IMMUNOREACT 7: Regular aspirin use is associated with immune surveillance activation in colorectal cancer.

BACKGROUND: Long-term daily use of aspirin reduces incidence and mortality due to colorectal cancer (CRC). This study aimed to analyze the effect of aspirin on the tumor microenvironment, systemic immunity, and on the healthy mucosa surrounding cancer. METHODS: Patients with a diagnosis of CRC operated on from 2015 to 2019 were retrospectively analyzed (METACCRE cohort). Expression of mRNA of immune surveillance-related genes (PD-L1, CD80, CD86, HLA I, and HLA II) in CRC primary cells treated with aspirin were extracted from Gene Expression Omnibus-deposited public database (GSE76583). The experiment was replicated in cell lines. The mucosal immune microenvironment of a subgroup of patients participating in the IMMUNOREACT1 (ClinicalTrials.gov NCT04915326) project was analyzed with immunohistochemistry and flow cytometry. RESULTS: In the METACCRE Cohort, 12% of 238 patients analyzed were aspirin users. Nodal metastasis was significantly less frequent (p = .008) and tumor-infiltrating lymphocyte infiltration was higher (p = .02) among aspirin users. In the CRC primary cells and selected cell lines, CD80 mRNA expression was increased following aspirin treatment (p = .001). In the healthy mucosa surrounding rectal cancer, the ratio of CD8/CD3 and epithelial cells expressing CD80 was higher in aspirin users (p = .027 and p = .034, respectively). CONCLUSIONS: These data suggested that regular aspirin use may have an active role in enhancing immunosurveillance against CRC.

Gene and MicroRNA Expression Are Predictive of Tumor Response in Rectal Adenocarcinoma Patients Treated With Preoperative Chemoradiotherapy.

Preoperative chemoradiotherapy (pCRT) followed by surgery is the standard treatment for locally advanced rectal cancer (LARC). However, tumor response to pCRT is not uniform, and there are no effective predictive methods. This study investigated whether specific gene and miRNA expression are associated with tumor response to pCRT. Tissue biopsies were obtained from patients before pCRT and resection. Gene and miRNA expression were analyzed using a one-color microarray technique that compares signatures between responders (R) and non-responders (NR), as measured based on tumor regression grade. Two groups composed of 38 "exploration cohort" and 21 "validation cohort" LARC patients were considered for a total of 32 NR and 27 R patients. In the first cohort, using SAM Two Class analysis, 256 genes and 29 miRNAs that were differentially expressed between the NR and R patients were identified. The anti-correlation analysis showed that the same 8 miRNA interacted with different networks of transcripts. The miR-630 appeared only with the NR patients and was anti-correlated with a single transcript: RAB5B. After PAM, the following eight transcripts were strong predictors of tumor response: TMEM188, ITGA2, NRG, TRAM1, BCL2L13, MYO1B, KLF7, and GTSE1. Using this gene set, an unsupervised cluster analysis was applied to the validation cohort and correctly assigned the patients to the NR or R group with 85.7% accuracy, 90% sensitivity, and 82% specificity. All three parameters reached 100% when both cohorts were considered together. In conclusion, gene and miRNA expression profiles may be helpful for predicting response to pCRT in LARC patients. J. Cell. Physiol. 232: 426-435, 2017. © 2016 Wiley Periodicals, Inc.

Prediction of N0 Irradiated Rectal Cancer Comparing MRI Before and After Preoperative Chemoradiotherapy.

