Total circulating cell-free DNA as a prognostic biomarker in metastatic colorectal cancer before first-line oxaliplatin-based chemotherapy.

BACKGROUND: Metastatic colorectal cancer (mCRC) is a heterogeneous disease where prognosis is dependent both on tumor biology and host factors. Total circulating cell-free DNA (cfDNA) has shown to harbor prognostic information in mCRC, although less is known about the biological correlates of cfDNA levels in this patient group. The primary objective was to evaluate the prognostic value of pretreatment cfDNA in patients receiving the first-line oxaliplatin-based chemotherapy for mCRC, by using a predefined upper limit of normal (ULN) from a cohort of presumed healthy individuals. The secondary objective was to model cfDNA levels as a function of predefined tumor and host factors. PATIENTS AND METHODS: This was a retrospective post hoc study based on a prospective multicenter phase III trial, the NORDIC-VII study. DNA was purified from 547 plasma samples and cfDNA quantified by a droplet digital PCR assay (B2M, PPIA) with controls for lymphocyte contamination. Main clinical end point was overall survival (OS). RESULTS: cfDNA was quantified in 493 patients, 54 were excluded mainly due to lymphocyte contamination. Median cfDNA level was 7673 alleles/ml (1050-1 645 000) for B2M and 5959 alleles/ml (555-854 167) for PPIA. High cfDNA levels were associated with impaired outcome; median OS of 16.6 months for levels above ULN and 25.9 months for levels below ULN (hazard ratio = 1.83, 95% confidence interval 1.51-2.21, P < 0.001). The result was confirmed in multivariate OS analysis adjusting for established clinicopathological characteristics. A linear regression model predicted cfDNA levels from sum of longest tumor diameters by RECIST, the presence of liver metastases and systemic inflammatory response as measured by interleukin 6 (F(6, 357) = 62.7, P < 0.001). CONCLUSION: cfDNA holds promise as a minimally invasive and clinically relevant prognostic biomarker in mCRC before initiating first-line oxaliplatin-based chemotherapy and may be a complex entity associated with tumor burden, liver metastases and systemic inflammatory response. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00145314.

AGXT and ERCC2 polymorphisms are associated with clinical outcome in metastatic colorectal cancer patients treated with 5-FU/oxaliplatin.

The objective of the study was to investigate whether specific single nucleotide polymorphisms (SNPs) with influence on drug transport, biotransformation and repair mechanisms are associated with treatment outcome and toxicity in metastatic colorectal cancer (mCRC). We genotyped blood samples from 519 mCRC patients treated with first-line 5-fluorouracil and oxaliplatin +/- cetuximab for 17 SNPs in 10 genes involved in membrane transport (ABCC1 and ABCC2), drug biotransformation (GSTP1 and AGXT) and DNA repair (ERCC1, ERCC2, XRCC1, XRCC3, XPG and MSH6). The AGXT-rs34116584 and the ERCC2-rs238406 polymorphisms were significantly associated with progression-free survival (P=0.002 and P=0.001, respectively). Associations between 18 toxicity variables and SNPs were identified, although none were significant after Bonferroni correction for multiple comparisons. The study identified SNPs of potential use as markers of clinical outcome in oxaliplatin-treated mCRC patients. If validated in other studies, they could improve the selection of therapy in mCRC.

Plasma microRNAs predicting clinical outcome in metastatic colorectal cancer patients receiving first-line oxaliplatin-based treatment.

