ctDNA-Response evaluation criteria in solid tumors - a new measure in medical oncology.

In the metastatic setting, most decisions during systemic palliative therapies are based on the imaging-based Response Evaluation Criteria in Solid Tumors (RECIST), which is, however, known to be a suboptimal surrogate marker for the clinical outcome overall survival. Over the past decade, research has brought focus to the potential of circulating tumour DNA in cancer. However, at present, there is no generally accepted classification of quantitative changes during the treatment course, and prospective investigations can therefore not be validated. We here propose, for the first time, a response classification based on circulating tumour DNA measurements and its confidence intervals, a "ctDNA-RECIST" that has proven valuable in retrospective studies and goes along with the conventional RECIST classification. We aim to raise the topic for discussion and to encourage analyses of ctDNA data along this line.

Circulating DNA in patients undergoing loco-regional treatment of colorectal cancer metastases: a systematic review and meta-analysis.

Background: Loco-regional treatment strategies of colorectal cancer (CRC) metastases are evolving, but biological markers that can benefit patients and assist physicians in clinical decisions are lacking. The primary objective of this systematic review and meta-analysis is to investigate the current knowledge on circulating DNA and its clinical utility in predicting outcomes in patients undergoing loco-regional treatment of CRC metastases. Methods: A systematic search of PubMed, Embase, and Cochrane Central Register of Controlled Trials was conducted on March 22, 2022. We included studies on patients undergoing loco-regional treatment of CRC metastases reporting the predictive or prognostic value of circulating DNA in the blood. Hazard ratios (HR) were pooled in separate random-effects meta-analyses to investigate if pre- or post-ablation measurements of circulating DNA were associated with survival. The risk of bias was assessed according to the Quality in Prognosis Studies tool. Results: Twenty-eight studies with 2868 patients were included, of which 16 studies were eligible for meta-analyses. As expected in this new research field, a majority of included studies (n = 21/28) had a high risk of bias in at least one domain. Circulating DNA above the cutoff in a plasma sample taken before loco-regional treatment was associated with a short recurrence-free survival [pooled HR = 2.8, 95% confidence interval (CI) 1.4-5.7, n = 162] and overall survival (pooled HR = 4.7, 95% CI 1.1-20.6, n = 105). Circulating DNA above the cutoff in a plasma sample taken after loco-regional treatment was associated with a short recurrence-free survival (pooled HR = 4.5, 95% CI 3.4-6.1, n = 569) and overall survival (pooled HR = 7.5, 95% CI 2.0-27.3, n = 161). There was limited data on the association between dynamics in circulating DNA and outcome. Conclusions: Measurements of circulating DNA can be valuable when selecting and monitoring patients undergoing loco-regional treatment of CRC metastases. Studies designed to investigate the true clinical utility of circulating DNA in the context of various ablation modalities are warranted.The review has been registered at PROSPERO (ID: CRD42022320032).

Circulating tumour DNA and its clinical utility in predicting treatment response or survival in patients with metastatic colorectal cancer: a systematic review and meta-analysis.

BACKGROUND: We investigate the current knowledge on circulating tumour DNA (ctDNA) and its clinical utility in predicting outcomes in patients with metastatic colorectal cancer (mCRC). METHODS: PubMed, Embase, Cochrane Database of Systematic Reviews and Cochrane Central Register of Controlled Trials were searched. Last search 16/12/2020. We included studies on patients with mCRC reporting the predictive or prognostic value of ctDNA. We performed separate random-effects meta-analyses to investigate if baseline ctDNA and early changes in ctDNA levels during treatment were associated with survival. The risk of bias was assessed according to the Quality in Prognosis Studies tool. RESULTS: Seventy-one studies were included with 6930 patients. Twenty-four studies were included in meta-analyses. High baseline ctDNA level was associated with short progression-free survival (PFS) (HR = 2.2; 95% CI 1.8-2.8; n = 509) and overall survival (OS) (HR = 2.4; 95% CI 1.9-3.1; n = 1336). A small or no early decrease in ctDNA levels during treatment was associated with short PFS (HR = 3.0; 95% CI 2.2-4.2; n = 479) and OS (HR = 2.8; 95% CI 2.1-3.9; n = 583). Results on clonal evolution and lead-time were inconsistent. A majority of included studies (n = 50/71) had high risk of bias in at least one domain. CONCLUSIONS: Plasma ctDNA is a strong prognostic biomarker in mCRC. However, true clinical utility is lacking.

