Effect of Hyperthermic Intraperitoneal Chemotherapy on Cytoreductive Surgery in Gastric Cancer With Synchronous Peritoneal Metastases: The Phase III GASTRIPEC-I Trial.

PURPOSE: In patients with peritoneal metastasis (PM) from gastric cancer (GC), chemotherapy is the treatment of choice. Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) are still being debated. This randomized, controlled, open-label, multicenter phase III trial (EudraCT 2006-006088-22; ClinicalTrials.gov identifier: NCT02158988) explored the impact on overall survival (OS) of HIPEC after CRS. PATIENTS AND METHODS: Adult patients with GC and histologically proven PM were randomly assigned (1:1) to perioperative chemotherapy and CRS alone (CRS-A) or CRS plus HIPEC (CRS + H). HIPEC comprised mitomycin C 15 mg/m2 and cisplatin 75 mg/m2 in 5 L of saline perfused for 60 minutes at 42°C. The primary end point was OS; secondary endpoints included progression-free survival (PFS), other distant metastasis-free survival (MFS), and safety. Analyses followed the intention-to-treat principle. RESULTS: Between March 2014 and June 2018, 105 patients were randomly assigned (53 patients to CRS-A and 52 patients to CRS + H). The trial stopped prematurely because of slow recruitment. In 55 patients, treatment stopped before CRS mainly due to disease progression/death. Median OS was the same for both groups (CRS + H, 14.9 [97.2% CI, 8.7 to 17.7] months v CRS-A, 14.9 [97.2% CI, 7.0 to 19.4] months; P = .1647). The PFS was 3.5 months (95% CI, 3.0 to 7.0) in the CRS-A group and 7.1 months (95% CI, 3.7 to 10.5; P = .047) in the CRS + H group. The CRS + H group showed better MFS (10.2 months [95% CI, 7.7 to 14.7] v CRS-A, 9.2 months [95% CI, 6.8 to 11.5]; P = .0286). The incidence of grade ≥3 adverse events (AEs) was similar between groups (CRS-A, 38.1% v CRS + H, 43.6%; P = .79). CONCLUSION: This study showed no OS difference between CRS + H and CRS-A. PFS and MFS were significantly better in the CRS + H group, which needs further exploration. HIPEC did not increase AEs.

Gene-expression profiles of pretreatment biopsies predict complete response of rectal cancer patients to preoperative chemoradiotherapy.

PURPOSE: Preoperative (neoadjuvant) chemoradiotherapy (CRT) and total mesorectal excision is the standard treatment for rectal cancer patients (UICC stage II/III). Up to one-third of patients treated with CRT achieve a pathological complete response (pCR). These patients could be spared from surgery and its associated morbidity and mortality, and assigned to a "watch and wait" strategy. However, reliably identifying pCR based on clinical or imaging parameters remains challenging. EXPERIMENTAL DESIGN: We generated gene-expression profiles of 175 patients with locally advanced rectal cancer enrolled in the CAO/ARO/AIO-94 and -04 trials. One hundred and sixty-one samples were used for building, training and validating a predictor of pCR using a machine learning algorithm. The performance of the classifier was validated in three independent cohorts, comprising 76 patients from (i) the CAO/ARO/AIO-94 and -04 trials (n = 14), (ii) a publicly available dataset (n = 38) and (iii) in 24 prospectively collected samples from the TransValid A trial. RESULTS: A 21-transcript signature yielded the best classification of pCR in 161 patients (Sensitivity: 0.31; AUC: 0.81), when not allowing misclassification of non-complete-responders (False-positive rate = 0). The classifier remained robust when applied to three independent datasets (n = 76). CONCLUSION: The classifier can identify >1/3 of rectal cancer patients with a pCR while never classifying patients with an incomplete response as having pCR. Importantly, we could validate this finding in three independent datasets, including a prospectively collected cohort. Therefore, this classifier could help select rectal cancer patients for a "watch and wait" strategy. TRANSLATIONAL RELEVANCE: Forgoing surgery with its associated side effects could be an option for rectal cancer patients if the prediction of a pathological complete response (pCR) after preoperative chemoradiotherapy would be possible. Based on gene-expression profiles of 161 patients a classifier was developed and validated in three independent datasets (n = 76), identifying over 1/3 of patients with pCR, while never misclassifying a non-complete-responder. Therefore, the classifier can identify patients suited for "watch and wait".