BACKGROUND: The prediction of lymph node status using MRI has an impact on the management of rectal cancer, both before and after preoperative chemoradiotherapy. OBJECTIVE: The purpose of this study was to maximize the negative predictive value and sensitivity of mesorectal lymph node imaging after chemoradiotherapy because postchemoradiation node-negative patients may be treated with rectum-sparing approaches. DESIGN: This was a retrospective study. SETTINGS: The study was conducted at a tertiary care hospital. PATIENTS: Sixty-four patients with locally advanced rectal cancer who underwent preoperative chemoradiotherapy and MRI for staging and the assessment of response were evaluated. MAIN OUTCOME MEASURES: The sums of the sizes of all mesorectal lymph nodes in each patient on both prechemoradiotherapy and postchemoradiotherapy imaging data sets were calculated to determine the lymph node global size reduction rates, taking these to be the outcomes of the histopathologic findings. Other included measures were interobserver agreement regarding the prediction of node status based on morphologic criteria and the diagnostic performance of contrast-enhanced images. RESULTS: Using a cutoff value of a 70% lymph node global size reduction rate with only 15 node-positive patients on histopathology, the sensitivity in the prediction of nodal status and negative predictive value were 93% (95% CI, 70.2%-98.8%) and 97% (95% CI, 82.9%-99.8%) for observer 1 and 100% (95% CI, 79.6%-100%) and 100% (95% CI, 62.9%-100%) for observer 2. The areas under the receiver operating characteristic curves for the 2 observers were 0.90 (95% CI, 0.82-0.98; p < 0.0001) for observer 1 and 0.65 (95% CI, 0.50-0.79; p = 0.08) for observer 2. The efficacy of the morphologic criteria and contrast-enhanced images in predicting node status was limited after chemoradiotherapy. LIMITATIONS: This study is limited by its small sample size and retrospective nature. CONCLUSIONS: Assessing the lymph node global size reduction rate value reduces the risk of undetected nodal metastases and may be helpful in better identifying suitable candidates for the local excision of early stage rectal cancer. See Video Abstract at http://links.lww.com/DCR/A412.

Clinical predictive circulating peptides in rectal cancer patients treated with neoadjuvant chemoradiotherapy.

Preoperative chemoradiotherapy is worldwide accepted as a standard treatment for locally advanced rectal cancer. Current standard of treatment includes administration of ionizing radiation for 45-50.4 Gy in 25-28 fractions associated with 5-fluorouracil administration during radiation therapy. Unfortunately, 40% of patients have a poor or absent response and novel predictive biomarkers are demanding. For the first time, we apply a novel peptidomic methodology and analysis in rectal cancer patients treated with preoperative chemoradiotherapy. Circulating peptides (Molecular Weight <3 kDa) have been harvested from patients' plasma (n = 33) using nanoporous silica chip and analyzed by Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometer. Peptides fingerprint has been compared between responders and non-responders. Random Forest classification selected three peptides at m/z 1082.552, 1098.537, and 1104.538 that were able to correctly discriminate between responders (n = 16) and non-responders (n = 17) before therapy (T0) providing an overall accuracy of 86% and an area under the receiver operating characteristic (ROC) curve of 0.92. In conclusion, the nanoporous silica chip coupled to mass spectrometry method was found to be a realistic method for plasma-based peptide analysis and we provide the first list of predictive circulating biomarker peptides in rectal cancer patients underwent preoperative chemoradiotherapy.

Rectal sparing approach after preoperative radio - and/or Chemo-therapy (ReSARCh): a prospective, multicentre, observational study.

BACKGROUND: Rectal-sparing approaches for patients with rectal cancer who achieved a complete or major response following neoadjuvant therapy constitute a paradigm of a potential shift in the management of patients with rectal cancer, however their role remains controversial. The aim of this study was to investigate the feasibility of rectal-sparing approaches to preserve the rectum without impairing the outcomes. METHODS: This prospective, multicentre, observational study investigated the outcomes of patients with clinical stage II-III mid-low rectal adenocarcinoma treated with any neoadjuvant therapy, and either transanal local excision or watch-and-wait approach, based on tumor response (major or complete) and patient/surgeon choice. The primary endpoint of the study was rectum preservation at a minimum follow-up of two years. Secondary endpoints were overall, disease-free, local and distant recurrence-free, and stoma-free survival at three years. RESULTS: Of 178 patients enrolled in 16 centres, 112 (62.9%) were managed with local excision and 66 (37.1%) with watch-and-wait. At a median (interquartile range) follow-up of 36.1 (30.6-45.6) months, the rectum was preserved in 144 (80.9%) patients. The 3-year rectum-sparing, overall, disease-free, local recurrence-free, distant recurrence-free survival was 80.6% (95%CI 73.9-85.8), 97.6% (95%CI 93.6-99.1), 90.0% (95%CI 84.3-93.7), 94.7% (95%CI 90.1-97.2), and 94.6% (95%CI 89.9-97.2), respectively. The 3-year stoma-free survival was 95.0% (95%CI 89.5-97.6). The 3-year regrowth-free survival in the watch-and-wait group was 71.8% (95%CI 59.9-81.2). CONCLUSIONS: In rectal cancer patients with major or complete clinical response after neoadjuvant therapy, the rectum can be preserved in about 80% of cases, without compromise the outcomes.