Management of late adverse effects after chemoradiation for anal cancer.

BACKGROUND AND PURPOSE: Significant improvements in the treatment of anal cancer have produced a growing population of anal cancer survivors. These patients often experience late adverse effects related to their treatment. Research has revealed substantial unmet needs because of long-term symptoms and functional impairments after treatment that may negatively affect health-related quality of life. The purpose of the present guidelines is to review the scientific evidence for the management of late adverse effects after (chemo)radiotherapy ([C]RT) for anal cancer and to extrapolate knowledge from other pelvic malignancies treated with pelvic (C)RT so that they may guide the clinical management of late adverse effects. MATERIALS AND METHODS: Relevant studies were systematically searched in four databases from their inception to June 2020 (no language limitation) and guidelines were searched in 16 databases, focussing on bowel dysfunction, psychosocial aspects, pain, and sexual and urinary dysfunction. The guidelines were developed by a panel of experts using the Oxford Centre for Evidence-based Medicine, levels of evidence, and grades of recommendations. SCIENTIFIC EVIDENCE: Late adverse effects after (C)RT for anal cancer are associated with a low overall quality of life among survivors. The most pronounced late adverse effects are bowel dysfunction (present in up to 78%), urinary dysfunction (present in up to 45%), and sexual dysfunction (present in up to 90% of men and up to 100% of women). Only indirect data on adequate treatment options of these late adverse effects for anal cancer are available. CONCLUSION: Quality of life and late adverse effects should be monitored systematically following treatment for anal cancer to identify patients who require further specialist evaluation or support. Increased awareness of the extent of the problem may serve to stimulate and facilitate multidisciplinary collaboration, which is often required.

The Clinical Value of Measuring Circulating HPV DNA during Chemo-Radiotherapy in Squamous Cell Carcinoma of the Anus.

BACKGROUND AND PURPOSE: Circulating tumor DNA (ctDNA) is investigated in various cancers. In squamous cell carcinoma of the anus (SCCA) infection with human papilloma virus (HPV) is found in around 90% of cases and here, plasma HPV (pHPV) can be used as ctDNA. Preliminary data have proved the ability to detect pHPV16 and -18 in SCCA. We have developed a highly sensitive method for measurement of six relevant pHPV subtypes, to investigate the elimination pattern of pHPV during chemo-radiotherapy (CRT) for SCCA and its clinical value. MATERIAL AND METHODS: Patients treated at Aarhus University Hospital from 2016-2020 were included. P16 status in the primary biopsy was measured and 82% of patients had P16 positive tumor. Blood samples were collected prior to treatment (PT), mid treatment (MT), end of therapy (EOT), and during follow-up (FU). An in-house multiplex digital droplet PCR method measured pHPV subtypes 16, 18, 31, 33, 51, 58. RESULTS: Samples from 88 patients were drawn PT (n = 73), MT (n = 72), EOT (n = 64) and during FU (n = 41). Plasma HPV was detectable in 52 patients and PT pHPV levels correlated to tumor stages. Three elimination patterns were observed during CRT with correlation to outcome: fast responders with no local or distant failures (0/12); slow responders with high risk of local failures (4/20), no distant failures; persistent molecular responders with high risk of distant failures (4/13), but no local failures, p < 0.01. CONCLUSION: During CRT, pHPV can divide patients with SCCA into three groups with significantly different risk of failure. The use of pHPV can potentially assist in clinical treatment decision.

Anorectal function and radiation dose to pelvic floor muscles after primary treatment for anal cancer.