KRAS mutation status concordance between the primary tumor and the corresponding metastasis in patients with rectal cancer.

INTRODUCTION: Oncogenic mutation within the KRAS gene represents a negative predictor for treatment response to anti-epidermal growth factor receptor (EGFR) in patients with colorectal cancer. Recently, we have shown no relevant heterogeneity for KRAS mutation status within and between pre- and posttherapeutic samples from the primary tumor in patients with locally advanced rectal cancer. The aim of this study was to evaluate the intertumoral heterogeneity of KRAS mutation status between the primary tumor and the corresponding metastasis or local recurrence in the similar cohort and to evaluate the ideal representative tissue for KRAS mutation testing. MATERIALS AND METHODS: KRAS mutation status was analyzed from 47 patients with locally advanced rectal cancer, which were enrolled in the CAO/ARO/AIO-94 or CAO/ARO/AIO-04 trial. Mutations in KRAS codons 12, 13, and 61 were analyzed by using the KRAS RGQ PCR Kit (therascreen® KRAS test). Six patients needed to be excluded due to incomplete follow up data. 11 patients showed a relapse of the disease during the follow up presented by distant metastases or local recurrence. DNA from representative areas of metastatic tissue was obtained from formalin-fixed paraffin-embedded specimens. RESULTS: The mean patient age was 64.13 ± 10.64 years. In total, 19 patients showed a KRAS mutation (46.34%) in the primary tumor. Of the eleven patients with a metastatic disease or local recurrence, five patients showed a KRAS mutation whereas six patients had a KRAS wildtype status. Metastatic localizations included the liver (n = 2), lung (n = 4), local recurrence (n = 1), liver + lung (n = 3), lung + local recurrence (n = 1). For these eleven patients with paired data available for the primary tumor and metastatic tissue, a significant KRAS mutation status concordance was detected in 81.18% (9/11) of the patients (p = 0.03271). Only two patients showed intertumoral heterogeneity, which harbored in one patient a KRAS G12C mutation status in the primary tumor, but a G12V KRAS mutation status in the corresponding lung lesion, and in the other patient a G12A mutation in the primary lesion and a WT in the lung metastasis. CONCLUSIONS: We show a significant concordance of the KRAS mutation status between tumor samples obtained from the primary tumor and the corresponding metastasis and/ or local recurrence in patients with rectal cancer indicating no relevant intertumoral heterogeneity. Our data suggest that sampling either the primary (pre- or posttherapeutical tumor tissue) or metastatic lesion may be valid for the initial evaluation of KRAS mutation status predicting the response to anti-EGFR treatment and guiding clinical decisions.

CA19-9 for detecting recurrence of pancreatic cancer.

CA19-9 values are regularly measured in patients with pancreatic cancer. Certainly, its potential as a biomarker has been compromised by false negative results in CA19-9 negative patients and false positive results in benign pancreatico-biliary diseases. For detection of PDAC recurrence, however, CA19-9 might play an important role. The aim of this study is to analyze the accuracy of CA19-9 for detecting recurrence of pancreatic cancer. All included patients were treated either at the University Medical Center Goettingen, or at the Department of Interdisciplinary Oncology and Pneumonology, DRK-Kliniken Nordhessen, Kassel. We analyzed data of 93 patients with pancreatic cancer in the training set and 41 in the validation set, both retrospectively. Pre- and postoperative CA19-9 values and results of imaging techniques were compared. We performed ROC-analysis. The association between longitudinally measured CA19-9 values and relapse was studied with a joint model between a random effects model for the longitudinal CA19-9 measurements and a Cox proportional hazards models for the survival data. In the test set (n = 93 patients) the median follow-up time was 644 days (22 months). Overall, 71 patients (76.3%) developed recurrence during follow-up. Patients with CA19-9 values of <10kU/l were considered as CA19-9 negative patients (n = 11) and excluded from further analysis. Among the rest, approximately 60% of the patients showed significantly elevated CA19-9 prior to detection of recurrence by imaging techniques. Recurrence was shown by 2.45 times elevated CA19-9 values with 90% positive predictive value. In the validation set, 2.45 times elevated CA19-9 values showed recurrence with 90% sensitivity and 83,33% specificity, with an area under the curve of 95%. Based on measured CA19-9 values during follow-up care, the joint model estimates in recurrence-free patients the probability of recurrence-free survival. CA19-9 elevation is an early and reliable sign for PDAC recurrence. On the strength of a very high accuracy in CA19-9 positive patients, it should be considered to use CA19-9 for therapy decision even without a correlate of imaging technics. Using the joint model, follow-up care of PDAC patients after curative therapy can be stratified.