Local excision after preoperative chemoradiotherapy for rectal cancer: results of a multicenter phase II clinical trial.

BACKGROUND: Transanal local excision has been suggested as an attractive approach for patients with rectal cancer who show a major clinical response after preoperative chemoradiotherapy. OBJECTIVE: To evaluate the impact of transanal local excision on the local recurrence of rectal cancer in patients who had a major clinical response after preoperative chemoradiotherapy. DESIGN: Sequential 2-stage phase II study for early efficacy. SETTING: Multicenter study. PATIENTS: Patients with clinical T3 or low-lying T2 rectal adenocarcinoma that showed a major clinical response after a preoperative chemoradiotherapy. Eligible patients underwent a full-thickness transanal local excision. According to their histopathology, the patients staged as ypT0-1 were observed, while the remaining patients were recommended to undergo a subsequent total mesorectal excision. MAIN OUTCOME MEASURES: A local recurrence rate of ≤5% was set as a successful rate for stopping the trial early after the first stage. RESULTS: The study group included 63 patients. Before chemoradiotherapy, patients were staged as clinical T3 (n = 42) and T2 (n = 21). After the local excision, 43 patients fulfilled the criteria to be observed with no further treatment. Nine of the remaining 20 patients for whom a subsequent total mesorectal excision was recommended refused surgery. Two of these patients who refused surgery had intraluminal local recurrence; both had a ypT2 tumor and underwent salvage surgery. The estimated cumulative 3-year overall survival, disease-free survival and local disease-free survival were 91.5% (95% CI: 75.9-97.2), 91.0% (95% CI: 77.0-96.6) and 96.9% (95% CI: 80.3-99.5), respectively. LIMITATIONS: The time of follow-up is still short and the sample size is limited. CONCLUSIONS: Our data suggest that local excision is a good option for patients with a major clinical response after chemoradiotherapy. A longer period of follow-up is required to confirm these findings.

Factors affecting the treatment of multiple colorectal adenomas.

BACKGROUND: Currently, no guidelines exist for the treatment of patients with multiple colorectal adenomas (MCRAs) (>10 but <100 synchronous nondiminutive polyps of the large bowel). This retrospective study aimed to investigate the clinical and molecular factors related to different treatments for MCRAs. METHODS: Patients with MCRAs were consecutively enrolled from January 2003 to June 2011. Sequencing of their APC and MutYH genes was performed. The clinical, molecular, and family histories of the patients were collected using the Progeny database. The patient treatments were divided into three groups of increasing clinical weight: endoscopic polypectomy, segmental resection, and total colectomy. A logistic regression analysis of clinicomolecular factors related to different treatment options was performed. RESULTS: The study comprised 80 patients (32 women, 40%) with a median age of 53 years (range 13-74 years). The median number of polyps was 33 (range 10-90).The cases included 62 diffuse polyposis, 18 segmental polyposis coli and synchronous colorectal carcinomas (CRC; 34 cases, 43%). The pathogenetic mutations were biallelic MutYH (n = 19, 24%) and APC (n = 4, 5%). The mean follow-up period was 74 months (median 43 months, range 1-468 months). Endoscopic polypectomy was performed in 25 cases (31%), segmental resection in 16 cases (20%), and total colectomy in 39 cases (49%). The logistics regression analysis, considering all the patients, showed that the number of polyps, the presence of CRC, and mutation were correlated with more intensive treatment. For the patients without CRC, only the number of polyps was correlated with the severity of the treatment (p > 0.0166). "On the ROC (receiver operating characteristic) curve, 25 was the number of polyps that best discriminated between surgical and endoscopic therapy. CONCLUSIONS: The majority of patients with MCRAs undergo surgery. For patients without CRC, only the number of polyps, and not the presence of a disease-causing mutation, is correlated with increased heaviness of treatment. Patients with more than 25 polyps are more likely to undergo a surgical resection.

