Monitoring the effect of first line treatment in RAS/RAF mutated metastatic colorectal cancer by serial analysis of tumor specific DNA in plasma.

BACKGROUND: Precision medicine calls for an early indicator of treatment efficiency. Circulating tumor DNA (ctDNA) is a promising marker in this setting. Our prospective study explored the association between disease development and change of ctDNA during first line chemotherapy in patients with RAS/RAF mutated metastatic colorectal cancer (mCRC). METHODS: The study included 138 patients with mCRC receiving standard first line treatment. In patients with RAS/RAF mutated tumor DNA the same mutation was quantified in the plasma using droplet digital PCR. The fractional abundance of ctDNA was assessed in plasma before treatment start and at every treatment cycle until radiologically defined progressive disease. RESULTS: RAS/RAF mutations were detected in the plasma from 77 patients. Twenty patients progressed on treatment and 57 stopped treatment without progression. The presence of mutated DNA in plasma was correlated with poor overall survival. A low level of ctDNA after the first cycle of chemotherapy was associated with a low risk of progression. On the other hand, a significant increase of ctDNA at any time during the treatment course was associated with a high risk of progression on continuous treatment. The first increase in ctDNA level occurred at a median of 51 days before radiologically confirmed progression. CONCLUSIONS: The results indicate that the ctDNA level holds potential as a clinically valuable marker in first line treatment of mCRC. A rapid decrease was associated with a prolonged progression free interval, whereas a significant increase gave notice of early progression with a relevant lead time.

Redefining high-risk patients with stage II colon cancer by risk index and microRNA-21: results from a population-based cohort.

BACKGROUND: The aim of the present study was to analyse the prognostic value of microRNA-21 (miRNA-21) in patients with stage II colon cancer aiming at a risk index for this group of patients. METHODS: A population-based cohort of 554 patients was included. MicroRNA-21 was analysed by qPCR based on tumour tissue. An index was created using the coefficients obtained from a collective multiple Cox regression. The entire procedure was cross-validated (10-fold). The performance of the index was quantified by time-dependent receiver operating characteristics curves. RESULTS: High miRNA-21 expression was associated with an unfavourable recurrence-free cancer-specific survival (RF-CSS), hazard ratio 1.35 (95% confidence interval, 1.03-1.76) (P=0.028). The generated RF-CSS index divided the traditional high-risk patients into subgroups with 5-year RF-CSS rates of 87% and 73%, respectively (P<0.001). The overall survival (OS) index identified three different subgroups (P<0.001). Cross-validated 5-year OS rates were 88%, 68%, and 50%, respectively. CONCLUSIONS: This population-based study supports miRNA-21 as an additional prognostic biomarker in patients with stage II colon cancer. Furthermore, the introduction of a risk index may guide the use of postoperative adjuvant treatment in a more appropriate way compared with current practice.

Ultrasound elastography in patients with rectal cancer treated with chemoradiation.

OBJECTIVE: The current literature has described several predictive markers in rectal cancer patients treated with chemoradiation, but so far none of them have been validated for clinical use. The purpose of the present study was to compare quantitative elastography based on ultrasound measurements in the course of chemoradiation with tumor response based on T stage classification and the Mandard tumor regression grading (TRG). MATERIALS AND METHODS: We prospectively examined 31 patients with rectal cancer planned for high dose radiochemotherapy. The tumor and the mesorectal fat elasticity were measured using the Acoustic Radiation Force Impulse to generate information on the mechanical properties of the tissue. The objective quantitative elastography shear wave velocity was compared to the T stage classification and TRG. RESULTS: The baseline mean tumor elasticity was 3.13 m/s. Two and six weeks after the start of chemoradiation the velocities were 2.17 m/s and 2.11 m/s, respectively. The difference between baseline velocity and velocities during the treatment course was statistically significant, (p<0.0001). Patients with tumor confined to the rectal wall at histopathology (ypT1-2) had a mean elasticity measurement after two weeks of treatment of 1.95 m/s, whereas tumors invading the mesorectal fat (ypT3-4) had a velocity of 2.47 m/s, (p<0.05). The mean elasticity tended to be lower (1.99m/s) after two weeks in patients with TRG 1-2 responses in contrast to 2.24 m/s in those with TRG 3-4. CONCLUSION: Ultrasound elastography after two weeks of chemoradiation seems to hold early predictive information to the pathological T stage.

The prognostic importance of miR-21 in stage II colon cancer: a population-based study.