The conventional first-line chemotherapy for metastatic colorectal cancer (mCRC) consists of fluorouracil (5-FU) in combination with either oxaliplatin or irinotecan. We have explored microRNAs (miRNAs) in plasma as potential predictive markers to oxaliplatin-based chemotherapy. The expression of 742 miRNAs was examined in plasma samples from 24 mCRC patients (12 responders and 12 non-responders) before onset and after four cycles of 5-FU/oxaliplatin. The top differentially expressed miRNAs between responders and non-responders were selected for further analysis in a validation cohort of 150 patients. In the validation cohort, there was a significant overrepresentation of miRNAs with higher mean expression in the non-responder group than in the responder group before treatment (p < 0.002). Moreover, we found three miRNAs (miR-106a, miR-484, and miR-130b) to be significantly differentially expressed before treatment (p = 0.008, 0.008, and 0.008, respectively). All three miRNAs were upregulated in non-responders. High expression of miR-27b, miR-148a, and miR-326 were associated with decreased progression-free survival (Hazard ratios (HR) of 1.4 (95% CI 1.1-1.8, p = 0.004), 1.3 (95% CI 1.1-1.6, p = 0.007), and 1.4 (95% CI 1.1-1.8, p = 0.008), respectively). miR-326 was also associated with decreased overall survival (HR 1.5 (95% CI 1.1-2.0, p = 0.003)). There were no significantly differentially expressed miRNAs in association with clinical outcome after four cycles of chemotherapy. The present study demonstrates that plasma miRNAs analyzed before treatment may serve as non-invasive markers predicting outcome in mCRC patients treated with 5-FU and oxaliplatin-based chemotherapy.

Expression of cyclin D1a and D1b as predictive factors for treatment response in colorectal cancer.

BACKGROUND: The aim of this study was to investigate the value of the cyclin D1 isoforms D1a and D1b as prognostic factors and their relevance as predictors of response to adjuvant chemotherapy with 5-fluorouracil and levamisole (5-FU/LEV) in colorectal cancer (CRC). METHODS: Protein expression of nuclear cyclin D1a and D1b was assessed by immunohistochemistry in 335 CRC patients treated with surgery alone or with adjuvant therapy using 5-FU/LEV. The prognostic and predictive value of these two molecular markers and clinicopathological factors were evaluated statistically in univariate and multivariate survival analyses. RESULTS: Neither cyclin D1a nor D1b showed any prognostic value in CRC or colon cancer patients. However, high cyclin D1a predicted benefit from adjuvant therapy measured in 5-year relapse-free survival (RFS) and CRC-specific survival (CSS) compared to surgery alone in colon cancer (P=0.012 and P=0.038, respectively) and especially in colon cancer stage III patients (P=0.005 and P=0.019, respectively) in univariate analyses. An interaction between treatment group and cyclin D1a could be shown for RFS (P=0.004) and CSS (P=0.025) in multivariate analysis. CONCLUSION: Our study identifies high cyclin D1a protein expression as a positive predictive factor for the benefit of adjuvant 5-FU/LEV treatment in colon cancer, particularly in stage III colon cancer.

Expression of EZH2 and Ki-67 in colorectal cancer and associations with treatment response and prognosis.

BACKGROUND: Enhancer of zeste homologue 2 (EZH2) is a member of the Polycomb group of genes that is involved in epigenetic silencing and cell cycle regulation. METHODS: We studied EZH2 expression in 409 patients with colorectal cancer stages II and III. The patients were included in a randomised study, and treated with surgery alone or surgery followed by adjuvant chemotherapy. RESULTS: EZH2 expression was significantly related to increased tumour cell proliferation, as assessed by Ki-67 expression. In colon cancer, strong EZH2 expression (P=0.041) and high proliferation (>or=40%; P=0.001) were both associated with better relapse-free survival (RFS). In contrast, no such associations were found among rectal cancers. High Ki-67 staining was associated with improved RFS in colon cancer patients who received adjuvant chemotherapy (P=0.001), but not among those who were treated by surgery alone (P=0.087). In colon cancers stage III, a significant association between RFS and randomisation group was found in patients with high proliferation (P=0.046), but not in patients with low proliferation (P=0.26). Multivariate analyses of colon cancers showed that stage III (hazard ratio (HR) 4.00) and high histological grade (HR 1.80) were independent predictors of reduced RFS, whereas high proliferation indicated improved RFS (HR 0.55). CONCLUSION: Strong EZH2 expression and high proliferation are associated features and both indicate improved RFS in colon cancer, but not so in rectal cancer.

Nationwide quality assurance of rectal cancer treatment.