BACKGROUND AND PURPOSE: Chemoradiotherapy is the primary treatment for localized anal cancer (AC). This treatment offers high rates of cure and organ preservation. Radiotherapy can however, result in late persisting anorectal dysfunction, with anal incontinence, urge and clustering. Correlation of radiation doses to pelvic substructures and functional outcome is not well described in AC. We correlated patient reported anorectal function to radiation doses to sphincters and pelvic floor muscles. MATERIALS AND METHODS: Patients treated with (chemo)radiotherapy for AC were asked to fill out LARS (lower anterior resection syndrome) questionnaires at follow-up. We compared patients with no LARS (score 0-19) and patients with major LARS (30-42) as well as individual LARS questions to specific radiation doses to sphincters, levators and puborectal muscles. RESULTS: Thirty-six patients were included, 18 with no LARS and 18 with major LARS. Gender, age, TNM stage, PTV, chemotherapy, time to LARS score (mean 660 and 749 days) were comparable between the two groups. LARS symptoms, occurring at least once per week, were reported between 25-55.7%, and poorer LARS outcome was associated to worse quality of life. Dose to sphincter complex (Dmean, V50Gy and D90%) differed significantly between patients with no and major LARS (p = 0.048, 0.035 and 0.02 respectively). Further, D90% to the sphincter complex was significantly higher in patients who had accidental leakage of stool, (p = 0.044). CONCLUSION: Patients treated with (chemo)radiotherapy for AC show high frequency of patient reported anorectal dysfunction. Specific doses to the sphincters could become a useful predictor of anal incontinence and major LARS and incorporated into future radiotherapy planning studies.

Prognostic and predictive value of circulating DNA for hepatic arterial infusion of chemotherapy for patients with colorectal cancer liver metastases.

Hepatic arterial infusion (HAI) of chemotherapy is an experimental treatment option for patients with colorectal cancer liver metastases (CRCLM). The current study aimed to investigate the predictive and prognostic value of cell free DNA (cfDNA) in patients with CRCLM receiving HAI with oxaliplatin and systemic capecitabine. Plasma samples from 62 patients were investigated who were included into a single arm phase II study investigating HAI treatment for patients with CRCLM. The clinical outcome of the trial has been presented previously. In brief, treatment consisted of intrahepatic infusion of oxaliplatin 100 mg/m2 every second week with concomitant oral capecitabine 3,500 mg/m2 every second week for up to 12 cycles. Blood samples were drawn at baseline and follow-up and plasma was analyzed for cell free DNA using a direct fluorescent assay. The baseline level of plasma cfDNA was 0.92 ng/µl (95% CI 0.84-1.00). Patients with a baseline value of cfDNA above the 75th quartile had a median overall survival of 2.4 years (95% CI 0.7-2.8), compared with 3.9 years (95% CI 2.8-5.9) for patients below the 75th quartile (P=0.02). The baseline level of cfDNA was significantly lower (0.91 ng/µl, 95% CI 0.76-0.98) in patients who achieved an objective response compared to non-responders (1.79 ng/µl; 95% CI 0.99-2.57; P=0.02). The current study demonstrated a possible prognostic and predictive value of cfDNA for patients with CRCLM undergoing HAI with oxaliplatin and concomitant capecitabine.

Circulating tumor DNA as a marker of minimal residual disease following local treatment of metastases from colorectal cancer.

BACKGROUND: Local treatment of liver and/or lung metastases from colorectal cancer (CRC) is increasingly used in daily practice and comprises resection, radiofrequency ablation (RFA) and stereotactic radiotherapy (SBRT). The need for prognostic markers for patients undergoing such treatment is currently unmet. We investigated post-treatment circulating tumor-specific DNA (ctDNA) analysis and address a possible prognostic value in a pilot study. MATERIALS: From July 2015 to September 2017, patients undergoing standard of care local treatment of liver and/or lung metastases were included in a prospective translational study. Blood samples were drawn 2 weeks after local treatment and during follow-up. CtDNA was detected by ddPCR and a mass spectrometry-based platform MassARRAY®. RESULTS: Post treatment blood samples were available for 35 patients including five with detectable ctDNA (KRAS mutation, n = 2; NRAS mutation, n = 2; BRAF mutation, n = 1) by ddPCR. 17 out of 35 patients (49%) developed recurrence within a median of 273 days (95%CI 95-NA) among patients positive for ctDNA, while the median time to recurrence was not reached for the group of patients negative for ctDNA (p = .03). CONCLUSION: The presence of ctDNA following local treatment of metastatic CRC is associated with an increased risk of recurrence and a short time to failure.

