Daniela Rega

Genetics, diagnosis and management of colorectal cancer (Review)

Colorectal cancer (CRC) is the third most common type of cancer worldwide and a leading cause of cancer death. Surgery represents the mainstay of treatment in early cases but often patients are primarily diagnosed in an advanced stage of disease and sometimes also distant metastases are present. Neoadjuvant therapy is therefore needed but drug resistance may influence response and concur to recurrent disease. At molecular level, it is a very heterogeneous group of diseases with about 30% of hereditary or familial cases. During colorectal adenocarcinomas development, epithelial cells from gastrointestinal trait acquire sequential genetic and epigenetic mutations in specific oncogenes and/or tumour suppressor genes, causing CRC onset, progression and metastasis. Molecular characterization of cancer associated mutations gives valuable information about disease prognosis and response to the therapy. Very early diagnosis and personalized care, as well as a better knowledge of molecular basis of its onset and progression, are therefore crucial to obtain a cure of CRC. In this review, we describe updated genetics, current diagnosis and management of CRC pointing out the extreme need for a multidisciplinary approach to achieve the best results in patient outcomes.

Synergistic effect of interleukin-10-receptor variants in a case of early-onset ulcerative colitis

AIMnTo investigated the molecular cause of very early-onset ulcerative colitis (UC) in an 18-mo-old affected child.nnnMETHODSnWe analysed the interleukin-10 (IL10) receptor genes at the DNA and RNA level in the proband and his relatives. Beta catenin and tumor necrosis factor-α (TNFα) receptors were analysed in the proteins extracted from peripheral blood cells of the proband, his relatives and familial adenomatous polyposis (FAP) and PTEN hamartoma tumor syndrome (PHTS) patients. Samples were also collected from the probands inflamed colorectal mucosa and compared to healthy and tumour mucosa collected from a FAP patient and patients affected by sporadic colorectal cancer (CRC). Finally, we examined mesalazine and azathioprine effects on primary fibroblasts stabilised from UC and FAP patients.nnnRESULTSnOur patient was a compound heterozygote for the IL10RB E47K polymorphism, inherited from his father, and for a novel point mutation within the IL10RA promoter (the -413G->T), inherited from his mother. Beta catenin and tumour necrosis factor α receptors-I (TNFRI) protein were both over-expressed in peripheral blood cells of the probands relatives more than the proband. However, TNFRII was over-expressed only in the proband. Finally, both TNFα-receptors were shown to be under-expressed in the inflamed colon mucosa and colorectal cancer tissue compared to healthy colon mucosa. Consistent with this observation, mesalazine and azathioprine induced, in primary fibroblasts, IL10RB and TNFRII over-expression and TNFRI and TNFα under-expression. We suggest that β-catenin and TNFRI protein expression in peripheral blood cells could represent molecular markers of sub-clinical disease in apparently healthy relatives of patients with early-onset UC.nnnCONCLUSIONnA synergistic effect of several variant alleles of the IL10 receptor genes, inherited in a Mendelian manner, is involved in UC onset in this young child.

The target sign in colorectal liver metastases: an atypical Gd-EOB-DTPA “uptake” on the hepatobiliary phase of MR imaging