BACKGROUND: Despite several years of research and attempts to develop prognostic models a considerable fraction of stage II colon cancer patients will experience relapse within few years from their operation. The aim of the present study was to investigate the prognostic importance of miRNA-21 (miR-21), quantified by in situ hybridisation, in a unique, large population-based cohort. PATIENTS AND METHODS: The study included 764 patients diagnosed with stage II colon cancer in Denmark in the year 2003. One section from a representative paraffin-embedded tumour tissue specimen from each patient was processed for analysis of miR-21 and quantitatively assessed by image analysis. RESULTS: The miR-21 signal was predominantly observed in fibroblast-like cells located in the stromal compartment of the tumours. We found that patients expressing high levels of miR-21 had significantly inferior recurrence-free cancer-specific survival (RF-CSS): HR=1.26; 95% CI: 1.15-1.60; P<0.001. In Cox regression analysis, a high level of miR-21 retained its prognostic importance and was found to be significantly related to poor RF-CSS: HR=1.41; 95% CI: 1.19-1.67; P<0.001. CONCLUSION: The present study showed that increasing miR-21 expression levels were significantly correlated to decreasing RF-CSS. Further investigations of the clinical importance of miR-21 in the selection of high-risk stage II colon cancer patients are merited.

Phase II marker-driven trial of panitumumab and chemotherapy in KRAS wild-type biliary tract cancer.

BACKGROUND: Combination chemotherapy has proven beneficial in biliary tract cancer and further improvements may be achieved by individualizing treatment based on biomarkers and by adding biological agents. We report the effect of chemotherapy with panitumumab as first-line therapy for KRAS wild-type irresectable biliary tract cancer. PATIENTS AND METHODS: Patients were treated with gemcitabine 1000 mg/m(2), oxaliplatin 60 mg/m(2), and panitumumab 6 mg/kg i.v. every 2 weeks followed by two daily administrations of capecitabine 1000 mg/m(2) in 7 days. RESULTS: During 22 months, 46 patients were included in a single institution. The primary end point, fraction of progression-free survival (PFS) at 6 months, was 31/42 [74%; 95% confidence interval (CI) 58% to 84%]. Forty-two patients had measurable disease. Response rate was 33% and disease control rate 86%. Median PFS was 8.3 months (95% CI 6.7-8.7 months) and median overall survival was 10.0 months (95% CI 7.4-12.7 months). Toxicity was manageable including eight cases of epidermal growth factor receptor-related skin adverse events of grade 2 or more. CONCLUSIONS: Marker-driven patient selection is feasible in the systemic treatment of biliary tract cancer. Combination chemotherapy with panitumumab in patients with KRAS wild-type tumors met the efficacy criteria for future testing in a randomized trial.

The predictive value of genetic variations in the vascular endothelial growth factor A gene in metastatic colorectal cancer.

Single-nucleotide polymorphisms (SNPs) in the vascular endothelial growth factor A (VEGF-A) gene may have clinical implications. The aim of this study was to investigate the possible predictive value of the VEGF-A SNPs, in patients with metastatic colorectal cancer (mCRC) treated with first-line capecitabine and oxaliplatin (XELOX). The study included 72 patients with mCRC. Genomic DNA was isolated from whole blood, and SNPs were analyzed by PCR. SNPs were correlated with response and progression-free survival (PFS). Haplotypes were estimated using the PHASE program. Response was observed in 21% of the patients with the -2578 CA genotype compared with 59% of the patients with CC+AA, P=0.002, in 26% of the patients with the -460 CT genotype compared with 57% with CC+TT, P=0.01, and in 27% of the patients with the +405 GC genotype compared with 54% with GG+CC, P=0.02. Two SNPs were significantly related to PFS. A haplotype with a significant relationship to response was identified. The results demonstrated obvious relationships between genetic variations in the VEGF-A gene and response to first-line XELOX in patients with mCRC, which translated to a significant difference in PFS. The results call for validation in a larger cohort of patients.

The relationship between serum vascular endothelial growth factor A and microsatellite instability in colorectal cancer.