IMMUNOREACT 5: female patients with rectal cancer have better immune editing mechanisms than male patients - a cohort study.

BACKGROUND: Studies evaluating sex differences in colorectal cancer (CRC) tumor microenvironment are limited, and no previous study has focused on rectal cancer patients' constitutive immune surveillance mechanisms. The authors aimed to assess gender-related differences in the immune microenvironment of rectal cancer patients. METHODS: A systematic review and meta-analysis were conducted up to 31 May 2021, including studies focusing on gender-related differences in the CRC tumor microenvironment. Data on the mutational profile of rectal cancer were extracted from the Cancer Genome Atlas (TCGA). A subanalysis of the two IMMUNOREACT trials (NCT04915326 and NCT04917263) was performed, aiming to detect gender-related differences in the immune microenvironment of the healthy mucosa in patients with early (IMMUNOREACT 1 cohort) and locally advanced rectal cancer following neoadjuvant therapy (IMMUNOREACT 2 cohort). In the retrospective IMMUNOREACT 1 cohort (therapy naive), the authors enrolled 442 patients (177 female and 265 male), while in the retrospective IMMUNOREACT 2 cohort (patients who had neoadjuvant therapy), we enrolled 264 patients (80 female and 184 male). In the prospective IMMUNOREACT 1 cohort (therapy naive), the authors enrolled 72 patients (26 female and 46 male), while in the prospective IMMUNOREACT 2 cohort (patients who had neoadjuvant therapy), the authors enrolled 105 patients (42 female and 63 male). RESULTS: Seven studies reported PD-L1 expression in the CRC microenvironment, but no significant difference could be identified between the sexes. In the TGCA series, mutations of SYNE1 and RYR2 were significantly more frequent in male patients with rectal cancer. In the IMMUNOREACT 1 cohort, male patients had a higher expression of epithelial cells expressing HLA class I, while female patients had a higher number of activated CD4+Th1 cells. Female patients in the IMMUNOREACT 2 cohort showed a higher infiltration of epithelial cells expressing CD86 and activated cytotoxic T cells (P=0.01). CONCLUSIONS: Male patients have more frequent oncogene mutations associated with a lower expression of T-cell activation genes. In the healthy mucosa of female patients, more Th1 cells and cytotoxic T cells suggest a potentially better immune response to the tumor. Sex should be considered when defining the treatment strategy for rectal cancer patients or designing prognostic scores.

Telomere-specific reverse transcriptase (hTERT) and cell-free RNA in plasma as predictors of pathologic tumor response in rectal cancer patients receiving neoadjuvant chemoradiotherapy.

PURPOSE: To investigate whether the plasma levels of cell-free RNA (cfRNA) and telomere-specific reverse transcriptase mRNA (hTERT) are associated with tumor response in rectal cancer patients who received preoperative chemoradiotherapy (pCRT). METHODS: Patients who underwent pCRT for rectal cancer and for whom baseline and paired post-pCRT blood samples were available were studied. On the basis of tumor regression score, patients were classified as having response or having no response. Clinical variables and plasma levels of cfRNA and hTERT before and after the pCRT were evaluated. The association between each predictor and tumor response was assessed by univariate and multivariate analyses. RESULTS: Of 98 eligible patients, 45 were determined to respond to therapy, and 53 did not respond to therapy. In univariate analysis, gender (P = 0.040), baseline levels of cfRNA (P = 0.026), post-pCRT levels of both hTERT and cfRNA (P < 0.0001 and P = 0.001, respectively), and the difference between the post- and pre-pCRT levels of both hTERT and cfRNA (P = 0.009 and P = 0.001, respectively) were found to be significant predictors of tumor response. In multivariate analysis, using variables that were available before pCRT, cfRNA levels and gender independently predicted the tumor response, while in multivariate analysis, which used all of the variables available before the surgical procedure, the post-pCRT levels of cfRNA and the difference between the post- and pre-pCRT levels of cfRNA independently predicted tumor response. CONCLUSIONS: Plasma levels of cfRNA and hTERT are promising markers of tumor response to pCRT for rectal cancer.