OBJECTIVE: The purpose of this prospective study was to examine the influence of the efforts for nationwide quality assurance of rectal cancer treatment. The study focuses on local recurrence and overall survival. METHODS: This study includes all 3388 Norwegian patients with a rectal cancer within 15 cm from the anal verge treated with curative intent in the period November 1993-December 1999. A comprehensive educational programme was established, and training courses were arranged in different Health Regions demonstrating the TME technique. A specific Rectal Cancer Registry enabled the monitoring of outcome of rectal cancer treatment for single hospitals. Radiotherapy was given to 10% of the patients. RESULTS: The risk of local recurrence has been significantly reduced, so that in 1999 the level was 50% below that observed in 1994 (Hazard ratio (HR)1999=0.5; 95% CI 0.4-0.8, P=0.002). Similarly, during 1998, the mean national overall survival was significantly improved, compared to the rate in 1994 (HR1998=0.8; 95% CI 0.6-1.0, P=0.014). CONCLUSION: The prognosis for rectal cancer can be improved by increased organizational focus on rectal cancer treatment and by establishing a rectal cancer registry monitoring treatment standards throughout the country.

Short-time infusion of oxaliplatin in combination with capecitabine (XELOX30) as second-line therapy in patients with advanced colorectal cancer after failure to irinotecan and 5-fluorouracil.

BACKGROUND: The efficacy of oxaliplatin combined with capecitabine (XELOX) as second-line therapy in patients with advanced colorectal cancer (ACRC) resistant to irinotecan is not well established. Oxaliplatin induces acute, cold-induced neuropathy in most patients. The incidence is claimed to be infusion rate-dependent and therefore a 2-h infusion is recommended. PATIENTS AND METHODS: For practical and economic reasons, but also for patient's convenience, we performed a phase II study to examine XELOX30 (capecitabine 1000 mg/m2 orally twice daily on days 1-14 and oxaliplatin 130 mg/m2 as a 30 min infusion on day 1) in patients with ACRC resistant to irinotecan. In addition the pharmacokinetics of oxaliplatin was studied. RESULTS: From November 2002 to September 2003, 70 patients with ACRC were treated with XELOX30. Median age was 62 (range 33-74 years) years and median performance status was 1 (range 0-2). The median number of courses was four (range 1-12) and median cumulative dose of oxaliplatin was 530 (range 125-1560) mg/m2. The response rate was 17% (95% CI 10-23), median time to progression (TTP) was 5.4 months (95% CI 4.6-6.4) and median survival 9.5 months (95% CI 8.5-11.2). White blood cell count (WBC) and performance status were significantly correlated to TTP. Neurotoxicity was moderate: grade 1 56%, grade 2 17% and grade 3 6%. Other grade 3 toxicities were nausea/vomiting 9%, diarrhoea 14% and PPE 8%. The maximum blood concentration and total body clearance of oxaliplatin was higher than previously reported in studies examining 2-h infusions, but the volume of distribution and terminal half-life was in close agreement with previous results. CONCLUSION: XELOX30 is a very convenient second-line regimen in ACRC with an activity and safety profile similar to other oxaliplatin schedules.

Quality of life and functional outcome following anterior or abdominoperineal resection for rectal cancer.

AIMS: The aims of the study were (1) to evaluate quality of life (QoL) and functional outcome in patients following anterior resection (AR) or abdominoperineal resection (APR) for rectal cancer, and (2) whether these outcomes were dependent on the level of anastomosis. METHODS: Patients who were without recurrent or metastatic disease were identified from the Norwegian Rectal Cancer Registry. QoL was assessed by the EORTC questionnaires QLQ-C30 and QLQ-CR38, and rectal function by a short questionnaire. Of 319 patients studied, 229 had undergone AR and 90 APR. The median age was 73 years, and the median time since surgery was 64 months. RESULTS: Mean QoL scores for body image and male sexual problems were better following AR than APR (P<0.01), also in patients with a low (< or = 3 cm) anastomosis. Patients who had undergone AR had higher mean scores for constipation (P<0.001) and more often used anti-diarrhoeal medication (P=0.005), than patients who had undergone APR. Patients with a low anastomosis (< or = 3 cm) had more incontinence for gas and solid stools (P<0.05), and had more incontinence (P=0.006) compared with patients with higher anastomosis, but there was no difference in QoL. Subgroup analysis showed that irradiated patients (n=34) had worse rectal function in terms of frequency, urgency, and incontinence (P<0.01). CONCLUSIONS: Although rectal function was impaired in patients with low anastomosis, patients who had undergone AR had better QoL than patients who had undergone APR.