AIM: It has been suggested that colorectal neoplasms with or without microsatellite instability (MSI) can stimulate angiogenesis in different ways. The vascular endothelial growth factor (VEGF) system is essential for the angiogenetic process and the growth of malignant tumours. The aim of this study was to analyse the relationship between serum VEGF-A and the MSI status of patients with colorectal cancer (CRC). METHOD: In the study, 249 patients with CRC were divided into a test cohort of 83 patients and a validation cohort of 166. MSI was determined using immunohistochemistry. Tumours lacking protein expression of any of the four mismatch repair genes (MLH1, PMS2, MSH2 or MSH6) were labelled as high MSI. The rest were considered to be microsatellite stable (MSS). The serum VEGF-A analyses were performed by ELISA. RESULTS: The tumours of 15 patients in the test cohort and 27 in the validation cohort were classified as MSI. In the test cohort, patients with an MSI tumour had a significantly higher median serum VEGF-A concentration [617 pg/ml (95% CI 445-863)], compared with patients with an MSS tumour, [317 pg/ml (95% CI 224-386)], P = 0.01. A similar relationship was confirmed in the validation cohort, P = 0.04. CONCLUSION: This study provides some evidence to suggest that patients with an MSI tumour have higher serum VEGF-A levels than patients with an MSS tumour. If further validated, these findings could be of importance when considering the effects of anti-VEGF-A treatment.

The importance of KRAS mutations and EGF61A>G polymorphism to the effect of cetuximab and irinotecan in metastatic colorectal cancer.

BACKGROUND: The effect of anti-epidermal growth factor receptor (EGFR) antibodies (mAb) in metastatic colorectal cancer seems limited to KRAS wild-type (wt) tumours, but still a major fraction of KRASwt patients are nonresponders and supplementary selection criteria are needed. We investigated methodological aspects of KRAS testing and the predictive and prognostic value of KRAS status combined with three EGFR-related gene polymorphisms [single-nucleotide polymorphisms (SNPs)] in patients treated with cetuximab and irinotecan. PATIENTS AND METHODS: The study included 71 patients referred to third-line cetuximab-irinotecan. Blood samples were analysed for SNPs. KRAS analysis was carried out by sequencing analysis and quantitative PCR (DxS kit) in primary tumour and distant metastases. RESULTS: There was a clear correlation between KRAS status in primary tumours and metastasis. The DxS kit presented the highest sensitivity. Response was confined to KRASwt patients (40% response rate versus 0%, P < 0.1(-3)), which translated into a significant difference in PFS. The EGF61A>G polymorphism showed relation to clinical outcome. A combined biomarker analysis showed a 19% progression rate in KRASwt-EGF61 homozygote patients and 60% in the EGF61A/G patients (P = 0.006) and a significant increase in overall survival (17.1 versus 5.9 months, log-rank, P = 0.002). CONCLUSION: The combined biomarker analysis maybe an attractive approach to selection of patients for third-line treatment including anti-EGFR mAbs.

The prognostic value of lymph node metastases and tumour regression grade in rectal cancer patients treated with long-course preoperative chemoradiotherapy.

OBJECTIVE: The purpose of the present study was to investigate the impact of tumour regression and the post-treatment lymph node status on the prognosis of rectal cancer treated by preoperative neoadjuvant chemoradiotherapy. METHOD: One hundred and thirty-five patients with locally advanced T3 and T4 rectal tumours received preoperative long-course chemoradiation, to a dose of 60 Gy external radiation and oral 5-fluorouracil 300 mg/m(2) daily and Leukovorin 22.5 mg/day 5 days a week. Surgery was performed 8 weeks after the end of treatment. The tumour response was evaluated according to the tumour regression grade system and lymph node status in the surgical specimen was assessed. The prognostic value of clinico-pathological parameters was analysed using univariate analysis and Kaplan-Meier methods for comparison of groups. RESULTS: All patients responded to treatment and 47% had a major response, including 25 (19%) complete responders. The median follow-up was 26 months (range 12-94 months). The cancer specific survival was 82% and there was a significant lower survival rate in the group of patients with post-treatment lymph node metastases compared to lymph-node negative patients [63% and 87% respectively (P = 0.007)]. Furthermore patients with a major tumour response and no lymph node metastases in the surgical specimen after treatment had a survival rate of 100% compared with 60% in the group of patients with major response but lymph node metastases after surgery (P < 0.01). CONCLUSION: The combined assessment of lymph-node status and tumour response has strong prognostic value in locally advanced rectal cancer patient treated with preoperative long-course chemoradiation.

Strategy in clinical practice for classification of unselected colorectal tumours based on mismatch repair deficiency.