PurposeTo describe the MRI findings in colorectal cancer liver metastases using gadoxetic acid (Gd-EOB-DTPA), with special emphasis on the target feature seen on the hepatobiliary phase.Material and methodsThe medical records of 45 colorectal cancer patients with an overall number of 150 liver metastases were reviewed. All patients underwent Gd-EOB-DTPA-enhanced MRI before any kind of treatment. We retrospectively evaluated, for each lesion, the signal intensity on the T1-weighted, T2-weighted, and diffusion-weighted images. Additionally, the enhancement pattern during the arterial-, portal-, equilibrium-, and hepatobiliary-phase was assessed. Fourteen lesions had a pathological correlation.ResultsLesions size was 5–40 mm (mean 15 mm). All metastases were hypointense on T1-w imaging. Ninety-nine lesions (66%) had a central area of very high signal intensity on T2-w imaging. Fifty-one metastases (34%) were hyperintense on the T2-w images. In DWI, all lesions had a restricted diffusion. The mean ADC value was 1.31xa0×xa010−3xa0mm2/s (range 1.10–1.45xa0×xa010−3xa0mm2/s). During the arterial-phase imaging, 61 lesions (41%) showed a rim enhancement, while 89 lesions (59%) appeared as hypointense. All lesions had low signal intensity in the portal and equilibrium phase. Thirty-nine percent of the lesions also showed an enhancing rim on the portal-phase images. During the hepatobiliary phase, 80 lesions (53.3%) were hypointense, while 70 lesions (46.7%) had a target appearance.ConclusionA number of metastases show an atypical contrast medium uptake during the hepatobiliary phase of gadoxetic acid-enhanced MRI, consisting in a target appearance.

Differential expression of PTEN gene correlates with phenotypic heterogeneity in three cases of patients showing clinical manifestations of PTEN hamartoma tumour syndrome

Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba syndrome (BRRS) and proteus syndrome are disorders known as PTEN hamartoma tumour syndrome (PHTS), that can show remarkable clinical overlap and are all caused by germline PTEN mutations.We here present two families, one affected by CS and the other affected by BRRS, both carriers of specific pathogenetic missense mutation in exon 5 of PTEN gene, within the catalitic domain. Both PHTS families exhibited extremely variable phenotypes, showing inter- and intra- familial variability. One of the two characterised mutations, the c.320A-u2009>u2009T; p.107Asp-u2009>u2009Val, identified in the CS family, was not previously described in the literature. Furthermore, the BRRS family, carrier of the c.406xa0T-u2009>u2009C; p.136Cys-u2009>u2009Arg mutation, shows a substantial alteration of PTEN protein expression that well correlates with intra-familial phenotypic variability.Finally, we describe an apparently sporadic case of an 80-year-old man, with a very low level of PTEN mRNA and protein expression, both in healthy and tumour colon mucosa, associated with a very atypical phenotype. He developed a metastatic colorectal carcinoma, macrocephaly and pheochromocytoma.According to literature data, our observations confirm that PTEN mutations of catalytic domain can cause different syndromes. We suggest that PTEN expression could represent one of the mechanisms involved in the remarkable heterogeneity of the clinical PHTS manifestations within affected families. Furthermore, constitutive strong decrease of PTEN expression in colon normal mucosa could be associated with late onset of colorectal cancer.

Role of endoscopic ultrasonography in the loco-regional staging of patients with rectal cancer

The prognosis of rectal cancer (RC) is strictly related to both T and N stage of the disease at the time of diagnosis. RC staging is crucial for choosing the best multimodal therapy: patients with high risk locally advanced RC (LARC) undergo surgery after neoadjuvant chemotherapy and radiotherapy (NAT); those with low risk LARC are operated on after a preoperative short-course radiation therapy; finally, surgery alone is recommended only for early RC. Several imaging methods are used for staging patients with RC: computerized tomography, magnetic resonance imaging, positron emission tomography, and endoscopic ultrasound (EUS). EUS is highly accurate for the loco-regional staging of RC, since it is capable to evaluate precisely the mural infiltration of the tumor (T), especially in early RC. On the other hand, EUS is less accurate in restaging RC after NAT and before surgery. Finally, EUS is indicated for follow-up of patients operated on for RC, where there is a need for the surveillance of the anastomosis. The aim of this review is to highlight the impact of EUS on the management of patients with RC, evaluating its role in both preoperative staging and follow-up of patients after surgery.