Significance of pulmonary nodules in patients with colorectal cancer.

OBJECTIVES: Radiographically small pulmonary nodules (PNs) in patients with colorectal cancer are troublesome because their discovery raises concern about metastases. This study sought to establish the appropriate timing of radiological follow-up for PNs detected at initial staging evaluation of colorectal carcinoma patients. METHODS: The medical records of 376 consecutive colorectal cancer patients who underwent curative surgery and had baseline and follow-up chest X-rays (CXR) and computed tomography (CT) were reviewed. RESULTS: The study included 92 patients who had all CXR and chest CT available for review, at least one PN found on baseline imaging, and no synchronous neoplasms. On baseline chest CT, these 92 patients had 170 PNs altogether and 77 (45.2 %) of them were greater than 5 mm in size. Baseline CXR detected 13 PNs in 12 patients and all but 2 were larger than 5 mm. Nodule size greater than 5 mm and irregular margins were predictors of nodule growth. The mean doubling time of 24/170 (14.1 %) growing PNs was about 4 months. CONCLUSIONS: Our findings suggest that baseline and follow-up CXR are pointless, and short-interval CT follow-up is warranted when PNs larger than 5 mm with irregular margins are detected on preoperative chest CT. KEY POINTS: • Pulmonary nodules in colorectal cancer patients raise concern about metastasis. • Baseline and follow-up chest X-ray in colorectal cancer can be abandoned. • CT is the best technique for assessing PNs in colorectal cancer. • Short-interval CT follow-up advisable for PNs larger than 5 mm with irregular margins.

Circulating cell-free DNA: a promising marker of pathologic tumor response in rectal cancer patients receiving preoperative chemoradiotherapy.

PURPOSE: The circulating cell-free DNA (cfDNA) in plasma has been reported to be a marker of cancer detection. The aim of this study was to investigate whether the cfDNA has a role as response biomarker in patients receiving preoperative chemoradiotherapy (CRT) for rectal cancer. METHODS: Sixty-seven patients (median age 61 years; male/female 42/25) who underwent CRT for rectal cancer were evaluated. After tumor regression grade (TRG) classification was made, the patients were classified as having disease that responded (TRG 1-2) and that did not respond (TRG 3-5) to therapy. Plasma samples were obtained from patients before and after CRT. The cfDNA levels were analyzed by quantitative real-time polymerase chain reaction of ß-globin. On the basis of the Alu repeats, the cfDNA was considered as either total (fragments of 115 bp, Alu 115) or tumoral (fragments of 247 bp, Alu 247). The association between the pre- or post-CRT levels and between variations during CRT of the Alu 247, Alu 115 repeat, and Alu 247/115 ratio (cfDNA integrity index) and the pathologic tumor response was analyzed. RESULTS: The baseline levels of cfDNA were not associated with tumor response. The post-CRT levels of the cfDNA integrity index were significantly lower in responsive compared to nonresponsive disease (P = 0.0009). Both the median value of the Alu 247 repeat and the cfDNA integrity index decreased after CRT in disease that responded to therapy (P < 0.005 and P < 0.005, respectively) compared to disease that did not respond to therapy (P = 0.83 and P = 0.726, respectively). The results of the multivariable logistic regression analysis showed that only the cfDNA integrity index was significantly and independently associated with tumor response to treatment. CONCLUSIONS: The plasma levels of the longer fragments (Alu 247) of cfDNA and the cfDNA integrity index are promising markers to predict tumor response after preoperative CRT for rectal cancer.

Gene and protein expression of mTOR and LC3 in hepatocellular carcinoma, colorectal liver metastasis and "normal" liver tissues.