OBJECTIVE: Deficiency of DNA mismatch repair (MMR) causes microsatellite instability (MSI) in a subset of colorectal cancers. Patients with these tumours have a better prognosis and may have an altered response to chemotherapy. Some of the tumours are caused by hereditary mutations (hereditary nonpolyposis colon cancer or Lynch syndrome), but most are epigenetic changes of sporadic origin. The aim of this study was to define a robust and inexpensive strategy for such classification in clinical practice. METHOD: Tumours and blood samples from 262 successive patients with colorectal adenocarcinomas were collected. Expression of the MMR proteins MLH1, MSH2, and MSH6 by immunohistochemistry (IHC) was compared with MSI DNA analysis. Methylation analysis of MLH1 and mutation analysis for BRAF V600E were compared in samples with MSI and/or lack of MLH1 expression to determine if the tumour was likely to be sporadic. RESULTS: Thirty-nine (14.9%) of the tumours showed MMR deficiency by IHC or by microsatellite analysis. Sporadic inactivation by methylation of MLH1 promoter was found in 35 patients whereby the BRAF activating V600E mutation, indicating sporadic origin, was found in 32 tumours. On the basis of molecular characteristics we found 223 patients with intact MMR, 35 patients with sporadic MMR deficiency, and four patients who were likely to have hereditary MMR deficiency. CONCLUSION: To obtain the maximal benefit for patients and clinicians, MMR testing should be supplemented with MLH1 methylation or BRAF mutation analysis to distinguish sporadic patients from likely hereditary ones. MMR deficient patients with sporadic disease can be reassured of the better prognosis and the likely hereditary cases should receive genetic counselling.

Molecular factors of 5-fluorouracil metabolism in colorectal cancer: analysis of primary tumor and lymph node metastasis.

Thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) are predictive markers for tumor response to 5-fluorouracil-based therapies. To determine whether gene expression values measured in primary cancer tissue would be useful for prediction of response of lymph node metastases, the expressions of these genes were quantitatively analyzed in 35 pairs of primary colorectal cancer (CRC) and corresponding lymph node metastases using real-time PCR. DPD and TP mRNA levels were significantly lower in the primary colorectal tumor and lymph node metastases compared with the normal adjacent stroma tissue (p<0.01), whereas TS mRNA levels were significantly higher in the primary tumor and lymph node metastases than in the normal adjacent tissue (p<0.001). Median gene expression levels of TP and TS did not differ significantly between primary colorectal tumor and corresponding lymph node metastasis but median DPD gene expression levels in the lymph node metastases were significantly higher compared to matched primary colorectal tumors (p=0.015). There was a significant correlation for DPD, TP and TS gene expression levels between primary colorectal tumor specimens and the matched lymph node metastasis. These results suggest that biopsies of the tumor of origin may be valid for determining predictive markers for chemotherapy response in patients with metastatic CRC.

Immunohistochemical expression of thymidylate synthase as predictor of response to capecitabine in patients with advanced colorectal adenocarcinoma.

Capecitabine is an oral prodrug to 5-fluorouracil (5-FU). The primary target of 5-FU is thymidylate synthase (TS). A mainstay of colorectal adenocarcinoma chemotherapy is inhibition of TS, which may be one of many determinant factors when predicting the outcome of chemotherapies based on fluoropyrimidine treatment. This retrospective study included 39 patients with advanced colorectal adenocarcinoma treated with capecitabine. Response was assessed by measuring the amount of tumour in the course of treatment. TS expression was evaluated by scoring the immunohistochemical (IHC) reaction and assessing the predominant IHC reaction pattern. This study showed significant correlation between the predominant IHC reaction pattern and response, but no correlation between IHC score and response. The predominant IHC reaction pattern may be a useful parameter in prediction of clinical outcome in patients treated with capecitabine.

[Celiac disease with subepithelial deposition of collagen]. / Cøliaki med subepitelial aflejring af kollagen.

In 1970 a case of malabsorption with flat small intestinal mucosa with subepithelial collagen deposition was described. There was no response to a gluten-free diet, and the condition was termed collagenous sprue. We report a case of coeliac disease with subepithelial deposition of collagen in duodenal biopsy, which responded to a gluten-free diet.

[Primary adenocarcinoma in appendiceal diverticulitis]. / Primaert adenokarcinom i appendixdivertikel.

A case of primary adenocarcinoma in appendiceal diverticulosis is reported. Such a case has never been mentioned before in the literature. It was not possible to diagnose the case preoperatively. This emphasizes the importance of histological examination of all appendiceal samples.