[Incidence and Predictive Factors for Bilaterality in Patients with Papillary Thyroid Carcinoma]. / Inzidenz und prädiktive Faktoren des bilateralen papillären Schilddrüsenkarzinoms.

BACKGROUND: The optimal extent of thyroidectomy for papillary thyroid cancer (PTC) ≥ 10 mm und < 10 mm is still controversial. Therefore, the purpose of this study was to investigate factors predictive for bilaterality in patients with papillary thyroid carcinoma (PTC). MATERIAL AND METHODS: We retrospectively reviewed 123 PTC patients in a single centre study who underwent either completion or total thyroidectomy and analysed the predictive value of tumour size, histological parameters, multifocality, and lymph node metastases with primary tumour size of ≥ 10 mm and < 10 mm as well as for ≥ 7 mm and < 7 mm. RESULTS: Out of 123 patients, 26 exhibited bilateral PTC. This was significantly more frequent in patients with a primary tumour size of ≥ 10 mm (77%) compared to a tumour size of < 10 mm (23%) (p = 0.004). Multifocality was found to be an independent predictive factor for bilaterality (p = 5.022e-18). Metachronous lymph node metastases showed a trend for bilateral PTCs (p = 0.0691). These findings were reproducible for the comparison between the ≥ 7 mm and < 7 mm group. CONCLUSION: The presence of bilateral PTC appears to be related to the size of the primary tumour ≥ 10 mm. Multifocality is a positive predictor for bilaterality. When multifocality, even with a primary tumour size of < 10 mm, is observed in patients with PTC, total thyroidectomy or completion thyroidectomy may be considered. If lobectomy is performed in patients with PTC, meticulous follow-up is needed to detect hidden malignancies in the contralateral lobe.

Colorectal cancer susceptibility loci as predictive markers of rectal cancer prognosis after surgery.

To understand the molecular mechanism of rectal cancer and develop markers for disease prognostication, we generated and explored a dataset from 243 rectal cancer patients by gene expression microarray analysis of cancer samples and matched controls, and SNP-arrays of germline DNA. We found that two of the loci most strongly linked with colorectal cancer (CRC) risk, 8q24 (upstream of MYC) and 18q21 (in the intron of SMAD7), as well as 20q13 (in the intron of LAMA5), are tightly associated with the prognosis of rectal cancer patients. For SNPs on 18q21 (rs12953717 and rs4464148) and 20q13 (rs4925386), alleles that correlate with higher risk for the development of CRC are associated with shorter disease free survival (DFS). However, for rs6983267 on 8q24, the low risk allele is associated with a higher risk for recurrence and metastasis after surgery, and importantly, is strongly correlated with the resistance of CRC cell lines to chemoradiotherapy (CRT). We also found that although MYC expression is dramatically increased in cancer, patients with higher levels of MYC have a better prognosis. The expression of SMAD7 is weakly correlated with DFS. Notably, the presence of the 8q24 and 18q21 SNP alleles is not correlated with expression levels of MYC and SMAD7. rs4464148, and probably rs6983267 and rs4925386, are linked with overall survival time of patients. In conclusion, we show that several CRC risk SNPs detect subpopulations of rectal cancer patients with poor prognosis, and that rs6983267 probably affects prognosis through interfering with the resistance of cancer cells to CRT.

Endovascular Treatment of Large and Giant Carotid Aneurysms with Flow-Diverter Stents Alone or in Combination with Coils: A Multicenter Experience and Long-Term Follow-up.