The physiological role of autophagy in the progression of liver diseases is still debated. To understand the clinical relevance of autophagy in primary e secondary hepatic tumors, we analyzed the expression of mTOR (mammalian target of rapamycin), a key regulator of autophagy; Raptor (regulatory-associated protein of mTOR); ULK1 (Unc-51 like kinase 1) determinant in the autophagy initiation; LC3 (microtubule-associated protein 1A/1B-light chain 3), a specific marker of autophagosomes; and p62, a selective autophagy receptor. Samples from subjects with chronic hepatitis (n.58), cirrhosis (n.12), hepatocellular carcinoma (HCC, n.56), metastases (n.48) from colorectal cancer and hyperplasia or gallbladder stones (n.7), the latter considered as controls, were examined. Gene expression analysis was carried out in n.213 tissues by absolute q-PCR, while protein expression by Western Blot in n.191 lysates, including tumoral, surrounding tumoral and normal tissues. Nonparametric statistical tests were used for comparing expression levels in the above-mentioned groups. Subgroup analysis was performed considering viral infection and chemotherapy treatment. The mTOR transcriptional level was significantly lower in metastases compared to HCC (P = 0.0001). p-mTOR(Ser2448) and LC3II/LC3I protein levels were significantly higher in metastases compared to HCC (P = 0.008 and P<0.0001, respectively). ULK(Ser757) levels were significantly higher in HCC compared to metastases (P = 0.0002) while the HCV- and HBV- related HCC showed the highest p62 levels. Chemotherapy induced a down-regulation of the p-mTOR(Ser2448) in metastases and in non-tumor surrounding tissues in treated patients compared to untreated (P = 0.001 and P = 0.005, respectively). Conclusions: the different expression of proteins considered, owning their interaction and diverse tissue microenvironment, indicate an impairment of the autophagy flux in primary liver tumors that is critical for the promotion of tumorigenesis process and a coexistence of autophagy inhibition and activation mechanisms in secondary liver tumors. Differences in mTOR and LC3 transcripts emerged in tumor-free tissues, therefore particular attention should be considered in selecting the control group.

Long-term oncologic results and complications after preoperative chemoradiotherapy for rectal cancer: a single-institution experience after a median follow-up of 95 months.

BACKGROUND: This study sought to evaluate the long-term outcome and complications, and occurrence of second malignancy after preoperative chemoradiotherapy (pCRT) for rectal cancer. METHODS: One hundred twenty-three consecutive patients (78 men, 45 women) with locally advanced mid-low rectal cancer underwent pCRT between 1994 and 2002. Patients were followed up by one surgeon with a standard protocol, and data were prospectively recorded in a dedicated database. No patient was lost to follow-up. Complications were defined as late if they occurred >6 months after surgery. Overall and disease-free survival were calculated by the Kaplan-Meier method. RESULTS: Of 123 patients, 111 underwent an R0 procedure. The rate of pathologic complete response was 16% (n = 20 patients). At a median follow-up of 95 (range, 56-160) months, 50 late complications occurred in 41 patients, 21 of whom required surgery. In seven cases, the complications were clearly CRT related and were significantly associated with the total dose of radiation delivered (P < .05). The estimated 5- and 10-year overall survival was 76% and 67%, respectively. The estimated 5- and 10-year disease-free survival was 83% and 82%, respectively. In 18 of 19 patients who experienced recurrence (local, n = 3; distant, n = 16), it occurred within 48 months from surgery. The most frequent site of metastasis as first site of recurrence was the lung (9 of 19). The most frequent second primary malignancy was lung cancer (3 of 8). CONCLUSIONS: Despite satisfactory oncological outcome, late morbidity after pCRT is relevant and related to the radiotherapy dose used. Most recurrences and second malignancies were located in the lung.