A randomised phase II multicentre trial of irinotecan (CPT-11) using four different schedules in patients with metastatic colorectal cancer.

The purpose of this phase II trial was to compare the efficacy, safety and pharmacokinetics of four irinotecan schedules for the treatment of metastatic colorectal cancer. In total, 174 5-fluorouracil pretreated patients were randomised to: arm A (n=41), 350 mg m(-2) irinotecan as a 90-min i.v. infusion q3 weeks; arm B (n=38), 125 mg m(-2) irinotecan as a 90-min i.v. infusion weekly x 4 weeks q6 weeks; arm C (n=46), 250 mg m(-2) irinotecan as a 90-min i.v. infusion q2 weeks; or arm D (n=49), 10 mg m(-2) day(-1) irinotecan as a 14-day continuous infusion q3 weeks. No significant differences in efficacy across the four arms were observed, although a shorter time to treatment failure was noted for arm D (1.7 months; P=0.02). Overall response rates were in the range 5-11%. Secondary end points included median survival (6.4-9.4 months), and time to progression (2.7-3.8 months) and treatment failure (1.7-3.2 months). Similarly, there were no significant differences in the incidence of grade 3-4 toxicities, although the toxicity profile between arms A, B, and C and D did differ. Generally, significantly less haematologic toxicity, alopecia and cholinergic syndrome were observed in arm D; however, there was a trend for increased gastrointestinal toxicity. Irinotecan is an effective and safe second-line treatment for colorectal cancer. The schedules examined yielded equivalent results, indicating that there is no advantage of the prolonged vs short infusion schedules.

Quality of life during radiotherapy for rectal cancer.

The aim of this study was to assess symptoms and health-related quality of life (HRQL) during (neo)adjuvant radiotherapy for rectal cancer. Patients receiving pelvic radiotherapy 50 Gy for rectal cancer, were studied prospectively (n=42). The European Organization for Research and Treatment of Cancer (EORTC) questionnaires quality of life-core 30 QLQ-C30 and QLQ-CR38 and a 5-day symptom diary were completed at the start and end of radiotherapy and 4-6 weeks later. At the end of radiotherapy, mean scores of diarrhoea, fatigue and appetite loss had significantly increased (P<0.01) compared with pretreatment scores, but this was not observed for scores for nausea or pain. At the end of radiotherapy, diarrhoea, fatigue, appetite loss, physical function, social function and global quality of life (QL) were significantly worse than the population-based norms. 64% of the patients reported an increase in fatigue and 52% an increase in diarrhoea during radiotherapy. HRQL scores had returned to pre-treatment levels 4-6 weeks after radiotherapy. Thus, diarrhoea, fatigue and appetite loss increased transiently during pelvic radiotherapy.

Irinotecan combined with bolus 5-fluorouracil and folinic acid Nordic schedule as first-line therapy in advanced colorectal cancer.