Pelvic insufficiency fractures, dose volume parameters and plan optimization after radiotherapy for rectal cancer.

Pelvic insufficiency fractures (PIF) is a known but under-acknowledged late effect of pelvic radiotherapy. In rectal cancer, studies describing incidence of PIF and relation to dose volume relationships are lacking. The aim of this study was (i) to analyse dose volume histograms (DVH) from pelvic bones in patients with and without PIF, and (ii) to determine bone sparing capacity of 2 and 3 arc volumetric arc therapy (VMAT), intensity modulated radiotherapy (IMRT) and proton beam therapy (PBT), in rectal cancer patients treated with chemoradiotherapy (CRT). MATERIAL AND METHODS: Patients treated with CRT for primary rectal cancer underwent a 3-year pelvic MRI for identification of PIFs. Bone structures were retrospectively delineated, and DVHs were re-calculated. Comparative planning was done with 2 (original) and 3 arc VMAT, fixed field IMRT and PBT plans. RESULTS: 27 patients (18 men, mean age 64 years) were included and PIFs were identified in 9 (33%), most (n = 6) had multiple fracture sites. In general, patients with PIFs received higher doses to pelvic bones, and V30 Gy to the sacroiliac joint was non-significantly higher in patients with PIF 68.5% (60.1-69.3 IQR) vs. 56% (54.1-66.6 IQR), p = 0.064. Comparative planning showed that especially 3 arc VMAT and proton beam therapy could be optimized for bone. CONCLUSIONS: Patients, treated with VMAT based CRT for rectal cancer, have high rates of PIFs after 3 years. Patients with PIFs tended to have received higher doses to sacroiliac joints. Comparative planning demonstrated most pronounced bone sparing capacity of 3 arc VMAT and with PBT having the potential to further lower doses. These results should be validated in larger and preferably prospective cohorts.

Robust dose planning objectives for mesorectal radiotherapy of early stage rectal cancer - A multicentre dose planning study.

BACKGROUND AND PURPOSE: Organ preservation strategies are increasingly being explored for early rectal cancer. This requires revision of target volumes according to disease stage, as well as new guidelines for treatment planning. We conducted an international, multicentre dose planning study to develop robust planning objectives for modern radiotherapy of a novel mesorectal-only target volume, as implemented in the STAR-TReC trial (NCT02945566). MATERIALS AND METHODS: The published literature was used to establish relevant dose levels for organ at risk (OAR) plan optimisation. Ten representative patients with early rectal cancer were identified. Treatment scans had mesorectal target volumes as well as bowel cavity, bladder and femoral heads outlined, and were circulated amongst the three participating institutions. Each institution produced plans for short course (SCRT, 5 × 5 Gy) and long course (LCRT, 25 × 2 Gy) treatment, using volumetric modulated arc therapy on different dose planning systems. Optimisation objectives for OARs were established by determining dose metric objectives achievable for ≥90% of plans. RESULTS: Sixty plans, all fulfilling target coverage criteria, were produced. The planning results and literature review suggested optimisation objectives for SCRT: V 10Gy < 180 cm3, V 18Gy < 110 cm3, V 23Gy < 85 cm3 for bowel cavity; V 21Gy < 15% and V 25Gy < 5% for bladder; and V 12.5Gy < 11% for femoral heads. Corresponding objectives for LCRT: V 20Gy < 180 cm3, V 30Gy < 130 cm3, V 45Gy < 90 cm3 for bowel cavity; V 35Gy < 22% and V 50Gy < 7% for bladder; and V 25Gy < 15% for femoral heads. Constraints were validated across all three institutions. CONCLUSION: We utilized a multicentre planning study approach to develop robust planning objectives for mesorectal radiotherapy for early rectal cancer.