Noninvasive Biomarkers of Colorectal Cancer: Role in Diagnosis and Personalised Treatment Perspectives.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. It has been estimated that more than one-third of patients are diagnosed when CRC has already spread to the lymph nodes. One out of five patients is diagnosed with metastatic CRC. The stage of diagnosis influences treatment outcome and survival. Notwithstanding the recent advances in multidisciplinary management and treatment of CRC, patients are still reluctant to undergo screening tests because of the associated invasiveness and discomfort (e.g., colonoscopy with biopsies). Moreover, the serological markers currently used for diagnosis are not reliable and, even if they were useful to detect disease recurrence after treatment, they are not always detected in patients with CRC (e.g., CEA). Recently, translational research in CRC has produced a wide spectrum of potential biomarkers that could be useful for diagnosis, treatment, and follow-up of these patients. The aim of this review is to provide an overview of the newer noninvasive or minimally invasive biomarkers of CRC. Here, we discuss imaging and biomolecular diagnostics ranging from their potential usefulness to obtain early and less-invasive diagnosis to their potential implementation in the development of a bespoke treatment of CRC.

Relationship between hospital volume and short-term outcomes: a nationwide population-based study including 75,280 rectal cancer surgical procedures.

There is growing interest on the potential relationship between hospital volume (HV) and outcomes as it might justify the centralization of care for rectal cancer surgery. From the National Italian Hospital Discharge Dataset, data on 75,280 rectal cancer patients who underwent elective major surgery between 2002 and 2014 were retrieved and analyzed. HV was grouped into tertiles: low-volume performed 1-12, while high-volume hospitals performed 33+ procedures/year. The impact of HV on in-hospital mortality, abdominoperineal resection (APR), 30-day readmission, and length of stay (LOS) was assessed. Risk factors were calculated using multivariate logistic regression. The proportion of procedures performed in low-volume hospitals decreased by 6.7 percent (p<0.001). The rate of in-hospital mortality, APR and 30-day readmission was 1.3%, 16.3%, and 7.2%, respectively, and the median LOS was 13 days. The adjusted risk of in-hospital mortality (OR = 1.49, 95% CI = 1.25-1.78), APR (OR 1.10, 95%CI 1.02-1.19), 30-day readmission (OR 1.49, 95%CI 1.38-1.61), and prolonged LOS (OR 2.29, 95%CI 2.05-2.55) were greater for low-volume hospitals than for high-volume hospitals. This study shows an independent impact of HV procedures on all short-term outcome measures, justifying a policy of centralization for rectal cancer surgery, a process which is underway.

Adenosquamous gallbladder carcinoma: Multigene hotspot mutational profiling reveals a monoclonal origin of the two components.

Adenosquamous carcinoma (ASC) of the gallbladder is a rare malignant tumor that is characterized by a coexisting of glandular and squamous components. In a case of ASC, we performed hotspot multigene mutational profiling of 164 hotspot regions of eleven cancer-associated genes (AKT1, APC, BRAF, CTNNB1, KIT, KRAS, NRAS, PDGFRA, PIK3CA, PTEN and TP53) in the two microdissected components. Both tumor phenotypes resulted characterized by a p.E542K point mutation in the PIK3CA gene, whereas adenocarcinoma component revealed also a TP53 Q331* homozygous stop mutation. Of note, coexisting high-grade dysplastic epithelium was characterized by a mixed cell population, with an upper part featuring a glandular differentiation and a basal layer of p63 positive (squamous committed) cells. Overall these data provide evidence of an early squamous differentiation of the lesion with a common genetic landscape of the two components.

Serum miR-125b is a non-invasive predictive biomarker of the pre-operative chemoradiotherapy responsiveness in patients with rectal adenocarcinoma.