BACKGROUND: This multicentre phase II study evaluated the efficacy and safety of irinotecan combined with the Nordic schedule of 5-fluorouracil (5-FU) and folinic acid (FA) as first-line therapy in patients with advanced colorectal cancer. PATIENTS AND METHODS: Seventy-four patients with measurable disease and a WHO performance status of 2 or less were treated with irinotecan 210 mg/m(2) as a 30-90 min intravenous infusion on day 1, followed by 5-FU 500 mg/m(2) and FA 60 mg/m(2) bolus on days 1 and 2, every 2 weeks, until disease progression or unacceptable toxicity. The primary end point was the objective response rate. RESULTS: Twenty-nine out of 68 evaluable patients achieved a complete (n = 7) or partial (n = 22) response, leading to an overall response rate of 43% [95% confidence interval (CI) 31% to 55%]. The median duration of response was 10 months. The estimated median time to progression and survival were 6.4 months (95% CI 5.4-9.0) and 15.6 months (95% CI 13.3-19.0), respectively, in the intention-to-treat population. A total of 860 cycles were administered to 74 patients. Neutropenia was the main adverse event with grade 3-4 toxicity in 66% of patients and 17.5% of cycles. Grade 3-4 non-haematological toxicities were infrequent and included diarrhoea in 16% of patients and 2% of cycles and nausea/vomiting in 10% of patients and 1% of cycles. CONCLUSIONS: Irinotecan combined with the bolus Nordic schedule of 5-FU/FA is active in advanced colorectal cancer with an easily managed safety profile which ensures good schedule compliance. The low incidence of grade 3-4 non-haematological toxicity justifies the further evaluation of this combination in the context of randomised clinical trials.

Design, organization and management of a controlled population screening study for detection of colorectal neoplasia: attendance rates in the NORCCAP study (Norwegian Colorectal Cancer Prevention).

BACKGROUND: In the past three decades, the incidence of colorectal cancer (CRC) in Norway has doubled, surpassing all other Nordic countries for both men and women to become the most frequently diagnosed cancer. A small-scale, randomized study on flexible sigmoidoscopy (FS) screening in Telemark, Norway, has shown a reduction in accumulated CRC incidence after 13 years. The aim of our study was to evaluate the effect on CRC mortality and morbidity by screen detection of CRC and removal of precursor lesions (polypectomy), and to test out the management and organization mimicking a countrywide screening service. A total of 13,823 men and women (1:1), age 55-64 years, were drawn randomly from the population registries in Oslo (urban) and the county of Telemark (mixed urban and rural) and invited to have a screening examination. The rest of the relevant age cohorts constituted the control groups. In the screening group, 535 individuals were excluded according to exclusion criteria, rendering 13,288 individuals eligible for screening examination. METHODS: A once only screening model was used. In the screening group, individuals were randomized to have a once only FS or a combination of FS and faecal occult blood test (FOBT). RESULTS: The overall attendance rate was 8,849 out of 13,288 (67%); 73% in Telemark and 60% in Oslo. Attendance for FS only was 68% and 65% for combined FS&FOBT. CONCLUSIONS: The present FSIFS&FOBT screening study obtained a high acceptance rate for both screening modalities. The attendance rate was stable throughout the trial, suggesting an acceptable model for management of future countrywide screening.

Quality of life in patients with urinary diversion after operation for locally advanced rectal cancer.

AIMS: When locally advanced or recurrent rectal cancer involves the bladder or prostate, curative treatment often requires pelvic exenteration. The aim was to assess the quality of life (QoL) in disease-free patients with urinary diversion after extensive surgery for advanced rectal cancer. METHODS: Twelve patients with urinary diversion (cases) were compared with 25 randomly selected patients given the same treatment, but without urinary diversion (controls). An age- and gender-adjusted general population was identified (reference). QoL was assessed with the EORTC questionnaires QLQ-C30, QLQ-CR38, and parts of the QLQ-BLM30. RESULTS: The cases did not report significantly worse overall QoL than the controls or the reference population. Both cases and controls had low mean scores of sexual function, and high mean scores of male sexual problems. In the nine cases that had two stomas, overall QoL was not worse than in the control or reference groups. CONCLUSIONS: Tumour-free patients did not report worse QoL scores than the controls or the general population, despite most having two stomas and low sexual function. Fear of reducing the patient's QoL should not be a major contraindication when surgery with urinary diversion is warranted to obtain curative resection.

Intra-operative irradiation (IORT) for primary advanced and recurrent rectal cancer. a need for randomised studies.