BACKGROUND: Although flow diverters (FDs) have been widely accepted by neurointerventionists, their safety has yet to be fully defined. Stratification by aneurysm size and location is essential for correct evaluation of treatment outcomes and risks of flow diversion. OBJECTIVE: To compare neurological and anatomic outcomes and evaluate the risk of complications after endovascular treatment of large or giant internal carotid artery (ICA) aneurysms with FD/FDs alone or together with coil embolization. METHODS: We reviewed all patients with large or giant, ruptured or unruptured ICA aneurysms who underwent endovascular treatment with flow diversion alone (group A) or with concomitant coiling (group B) in 4 international institutions between 2010 and 2015. Anatomic outcome was evaluated using the Byrne scale on digital subtraction angiography and/or angioMRI and/or angioCT scans 3, 6, 12, and 24 months postoperatively. RESULTS: We collected 44 patients with large or giant ICA aneurysms. Four patients (9%) presented with aneurysmal subarachnoid hemorrhage (SAH). FD/FDs were used alone in 26 patients and in combination with coil embolization in the 18 remaining patients. The mortality rate due to procedure-related and/or neurological complications was 2.2%. Twelve months after the procedure, 88.6% (n = 39) of patients had a favorable neurological outcome. One year after the procedure, the aneurysm was completely occluded in 72.7% of patients: 61.5% (16/18) in group A and 88.9% (16/26) in group B. CONCLUSION: Clinical outcomes and rates of intraoperative and postoperative complications did not differ significantly between the groups. Better anatomic results using FD/FDs combined with coils were documented 6 months after the procedure; this option seems to provide a higher aneurysm occlusion rate and reduce the need for retreatment.

Changes of Microrna Levels in Plasma of Patients with Rectal Cancer during Chemoradiotherapy.

Since the response to chemoradiotherapy in patients with locally advanced rectal cancer is heterogeneous, valid biomarkers are needed to monitor tumor response. Circulating microRNAs are promising candidates, however analyses of circulating microRNAs in rectal cancer are still rare. 111 patients with rectal cancer and 46 age-matched normal controls were enrolled. The expression levels of 30 microRNAs were analyzed in 17 pre-treatment patients' plasma samples. Differentially regulated microRNAs were validated in 94 independent patients. For 52 of the 94 patients a paired comparison between pre-treatment and post-treatment samples was performed. miR-17, miR-18b, miR-20a, miR-31, and miR-193a_3p, were significantly downregulated in pre-treatment plasma samples of patients with rectal cancer (p < 0.05). miR-29c, miR-30c, and miR-195 showed a trend of differential regulation. After validation, miR-31 and miR-30c were significantly deregulated by a decrease of expression. In 52 patients expression analyses of the 8 microRNAs in matched pre-treatment and post-treatment samples showed a significant decrease for all microRNAs (p < 0.05) after treatment. Expression levels of miR-31 and miR-30c could serve as valid biomarkers if validated in a prospective study. Plasma microRNA expression levels do not necessarily represent miRNA expression levels in tumor tissue. Also, expression levels of microRNAs change during multimodal therapy.

Effects of Overground Locomotor Training on Walking Performance in Chronic Cervical Motor Incomplete Spinal Cord Injury: A Pilot Study.

OBJECTIVE: To determine the effects of a novel overground locomotor training program on walking performance in people with chronic cervical motor incomplete spinal cord injury (iSCI). DESIGN: Before-after pilot study. SETTING: Human performance research laboratory. PARTICIPANTS: Adults (N=6, age >18y) with chronic cervical iSCI with American Spinal Injury Association Impairment Scale grades C and D. INTERVENTIONS: Overground locomotor training included two 90-minute sessions per week for 12 to 15 weeks. Training sessions alternated between uniplanar and multiplanar stepping patterns. Each session was comprised of 5 segments: joint mobility, volitional muscle activation, task isolation, task integration, and activity rehearsal. MAIN OUTCOME MEASURES: Overground walking speed, oxygen consumption (V˙o2), and carbon dioxide production (V˙co2). RESULTS: Overground locomotor training increased overground walking speed (.36±.20 vs .51±.24 m/s, P<.001, d=.68). Significant decreases in V˙o2 (6.6±1.3 vs 5.7±1.4mL·kg·min, P=.038, d=.67) and V˙co2 (753.1±125.5 vs 670.7±120.3mL/min, P=.036, d=.67) during self-selected constant work rate treadmill walking were also noted after training. CONCLUSIONS: The overground locomotor training program used in this pilot study is feasible and improved both overground walking speed and walking economy in a small sample of people with chronic cervical iSCI. Future studies are necessary to establish the efficacy of this overground locomotor training program and to differentiate among potential mechanisms contributing to enhanced walking performance in people with iSCI after overground locomotor training.