Lithium chloride induces mesenchymal‑to‑epithelial reverting transition in primary colon cancer cell cultures

Epithelial-to-mesenchymal transition (EMT) confers stem cell-like phenotype and more motile properties to carcinoma cells. During EMT, the expression of E-cadherin decreases, resulting in loss of cell-cell adhesion and increased migration. Expression of Twist1 and other pleiotropic transcription factors, such as Snail, is known to activate EMT. We established primary colon cancer cell cultures from samples of operated patients and validated cultures by cytogenetic and molecular biology approaches. Western blot assay, quantitative real-time PCR and immunofluorescence were performed to investigate the expression of E-cadherin, vimentin, β-catenin, cytokeratin-20 and -18, Twist1, Snail, CD44, cyclooxygenase-2 (COX2), Sox2, Oct4 and Nanog. Moreover, cell differentiation was induced by incubation with LiCl-containing medium for 10 days. We observed that these primary colorectal cancer (CRC) cells lost expression of the E-cadherin epithelial marker, which was instead expressed in cancer and normal colon mucosa of the same patient, while overexpressed vimentin (mesenchymal marker), Twist1, Snail (EMT markers) and COX2. Cytokeratin-18 was expressed both in tissues and cell cultures. Expression of stem cell markers, such as CD44, Oct4 and Nanog, were also observed. Following differentiation with the glycogen synthase kinase 3β (GSK3β) inhibitor LiCl, the cells began to express E-cadherin and, at once, Twist1 and Snail expression was strongly downregulated, suggesting a MET-reverting process. In conclusion, we established primary colon mesenchymal cancer cell cultures expressing mesenchymal and epithelial biomarkers together with high level of EMT transcription factors. We propose that they could represent a good model for studying EMT and its reverting mechanism, the mesenchymal-to-epithelial transition (MET). Our observation indicates that LiCl, a GSK3β inhibitor, induces MET in vitro, suggesting that LiCl and GSK3β could represent, respectively, interesting drug, and target for CRC therapy.

The biological complexity of colorectal cancer: Insights into biomarkers for early detection and personalized care

Colorectal cancer has been ranked the third and second most prevalent of all cancers in men and women, respectively, and it represents the fourth most common cause of cancer deaths. In 2012, there were 1.4 million estimated cases of colorectal cancer worldwide, and 700,000 estimated deaths, which implies significant impact on public health, especially in economically-developed countries. In recent years, there has been an increase in the number of tumors, although this has been accompanied by decreased mortality, due to more appropriate and available information, earlier diagnosis, and improvements in treatment. Colorectal cancers are characterized by great genotypic and phenotypic heterogeneity, including tumor microenvironment and interactions between healthy and cancer cells. All of these traits confer a unique peculiarity to each tumor, which can thus be considered as an individual disease. Well conducted molecular and clinical characterization of each colorectal cancer is essential with a view to the implementation of precision oncology, and thus personalized care. This last aims at standardization of therapeutic plans chosen according to the genetic background of each specific neoplasm, to increase overall survival and reduce treatment side effects. Thus, prognostic and predictive molecular biomarkers assume a critical role in the characterization of colorectal cancer and in the determination of the most appropriate therapy.

Evaluation of tumor response after short- course radiotherapy and delayed surgery for rectal cancer

Purpose Neoadjuvant therapy is able to reduce local recurrence in rectal cancer. Immediate surgery after short course radiotherapy allows only for minimal downstaging. We investigated the effect of delayed surgery after short-course radiotherapy at different time intervals before surgery, in patients affected by rectal cancer. Methods From January 2003 to December 2013 sixty-seven patients with the following characteristics have been selected: clinical (c) stage T3N0 ≤ 12 cm from the anal verge and with circumferential resection margin > 5 mm (by magnetic resonance imaging); cT2, any N, < 5 cm from anal verge; and patients facing tumors with enlarged nodes and/or CRM+ve who resulted unfit for chemo-radiation, were also included. Patients underwent preoperative short-course radiotherapy with different interval to surgery were divided in three groups: A (within 6 weeks), B (between 6 and 8 weeks) and C (after more than 8 weeks). Hystopatolgical response to radiotherapy was measured by Mandard’s modified tumor regression grade (TRG). Results All patients completed the scheduled treatment. Sixty-six patients underwent surgery. Fifty-three of which (80.3%) received a sphincter saving procedure. Downstaging occurred in 41 cases (62.1%). The analysis of subgroups showed an increasing prevalence of TRG 1–2 prolonging the interval to surgery (group A—16.7%, group B—36.8% and 54.3% in group C; p value 0.023). Conclusions Preoperative short-course radiotherapy is able to downstage rectal cancer if surgery is delayed. A higher rate of TRG 1–2 can be obtained if interval to surgery is prolonged to more than 8 weeks.