The aim of this study was to determine the impact of intra-operative irradiation (IORT) combined with pre-operative external beam irradiation (EBRT) and surgical resection in patients with locally advanced primary or recurrent rectal cancer. 64 patients with locally advanced primary cancer and 104 with recurrence had EBRT (46-50 Gy) before surgery. 80 patients received IORT (median dose 15 Gy energy 12 MeV). 80 patients had R0 resections, 47 R1 and 41 R2 resections. More R1 resections were performed in the IORT group, more R0 and R2 resections in the non-IORT group. Median follow-up was around 22 months. 146 patients were resected, 22 had exploratory laparotomy. The cumulative overall survival was similar for both the IORT and non-IORT groups. 5-year survival for primary cancers was 48% versus 28% for recurrences. No R2 resections survived 3.5 years. 5-year-survival for R0 resections was nearly 60% and around 30% for R1 resections. The survival curves of the patients given and not given IORT treatment was not statistically different when R0, R1 and R2 resections were analysed separately. IORT did not seem to influence the local recurrence rate when R0 and R1 resections were analysed separately or in a multivariate analysis. The IORT and non-IORT groups were not identical with regard to type of cancer and R-stage. Still the lack of an identifiable impact of IORT suggests that there is a need for randomised studies of the IORT effect.

Location of pelvic recurrence after 'curative' low anterior resection for rectal cancer.

AIMS: It has been emphasized that the mesorectum is the key to local recurrence after resection for rectal cancer. In view of this we studied the location of recurrences, relative to the bed of the primary tumour, the neorectum and the level of anastomoses, in patients referred for recurrences after low anterior resection (LAR) in the <>. METHODS: The relative level above the anal verge of the primary cancer, the anastomosis and the recurrence was registered by proctoscopy in 46 patients operated on for recurrent cancer after low anterior resection. The origin of the recurrence was determined from the operative specimen. RESULTS: The median level of the primary cancers was 10 cm above the anal verge, with the anastomoses 2 cm lower, the majority being within 2 cm. Most recurrences were within 1 cm of the anastomosis. No rectal cancer occurred more than 3 cm distal to the anastomosis. Seventy to 80% of recurrences started peri-rectally, most invading the anastomosis. CONCLUSIONS: The tumour bed is most often the origin of the recurrence. Recurrences were mostly due to inadequate radial, and in a few cases longitudinal, dissection of the mesorectum. Virtually all recurrences were within reach of the examining finger. At follow-up of rectal cancers most local recurrences can therefore be identified earlier by digital examination than by proctoscopy.

Type 1 protein tyrosine kinases in benign and malignant breast lesions.

AIMS: To determine their significance, we examined the expression pattern of the four epidermal growth factor receptor (EGFR) family members as well as the phosphotyrosine kinase activity in breast tumour tissues. METHODS AND RESULTS: Fifty-three malignant breast tumours, four breast cancer cell lines, and 10 benign breast tumours were investigated. Fifty-three per cent (28/53) of the malignant tumours expressed EGFR protein, and the majority of these positive tumours were strongly positive. Eighty per cent (8/10) of the benign tumours also expressed EGFR protein, but all in a lower or moderate level. An association between EGFR expression and increasing malignancy grade was found in the group of infiltrating ductal carcinomas. Of the malignant tumours, 35.8% (19/53) expressed c-erbB-2 protein and 17% (9/53) c-erbB-3 protein, while no expression of c-erbB-2 and c-erbB-3 proteins was found in the benign tumours. Contrary to previous reports, we observed c-erbB-4 receptor protein to be less expressed in the malignant breast tumours. The 'normal' breast epithelial cells adjacent to the malignant tumours and the benign tumours demonstrated intensified membrane staining for c-erbB-4, while a number of the malignant tumours demonstrated a weak cytoplasmic staining or were negative. However, several malignant tumours with strong membrane staining for the c-erbB-4 protein were also found. No simple association between the expression of the four receptors and phosphotyrosine kinase activity was found. CONCLUSION: Our study has revealed a complex expression pattern of the EGFR family members in breast tumour cells. While the data about EGFR, c-erbB-2, c-erbB-3 and phosphotyrosine are largely in line with what has been reported, we found the c-erbB-4 protein expression to be decreased in the malignant tumours.