Neoadjuvant Therapy in Rectal Cancer - Biobanking of Preoperative Tumor Biopsies.

Translational research relies on high-quality biospecimens. In patients with rectal cancer treated preoperatively with radiochemotherapy tissue based analyses are challenging. To assess quality challenges we analyzed tissue samples taken over the last years in a multicenter setting. We retrospectively evaluated overall 197 patients of the CAO/ARO/AIO-94- and 04-trial with locally advanced rectal cancer that were biopsied preoperatively at the University Medical Center Goettingen as well as in 10 cooperating hospitals in Germany. The cellular content of tumor, mucosa, stroma, necrosis and the amount of isolated DNA and RNA as well as the RNA integrity number (RIN) as quality parameters were evaluated. A high RNA yield (p = 2.75e-07) and the content of tumor (p = 0.004) is significantly associated to high RIN-values, whereas a high content of mucosa (p = 0.07) shows a trend and a high amount of necrosis (p = 0.01) is significantly associated with RNA of poor quality. Correlating biopsies from Goettingen and the cooperating centers showed comparable tumor content results. By taking small sized biopsies we could assess a clear correlation between a good RNA quality and a high amount of RNA and tumor cells. These results also indicate that specimens collected at different centers are of comparable quality.

Heterogeneity of KRAS Mutation Status in Rectal Cancer.

INTRODUCTION: Anti-EGFR targeted therapy is of increasing importance in advanced colorectal cancer and prior KRAS mutation testing is mandatory for therapy. However, at which occasions this should be performed is still under debate. We aimed to assess in patients with locally advanced rectal cancer whether there is intra-specimen KRAS heterogeneity prior to and upon preoperative chemoradiotherapy (CRT), and if there are any changes in KRAS mutation status due to this intervention. MATERIALS AND METHODS: KRAS mutation status analyses were performed in 199 tumor samples from 47 patients with rectal cancer. To evaluate the heterogeneity between different tumor areas within the same tumor prior to preoperative CRT, 114 biopsies from 34 patients (mean 3 biopsies per patient) were analyzed (pre-therapeutic intratumoral heterogeneity). For the assessment of heterogeneity after CRT residual tumor tissue (85 samples) from 12 patients (mean 4.2 tissue samples per patient) were analyzed (post-therapeutic intratumoral heterogeneity) and assessment of heterogeneity before and after CRT was evaluated in corresponding patient samples (interventional heterogeneity). Primer extension method (SNaPshot™) was used for initial KRAS mutation status testing for Codon 12, 13, 61, and 146. Discordant results by this method were reevaluated by using the FDA-approved KRAS Pyro Kit 24, V1 and the RAS Extension Pyro Kit 24, V1 Kit (therascreen® KRAS test). RESULTS: For 20 (43%) out of the 47 patients, a KRAS mutation was detected. With 12 out of 20, the majority of these mutations affected codon 35. We did not obtained evidence that CRT results in changes of the KRAS mutation pattern. In addition, no intratumoral heterogeneity in the KRAS mutational status could be proven. This was true for both the biopsies prior to CRT and the resection specimens thereafter. The discrepancy observed in some samples when using the SNaPshot™ assay was due to insufficient sensitivity of this technique upon massive tumor regression by CRT as application of the therascreen® KRAS test revealed concordant results. CONCLUSION: Our results indicate that the KRAS mutation status at the primary tumor site of rectal cancer is homogenous. Its assessment for therapeutic decisions is feasible in pre-therapeutic biopsies as well as in post-therapeutic resected specimens. The amount of viable tumor cells seems to be an important determinant for assay sensitivity and should thus be considered for selection of the analytical method.

Prognostic Value of MicroRNAs in Preoperative Treated Rectal Cancer.