Prospective evaluation of acute toxicity and patient reported outcomes in anal cancer and plan optimization.

BACKGROUND AND PURPOSE: Chemoradiotherapy (CRT) is the standard therapy for localized anal cancer (AC), but this treatment is associated with substantial toxicity. However, there is a lack of prospectively collected toxicity and patient reported outcome (PRO) data from larger cohorts. The purpose was to prospectively collect and determine agreement between physician assessed toxicity (CTCAE) and PRO during and after CRT and to compare IMRT, VMAT and proton-based planning in a subgroup of patients. MATERIAL AND METHODS: Patients, treated with CRT for AC, were included between 2015 and 2017. NCI-CTCAE v.4.0, EORTC QLQ-C30 and CR29 data were collected baseline, mid-therapy, end-of therapy and 2-4 weeks posttherapy. Treatment planning with 5- or 6-fixed field IMRT, 2 and 3 arc VMAT, and 3- and 4-field proton plans were compared. RESULTS: One-hundred patients were included. Both CTCAE and PROs related to acute toxicity reached a maximum at end of therapy. Incidences of PROs were markedly higher with only slight to fair agreement to CTCAE, (κ 13-37). Comparative planning revealed dosimetric equality of IMRT and VMAT plans, but superiority of proton plans. CONCLUSIONS: The high incidence of PRO scores and weak agreement to CTCAE suggest that PROs are important tools complementary to CTCAE in evaluating patient symptoms during and after CRT. Proton therapy has the potential to lower radiation doses to most organs at risk.

Cell-Free DNA in Metastatic Colorectal Cancer: A Systematic Review and Meta-Analysis.

BACKGROUND: Circulating DNA can be detected and quantified in the blood of cancer patients and used for detection of tumor-specific genetic alterations. The clinical utility has been intensively investigated for the past 10 years. The majority of reports focus on analyzing the clinical potential of tumor-specific mutations, whereas the use of total cell-free DNA (cfDNA) quantification is somehow controversial and sparsely described in the literature, but holds important clinical information in itself. The purpose of the present report was to present a systematic review and meta-analysis of the prognostic value of total cfDNA in patients with metastatic colorectal cancer (mCRC) treated with chemotherapy. In addition, we report on the overall performance of cfDNA as source for KRAS mutation detection. MATERIALS AND METHODS: A systematic literature search of PubMed and Embase was performed by two independent investigators. Eligibility criteria were (a) total cfDNA analysis, (b) mCRC, and (c) prognostic value during palliative treatment. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines were followed, and meta-analysis applied on both aggregate data extraction and individual patients' data. RESULTS: Ten eligible cohorts were identified, including a total of 1,076 patients. Seven studies used quantitative polymerase chain reaction methods, two BEAMing [beads, emulsification, amplification, and magnetics] technology, and one study digital droplet polymerase chain reaction. The baseline levels of cfDNA was similar in the presented studies, and all studies reported a clear prognostic value in favor of patients with lowest levels of baseline cfDNA. A meta-analysis revealed a combined estimate of favorable overall survival hazard ratio (HR) in patients with levels below the median cfDNA (HR = 2.39, 95% confidence interval 2.03-2.82, p < .0001). CONCLUSION: The total cfDNA levels are high in patients with mCRC and bear strong prognostic information, which should be tested prospectively by using a predefined cut-off value based on normal values in healthy cohorts. Finally, the potential use of cfDNA for detection of tumor-specific mutations was emphasized in a large individual patients' data meta-analysis. IMPLICATIONS FOR PRACTICE: Reliable prognostic markers could help to guide patients and treating physicians regarding the relevance and choice of systemic therapy. Small fragments of circulating cell-free DNA (cfDNA) can be measured in a simple blood sample. This report presents the first meta-analysis of the prognostic value of total cfDNA measurement in patients with metastatic colorectal cancer. Data from 1,076 patients confirmed that patients with the lowest pre-treatment levels of cfDNA had a significantly higher chance of longer survival than those with higher levels. Cell-free DNA analysis can also be used for detection of tumor-specific mutations, and hold potential as a valuable tool in colorectal cancer treatment.

