cancer leading to increasing numbers of local excisions and polyp cancers.

Using a high-resolution wall-sized virtual microscope to teach undergraduate medical students

The Leeds Virtual Microscope is an interactive visualization system, capable of rendering gigapixel virtual slides onto high-resolution, wall-sized displays. We describe the evaluation of this technology for teaching pathology to undergraduate medical students, providing insights into the use of high-resolution, wall-sized displays in an educational context. Students were quickly able to become confident in using the technology, collaboratively exploring virtual slides in order to understand the mechanisms of disease. Being able to point with a finger to features on virtual slides promoted multi-way interaction between the students and tutor, led to the spontaneous expansion of the tutorials scope, and was indicative of a high level of engagement. Students were very positive about being able to interact with the virtual slides and described their increased enthusiasm for pathology as a subject.

Prognostic and predictive significance of proliferation in 867 colorectal cancers

Aim Recently, the Oncotype DX recurrence score, which measures a gene expression signature including markers of tumour proliferation, was validated as a prognostic signature in colorectal cancer. This study aimed to evaluate whether the Ki67 proliferation index can provide similar prognostic and predictive information. Methods Tissue microarrays were constructed from triplicate cores of colorectal cancer. Immunohistochemistry for Ki67 was performed with the SP6 antibody and the percentage of positive tumour cells scored. Prognostic significance was evaluated in 867 cancers (601 events) using Cox proportional hazards models. Results The Ki67 labelling index, divided at the median, was not a statistically or clinically significant prognostic factor in univariate analyses of 5-year overall survival (HR 0.98, 95% CI 0.84 to 1.15, p=0.84). Multivariate analyses were similarly non-significant. However, in Dukes’ stage C patients, the high Ki67 subgroup derived a greater 5-year overall survival benefit from chemotherapy (HR 0.32, 95% CI 0.21 to 0.51, p<0.0001) than the low subgroup (HR 0.57, 95% CI 0.37 to 0.89, p=0.011). Conclusions The Ki67 proliferation index is not a useful prognostic factor in colorectal cancer, but deserves further evaluation as a predictive factor for the incremental benefit derived from adjuvant chemotherapy.

What can the molecular pathologist offer for optimal decision making

As a consequence of new innovative therapies and therapeutic combinations, the treatment of advanced colorectal cancer is becoming increasingly complex. Validated molecular biomarkers could contribute to patient management, but until recently, none has been routinely used. With the recognition that activating mutations of the KRAS oncogene can predict resistance to anti-epidermal growth factor receptor agents, the clinical value of biomarkers in advanced colorectal cancer has been brought to the fore. Prognostic and predictive biomarkers that reflect the molecular and therapeutic complexities of advanced colorectal cancer may provide valuable information regarding likely clinical outcome and therapeutic response and thus may improve patient management and therapeutic agent selection. Such biomarkers are discussed herein.