BACKGROUND: Patients with locally advanced rectal cancer are treated with preoperative chemoradiotherapy followed by surgical resection. Despite similar clinical parameters (uT2-3, uN+) and standard therapy, patients' prognoses differ widely. A possible prediction of prognosis through microRNAs as biomarkers out of treatment-naïve biopsies would allow individualized therapy options. METHODS: Microarray analysis of 45 microdissected preoperative biopsies from patients with rectal cancer was performed to identify potential microRNAs to predict overall survival, disease-free survival, cancer-specific survival, distant-metastasis-free survival, tumor regression grade, or nodal stage. Quantitative real-time polymerase chain reaction (qPCR) was performed on an independent set of 147 rectal cancer patients to validate relevant miRNAs. RESULTS: In the microarray screen, 14 microRNAs were significantly correlated to overall survival. Five microRNAs were included from previous work. Finally, 19 miRNAs were evaluated by qPCR. miR-515-5p, miR-573, miR-579 and miR-802 demonstrated significant correlation with overall survival and cancer-specific survival (p < 0.05). miR-573 was also significantly correlated with the tumor regression grade after preoperative chemoradiotherapy. miR-133b showed a significant correlation with distant-metastasis-free survival. miR-146b expression levels showed a significant correlation with nodal stage. CONCLUSION: Specific microRNAs can be used as biomarkers to predict prognosis of patients with rectal cancer and possibly stratify patients' therapy if validated in a prospective study.

Identification of Predictive Markers for Response to Neoadjuvant Chemoradiation in Rectal Carcinomas by Proteomic Isotope Coded Protein Label (ICPL) Analysis.

Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%-15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients' informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort.

Cancer Gene Profiling for Response Prediction.

The revolution of genomic technologies, including gene expression profiling, high-resolution mapping of genomic imbalances, and next-generation sequencing, allows us to establish molecular portraits of cancer cells with unprecedented accuracy. This generates hope and justifies anticipation that disease diagnosis, prognosis, and the choice of treatment will be adapted to the individual needs of patients based on molecular evidence. Preoperative treatment strategies are now recommended for a variety of human cancers. Unfortunately, the response of individual tumors to a preoperative treatment is not uniform, and ranges from complete regression to resistance. This poses a considerable clinical dilemma, as patients with a priori resistant tumors could either be spared exposure to radiation or DNA-damaging drugs, i.e., could be referred to primary surgery, or dose-intensified protocols could be pursued. Because the response of an individual tumor as well as therapy-induced side effects represent the major limiting factors of current treatment strategies, identifying molecular markers of response or for treatment toxicity has become exceedingly important. However, complex phenotypes such as tumor responsiveness to multimodal treatments probably do not depend on the expression levels of just one or a few genes and proteins. Therefore, methods that allow comprehensive interrogation of genetic pathways and networks hold great promise in delivering such tumor-specific signatures, since expression levels of thousands of genes can be monitored simultaneously. Over the past few years, microarray technology has emerged as a central tool in addressing pertinent clinical questions, the answers to which are critical for the realization of a personalized genomic medicine, in which patients will be treated based on the biology of their tumor and their genetic profile (Quackenbush, N Engl J Med 354:2463-72, 2006; Jensen et al., Curr Opin Oncol 18:374-380, 2006; Bol and Ebner, Pharmacogenomics 7:227-235, 2006; Nevins and Potti, Nat Rev Genet 8:601-609, 2007).

Preoperative Prediction of Lymph Node Status by Circulating Mir-18b and Mir-20a During Chemoradiotherapy in Patients with Rectal Cancer.

BACKGROUND: In locally advanced rectal cancer, therapeutic success of preoperative chemoradiotherapy (CRT) ranges from resistance to complete regression. For those patients that respond well to CRT, local resection (LR) procedures are currently under investigation to minimize surgical morbidity and to improve functional outcome. To maintain the oncologic benefit appropriate staging procedures are essential. However, current clinical assessment and imaging techniques need further improvement. METHODS: Five miRNAs associated with rectal cancer (miR-17, miR-18b, miR-20a, miR-31, and miR-193-3p) were analyzed in the plasma of rectal cancer patients (n = 42) using qPCR. Expression levels were assessed before, during and after CRT and analyzed in regard to patients' lymph node status obtained after total mesorectal excision and intensive histopathological work-up. RESULTS: Four of the five miRNAs revealed reliable results in the plasma. miR-31 was excluded due to its low expression. MicroRNA-17, 18b, 20a, and 193-3p showed altering expression levels at different time points. Only 43% (miR-17), 43% (miR-18b), 53% (miR-20a), and 60% (miR-193-3p) showed a continuous in- or decrease of miRNA expression. The reduced expression of miR-18b and miR-20a during CRT was found to be significantly associated with postoperative lymph node negativity (p < 0.05). CONCLUSION: MicroRNA expression in patient plasma changes during preoperative CRT. The alteration is not continuous and the meaning requires additional analysis on a larger patient cohort. The co-occurrence of reduced miR-18b and miR-20a expression with lymph node negativity after preoperative CRT could help to stratify the surgical procedure with respect to total mesorectal excision and LR if validated prospectively.