Methodological, biological and clinical aspects of circulating free DNA in metastatic colorectal cancer.

BACKGROUND: Circulating DNA can be used to measure the total cell-free DNA (cfDNA) and for detection and quantification of tumor-specific genetic alterations in the peripheral blood, and the broad clinical potential of circulating DNA has attracted increasing focus over the past decade. Concentrations of circulating DNA are high in metastatic colorectal cancer (CRC), and the total levels of cfDNA have been reported to hold strong prognostic value. Colorectal tumors are characterized by a high frequency of well known, clinically relevant genetic alteration, which is readily detected in the cfDNA and holds potential for tailoring of palliative therapy and for monitoring during treatment. This review aims to present the current literature which has specifically reported data on the potential utility of cfDNA and on tumor-specific mutations in metastatic colorectal cancer (mCRC). METHOD: Methodological, biological and clinical aspects are discussed based on the most recent development in this specific setting, and eligible studies were identified by systematic literature searched from Pubmed and EMBASE in addition to conference papers and communications. RESULTS: The literature regarding cfDNA in CRC is broad and heterogeneous concerning aims, nomenclature, methods, cohorts and clinical endpoints and consequently difficult to include in a single systematic search. However, the available data underline a strong clinical value of measuring both total cfDNA levels and tumor-specific mutations in the plasma of patients with mCRC, pre- and during systemic therapy. CONCLUSION: This paper had gathered the most recent literature on several aspects of cfDNA in mCRC, including methodological, biological and clinical aspects, and discussed the large clinical potential in this specific setting, which needs to be validated in carefully designed prospective studies in statistically relevant cohorts.

Gemcitabine and capecitabine for heavily pre-treated metastatic colorectal cancer patients--a phase II and translational research study.

AIM: We investigated the efficacy and safety of capecitabine and gemcitabin (GemCap) in heavily pre-treated, therapy-resistant metastatic colorectal cancer (mCRC) patients and the clinical importance of cell-free DNA (cfDNA) measurement. PATIENTS AND METHODS: Patients' inclusion criteria included histopathologically-verified mCRC refractory to standard chemotherapy, adequate organ function and performance status. Treatment included capecitabine (2,000 mg/m(2) day on days 1-7 q2w) and gemcitabine (1,000 mg/m(2) on day 1). The number of DNA alleles was measured in pre-treatment plasma samples using an in-house developed qPCR. RESULTS: Forty-nine patients were included in the study. GemCap was well-tolerated in the majority of patients. Disease control rate was 30%, median progression-free survival (PFS) and overall survival (OS) by intention-to-treat were 2.7 (95%CI=2.6-2.8) and 6.8 (95%CI=5.0-7.7) months. Median OS in patients with cfDNA concentrations above the median (13,200 alleles/ml) was 4.7 (3.7-9.6) months compared to 7.8 months in the remaining patients (HR=2.22; 1.07-3.9; p=0.0186). The prognostic value of the cell-free DNA (cfDNA) was confirmed by multivariate analysis. CONCLUSION: GemCap was well-tolerated with encouraging efficacy, and cfDNA was shown to hold a strong prognostic value.

Phase II trial of temsirolimus alone and in combination with irinotecan for KRAS mutant metastatic colorectal cancer: outcome and results of KRAS mutational analysis in plasma.