BACKGROUND: Therapeutic management of Locally Advanced Rectal Cancer (LARC) involves pre-operative chemoradiotherapy (pCRT) followed by surgery. However, after pCRT the complete pathological response is approximately 20%, whereas in 20 to 40% of patients the response is poor or absent. METHODS: Cancer biopsy specimens (n= 38) and serum samples (n= 34) obtained before pCRT from 38 LARC patients were included in the study. Patients were classified in responders (R, tumor regression grade [TRG] 1-2; n= 16) and non-responders (NR, TRG 3-5; n= 22) according to the pathological response observed upon surgery. We performed miRNA microarrays analysis on biopsy specimens, and validated the selected candidates both by qRT-PCR (tissue and serum) and by in situ hybridization (tissue, miR-125b) analyses. RESULTS: Eleven miRNAs were significantly different between R and NR (miR-154, miR-409-3p, miR-127-3p, miR-214*, miR-299-5p and miR-125b overexpressed in NR; miR-33a, miR-30e, miR-338-3p, miR-200a and miR-378 decreased). In particular, miR-125b resulted to be the best candidate to discriminate the two groups (AUC of 0.9026; 95% CI, 0.7618-1.043). Additionally, miR-125b serum levels were significantly overexpressed in NR patients compared to R (p-value=0.0087), with an excellent discriminating power (AUC of 0.782; 95% CI, 0.6123-0.9518). CONCLUSIONS: The obtained results further support the clinical impact of miRNA analysis. High miR-125b expression in tissue and serum were associated with a poor treatment response in LARC patients, therefore miR-125b could be considered as a possible novel non-invasive biomarker of response in LARC treatment.

An integrative approach for the identification of prognostic and predictive biomarkers in rectal cancer.

INTRODUCTION: Colorectal cancer is the third most common cancer in the world, a small fraction of which is represented by locally advanced rectal cancer (LARC). If not medically contraindicated, preoperative chemoradiotherapy, represent the standard of care for LARC patients. Unfortunately, patients shows a wide range of response rates in which approximately 20% has a complete pathological response, whereas in 20 to 40% the response is poor or absent. RESULTS: The following specific gene signature, able to discriminate responders' patients from non-responders, were founded: AKR1C3, CXCL11, CXCL10, IDO1, CXCL9, MMP12 and HLA-DRA. These genes are mainly involved in immune system pathways and interact with drugs traditionally used in the adjuvant treatment of rectal cancer. DISCUSSION: The present study suggests that new ideas for therapy could be found not only limited to studying genes differentially expressed between the two groups of patients but deepening the mechanisms, associated to response, in which they are involved. METHODS: Gene expression studies performed by: Agostini et al., Rimkus et al. and Kim et al. have been merged through a meta-analysis of the raw data. Gene expression data-sets have been processed using A-MADMAN. Common differentially expressed gene (DEG) were identified through SAM analysis. To further characterize the identified DEG we deeply investigated its biological role using an integrative computational biology approach.

A functional biological network centered on XRCC3: a new possible marker of chemoradiotherapy resistance in rectal cancer patients.

Preoperative chemoradiotherapy is widely used to improve local control of disease, sphincter preservation and to improve survival in patients with locally advanced rectal cancer. Patients enrolled in the present study underwent preoperative chemoradiotherapy, followed by surgical excision. Response to chemoradiotherapy was evaluated according to Mandard's Tumor Regression Grade (TRG). TRG 3, 4 and 5 were considered as partial or no response while TRG 1 and 2 as complete response. From pretherapeutic biopsies of 84 locally advanced rectal carcinomas available for the analysis, only 42 of them showed 70% cancer cellularity at least. By determining gene expression profiles, responders and non-responders showed significantly different expression levels for 19 genes (P < 0.001). We fitted a logistic model selected with a stepwise procedure optimizing the Akaike Information Criterion (AIC) and then validated by means of leave one out cross validation (LOOCV, accuracy = 95%). Four genes were retained in the achieved model: ZNF160, XRCC3, HFM1 and ASXL2. Real time PCR confirmed that XRCC3 is overexpressed in responders group and HFM1 and ASXL2 showed a positive trend. In vitro test on colon cancer resistant/susceptible to chemoradioterapy cells, finally prove that XRCC3 deregulation is extensively involved in the chemoresistance mechanisms. Protein-protein interactions (PPI) analysis involving the predictive classifier revealed a network of 45 interacting nodes (proteins) with TRAF6 gene playing a keystone role in the network. The present study confirmed the possibility that gene expression profiling combined with integrative computational biology is useful to predict complete responses to preoperative chemoradiotherapy in patients with advanced rectal cancer.