Identification of a DNA methylation signature to predict disease-free survival in locally advanced rectal cancer.

In locally advanced rectal cancer a preoperative predictive biomarker is necessary to adjust treatment specifically for those patients expected to suffer relapse. We applied whole genome methylation CpG island array analyses to an initial set of patients (n=11) to identify differentially methylated regions (DMRs) that separate a good from a bad prognosis group. Using a quantitative high-resolution approach, candidate DMRs were first validated in a set of 61 patients (test set) and then confirmed DMRs were further validated in additional independent patient cohorts (n=71, n=42). We identified twenty highly discriminative DMRs and validated them in the test set using the MassARRAY technique. Ten DMRs could be confirmed which allowed separation into prognosis groups (p=0.0207, HR=4.09). The classifier was validated in two additional cohorts (n=71, p=0.0345, HR=3.57 and n=42, p=0.0113, HR=3.78). Interestingly, six of the ten DMRs represented regions close to the transcriptional start sites of genes which are also marked by the Polycomb Repressor Complex component EZH2. In conclusion we present a classifier comprising 10 DMRs which predicts patient prognosis with a high degree of accuracy. These data may now help to discriminate between patients that may respond better to standard treatments from those that may require alternative modalities.

Identification of a microRNA expression signature for chemoradiosensitivity of colorectal cancer cells, involving miRNAs-320a, -224, -132 and let7g.

BACKGROUND AND PURPOSE: Preoperative chemoradiotherapy (CRT) represents the standard treatment for locally advanced rectal cancer. Tumor response and progression vary considerably. MicroRNAs represent master regulators of gene expression, and may therefore contribute to this diversity. MATERIAL AND METHODS: Genome-wide microRNA (miRNA) profiling was performed for 12 colorectal cancer (CRC) cell lines and an individual in vitro signature of chemoradiosensitivity was established. Functional relevance of selected miRNAs was established by transfecting miRNA-mimics into SW480 and SW837 cells. The prognostic value of selected miRNAs was assessed in 128 pretherapeutic patient biopsies. RESULTS: Thirty-six miRNAs were identified to significantly correlate with sensitivity to CRT (Q < 0.05) including miR-320a and other miRNAs involved in the MAPK-, TGF- and Wnt-pathway. Transfection of selected miRNAs (let-7g, miR-132, miR-224, miR-320a) each induced a shift of sensitivity. High expression of let-7 g was associated with a good prognosis in rectal cancer patients (P = 0.03). CONCLUSIONS: This is the first report of a miRNA expression signature for in vitro chemoradiosensitivity of CRC cell lines. Many of the identified miRNAs have not been linked to the response to CRT and may represent potential molecular targets to sensitize resistant cancers. If further validated, let7g expression may serve as predictive biomarker.

Molecular patterns in the evolution of serrated lesion of the colorectum.