BACKGROUND: Patients with chemotherapy refractory metastatic colorectal cancer and KRAS mutations have no effective treatment option. The present study evaluated the efficacy of temsirolimus in chemotherapy refractory mCRC with KRAS mutations. Furthermore, we wanted to investigate if resistance to temsirolimus could be reversed by the addition of irinotecan. Finally, we analyzed pre-treatment blood samples for KRAS mutations to investigate the association between quantitative measures of KRAS mutated alleles and clinical outcome. MATERIAL AND METHODS: Patients received weekly temsirolimus 25 mg until progression. Thereafter patients were treated with combination therapy comprising biweekly irinotecan 180 mg/m(2) and weekly temsirolimus. A polymerase chain reaction method was used to quantify the KRAS mutated alleles in plasma (pKRAS). RESULTS: Sixty-four patients were included. Treatment was well tolerated. Thirty-eight percent achieved stable disease on monotherapy and 63% on combination therapy. Four and eight patients had a minimal response, respectively. Median overall survival was 160 days. Median time to progression was 45 and 84 days, respectively. The concordance between KRAS status in tumor and plasma was 82%. All patients with tumor reduction had low levels of pKRAS. Patients with high pKRAS had a 77% risk of early progression on monotherapy compared to 43% in patients with lower levels. Multivariate survival analysis confirmed that pKRAS was a strong prognostic factor. CONCLUSION: Temsirolimus has limited efficacy in chemotherapy resistant KRAS mutant disease, but plasma KRAS quantification is a strong predictor of outcome.

The influence of tissue ischemia on biomarker expression in colorectal cancer.

The aim of the present study was to investigate the influence of fixation delay and the perioperative ischemia on hypoxia inducible factor (HIF)-1α gene expression, HIF-1α protein expression, and immunohistochemical (IHC) expression of HIF-1α, GLUT-1, Bcl-2, and Ki-67 in colorectal cancer. The study included 25 surgically removed colorectal tumors. Three sets of samples were collected readily after removal and exposed to 0, 30, and 60 minutes of delay of fixation or freezing. The perioperative ischemia time was registered. In each set of the samples, HIF-1α gene expression was analyzed by quantitative real time polymerase chain reaction, protein concentration of HIF-1α was assessed by enzyme-linked immunosorbent assay, and IHC staining of HIF-1α, GLUT-1, Bcl-2, and Ki-67 was performed. Preoperative formalin-fixed paraffin-embedded biopsies and whole sections of the entire tumor were analyzed by IHC. We found that the HIF-1α gene expression, HIF-1α protein concentration, and IHC expression of HIF-1α, GLUT-1, Ki-67, and Bcl-2 were not systematically affected by either the fixation or freezing delay of the tissue, the perioperative ischemia time, or the total ischemia time (perioperative ischemia+delay of fixation or freezing) in colorectal tumors. However, the intraindividual variation was quite large, which may question the use of individually, non-standardized-handled single biopsies or small tissue samples for analysis of often rather heterogenously expressed biomarkers.

Tumour hypoxia imaging with 18F-fluoroazomycinarabinofuranoside PET/CT in patients with locally advanced rectal cancer.

OBJECTIVE: The aim of this study was to investigate the feasibility of F-fluoroazomycinarabinofuranoside (F-FAZA) positron emission tomography (PET)/computed tomography (CT) in patients with locally advanced rectal cancer. MATERIALS AND METHODS: The study included 14 patients with locally advanced rectal cancer. Before chemoradiotherapy, PET/CT with F-FAZA was performed with static 15 min images 2 h after injection of F-FAZA. Attenuation correction was obtained with a low-dose CT, and a contrast-enhanced CT was performed immediately after the PET scan. RESULTS: F-FAZA uptake [mean and maximum standardized uptake value (SUVmean) and (SUVmax)] was significantly higher in rectal tumours than in both muscles (P<0.003) and normal intestinal walls (P<5×10). The tumour to muscle (T/M) ratios ranged from 1.19 to 3.05 with a mean of 1.97, whereas the tumour to intestinal wall (T/I) ratios had values of 1.73-5.81 with a mean of 2.83. Intense activity accumulating in the bladder produced obvious scattered activity, which spread into the surrounding tissue. Tumour volumes excluding scatter were therefore determined, in which the SUVmax and SUVmean were also significantly higher than in both muscles (P<0.004) and normal intestinal walls (P<2×10) and had T/M ratios of 1.19-2.72 with a mean of 1.85 and T/I ratios of 1.71-5.40 with a mean of 2.67. The individual SUVmax, SUVmean, T/M and T/I values were significantly higher in the entire tumour volume compared with the tumour volume adjusted for scatter from the urinary bladder (P<0.005), although the absolute differences were small. CONCLUSION: F-FAZA PET/CT is feasible for visualization of hypoxia in patients with rectal cancer, but scattered activity from the urinary bladder should be taken into consideration.

EGFR related mutational status and association to clinical outcome of third-line cetuximab-irinotecan in metastatic colorectal cancer.

BACKGROUND: As supplement to KRAS mutational analysis, BRAF and PIK3CA mutations as well as expression of PTEN may account for additional non-responders to anti-EGFR-MoAbs treatment. The aim of the present study was to investigate the utility as biomarkers of these mutations in a uniform cohort of patients with metastatic colorectal cancer treated with third-line cetuximab/irinotecan. METHODS: One-hundred-and-seven patients were prospectively included in the study. Mutational analyses of KRAS, BRAF and PIK3CA were performed on DNA from confirmed malignant tissue using commercially available kits. Loss of PTEN and EGFR was assessed by immunohistochemistry. RESULTS: DNA was available in 94 patients. The frequency of KRAS, BRAF and PIK3CA mutations were 44%, 3% and 14%, respectively. All were non-responders. EGF receptor status by IHC and loss of PTEN failed to show any clinical importance. KRAS and BRAF were mutually exclusive. Supplementing KRAS analysis with BRAF and PIK3CA identified additional 11% of non-responders. Patient with any mutation had a high risk of early progression, whereas triple-negative status implied a response rate (RR) of 41% (p<0.001), a disease control (DC) rate of 73% (p<001), and a significantly higher PFS of 7.7(5.1-8.6 95%CI) versus 2.3 months (2.1-3.695%CI) (p<0.000). CONCLUSION: Triple-negative status implied a clear benefit from treatment, and we suggest that patient selection for third-line combination therapy with cetuximab/irinotecan could be based on triple mutational testing.

The importance of -460 C/T and +405 G/C single nucleotide polymorphisms to the function of vascular endothelial growth factor A in colorectal cancer.

PURPOSE: The present study investigated the functional influence of the single nucleotide polymorphisms (SNPs) -460 C/T and +405 G/C at vascular endothelial growth factor A (VEGF-A), mRNA and protein levels in colorectal cancer (CRC) and normal colorectal tissue. METHODS: Blood and tissue were collected from 113 patients surgically resected for colorectal cancer. SNPs were analysed from genomic DNA by PCR, the VEGF-A gene expression analysis was performed by RT-PCR and protein analysis by ELISA. RESULTS: The T-allele in the -460 C/T SNP and the C-allele in the +405 G/C SNP were associated with significantly lower VEGF-A protein levels in normal colorectal tissue. There were no differences in protein levels in the malignant tissue according to genotypes. No differences were observed at the gene expression levels either. CONCLUSION: The results indicate that the two SNPs have a functional influence on the VEGF-A protein levels in normal colorectal tissue. The possible clinical implications of the findings need further investigation.

The prognostic value of haplotypes in the vascular endothelial growth factor a gene in colorectal cancer.

New prognostic markers in patients with colorectal cancer (CRC) are a prerequisite for individualized treatment. Prognostic importance of single nucleotide polymorphisms (SNPs) in the vascular endothelial growth factor A (VEGF-A) gene has been proposed. The objective of the present study was to investigate the prognostic importance of haplotypes in the VEGF-A gene in patients with CRC. The study included 486 patients surgically resected for stage II and III CRC, divided into two independent cohorts. Three SNPs in the VEGF-A gene were analyzed by polymerase chain reaction. Haplotypes were estimated using the PHASE program. The prognostic influence was evaluated using Kaplan-Meir plots and log rank tests. Cox regression method was used to analyze the independent prognostic importance of different markers. All three SNPs were significantly related to survival. A haplotype combination, responsible for this effect, was present in approximately 30% of the patients and demonstrated a significant relationship with poor survival, and it remained an independent prognostic marker after multivariate analysis, hazard ratio 2.46 (95% confidence interval 1.49-4.06), p < 0.001. Validation was provided by consistent findings in a second and independent cohort. Haplotype combinations call for further investigation.