Colorectal cancer (CRC) mostly develops from a variety of polyps following mainly three different molecular pathways: chromosomal instability (CIN), microsatellite instability (MSI) and CpG island methylation (CIMP). Polyps are classified histologically as conventional adenomas, hyperplastic polyps, sessile serrated adenomas/polyps (SSA/P) and traditional serrated adenomas (TSA). However, the association of these polyps with the different types of CRCs and the underlying genetic and epigenetic aberrations has yet to be resolved. In order to address this question we analyzed 140 tumors and 20 matched mucosae by array comparative genomic hybridization, by sequence analysis of the oncogenes BRAF, KRAS, PI3K3CA and by methylation arrays. MSI was tested indirectly by immunohistochemistry (IHC) and a loss of MLH1, MSH2, MSH6 or PMS2 was assigned as high microsatellite instability (MSI-H), while low microsatellite instability (MSI-L) was defined as MGMT IHC negativity only. CIN was detected in 78% of all MSI-H CRCs, most commonly as a gain of chromosome 8. Methylation data analyses allowed classification of samples into four groups and detected similar methylation profiles in SSA/P and MSI-H CRC. TSA also revealed aberrant methylation pattern, but clustered more heterogeneously and closer to microsatellite stable (MSS) CRCs. SSA/P, TSA and MSI-H CRCs had the highest degree of promotor methylation (CIMP pathway). Chromosomal instability, in contrast to the established doctrine, is a common phenomenon in MSI CRCs, yet to a lower extent and at later stages than in MSS CRCs. Methylation analyses suggest that SSA/P are precursors for MSI-H CRCs and follow the CIMP pathway.

CpG island methylator phenotype infers a poor disease-free survival in locally advanced rectal cancer.

BACKGROUND: Locally advanced rectal cancers are treated with preoperative radiochemotherapy (RCT). However, subsets of patients have no benefit from preoperative treatment. Since epigenetic modifications, including DNA methylation, may influence response to neoadjuvant treatment we studied the CpG island methylator phenotype (CIMP) in patients who received a 5-fluouracil based RCT. METHODS: One hundred fifty patients with locally advanced rectal cancer, treated within a phase III clinical trial (CAO/ARO/AIO-94 and -04), were included in this analysis. CIMP was assessed by methylation specific PCR (MSP) using RUNX3, SOCS1, NEUROG1, IGF2, and CACNA1G as a marker panel. Loss of mismatch repair gene (MMR) expression was assessed by immunohistochemistry for a subset of patients. KRAS and BRAF mutation status were assessed using Sanger sequencing. RESULTS: The CIMP status could be established in all 150 patients. Fifteen (10%) revealed CIMP positivity (≥3 methylated promoters), whereas 135 patients (90%) where classified as CIMP negative. Analysis for MMR status did not reveal any microsatellite instability (MSI). A single mutation of the BRAF gene (D594G) was detected. The KRAS gene (exon 1, 2, and 3) was mutated in 65 tumors (43%) but was not correlated to a specific CIMP status. Three- and 5-year disease-free survival was notably worse in CIMP positive patients (56% and 0% vs 80% and 75%; P < .01) suggesting an increased likelihood of poor clinical outcome (HR 5.5; 95%CI: [2.1, 13.9]). CONCLUSION: CIMP positivity, defined by methylation of at least 3 specific gene promoters, is an infrequent event in locally advanced rectal cancer. However, it increases the likelihood of distant metastases. Therefore, the CIMP status may be included as a molecular marker for the identification of high-risk patients and might contribute to individual treatment stratification.

Acute toxicity of radiochemotherapy in rectal cancer patients: a risk particularly for carriers of the TGFB1 Pro25 variant.

PURPOSE: Transforming growth factor-beta1 is related to adverse events in radiochemotherapy. We investigated TGFB1 genetic variability in relation to quality of life-impairing acute organ toxicity (QAOT) of neoadjuvant radiochemotherapy under clinical trial conditions. METHODS AND MATERIALS: Two independent patient cohorts (n = 88 and n = 75) diagnosed with International Union Against Cancer stage II/III rectal cancer received neoadjuvant radiation doses of 50.4 Gy combined with 5-fluorouracil-based chemotherapy. Toxicity was monitored according to Common Terminology Criteria for Adverse Events. QAOT was defined as a CTCAE grade ≥2 for at least one case of enteritis, proctitis, cystitis, or dermatitis. Nine germline polymorphisms covering the common genetic diversity in the TGFB1 gene were genotyped. RESULTS: In both cohorts, all patients carrying the TGFB1 Pro25 variant experienced QAOT (positive predictive value of 100%, adjusted p = 0.0006). In a multivariate logistic regression model, gender, age, body mass index, type of chemotherapy, or disease state had no significant impact on QAOT. CONCLUSION: The TGFB1 Pro25 variant could be a relevant marker for individual treatment stratification and carriers may benefit from adaptive clinical care or specific radiation techniques.