Sequential PET/CT with [18F]-FDG Predicts Pathological Tumor Response to Preoperative Short Course Radiotherapy with Delayed Surgery in Patients with Locally Advanced Rectal Cancer Using Logistic Regression Analysis

Previous studies indicate that FDG PET/CT may predict pathological response in patients undergoing neoadjuvant chemo-radiotherapy for locally advanced rectal cancer (LARC). Aim of the current study is evaluate if pathological response can be similarly predicted in LARC patients after short course radiation therapy alone. Methods: Thirty-three patients with cT2-3, N0-2, M0 rectal adenocarcinoma treated with hypo fractionated short course neoadjuvant RT (5x5 Gy) with delayed surgery (SCRTDS) were prospectively studied. All patients underwent 3 PET/CT studies at baseline, 10 days from RT end (early), and 53 days from RT end (delayed). Maximal standardized uptake value (SUVmax), mean standardized uptake value (SUVmean) and total lesion glycolysis (TLG) of the primary tumor were measured and recorded at each PET/CT study. We use logistic regression analysis to aggregate different measures of metabolic response to predict the pathological response in the course of SCRTDS. Results: We provide straightforward formulas to classify response and estimate the probability of being a major responder (TRG1-2) or a complete responder (TRG1) for each individual. The formulas are based on the level of TLG at the early PET and on the overall proportional reduction of TLG between baseline and delayed PET studies. Conclusions: This study demonstrates that in the course of SCRTDS it is possible to estimate the probabilities of pathological tumor responses on the basis of PET/CT with FDG. Our formulas make it possible to assess the risks associated to LARC borne by a patient in the course of SCRTDS. These risk assessments can be balanced against other health risks associated with further treatments and can therefore be used to make informed therapy adjustments during SCRTDS.

MR imaging perfusion and diffusion analysis to assess preoperative Short Course Radiotherapy response in locally advanced rectal cancer: Standardized Index of Shape by DCE-MRI and intravoxel incoherent motion-derived parameters by DW-MRI

Our aim is to assess preoperative Short Course Radiotherapy (SCR) tumor response in locally advanced rectal cancer (LARC) through Standardized Index of Shape (SIS) by DCE-MRI, apparent diffusion coefficient (ADC) and intravoxel incoherent motion-derived parameters by DW-MRI. 35 patients with LARC underwent MR scan before and after SCR followed by delayed surgery, retrospectively, were enrolled. SIS, ADC, tissue diffusion (Dt), pseudodiffusion (Dp), and perfusion fraction (f) were extracted by MRI for each patient before and after SCR. Tumor regression grade (TRG) was estimated. Receiver operating characteristic curve and linear classification were performed. Sixteen patients were classified as responders (TRGxa0≤xa02) and 19 as non-responders. Seven patients had TRG1 [pathological complete response (pCR)]. The best parameter to discriminate responders by non-responders was SIS (sensitivity 94%, specificity 84%, accuracy 89%, cutoff valuexa0=xa0−xa07.8%). SIS obtained the best diagnostic performance also to discriminate pCR (sensitivity 86%, specificity 89%, accuracy 89%, cutoff valuexa0=xa068.2%). No accuracy increase was obtained combining linearly each possible parameters couple or all functional MR-derived parameters. SIS is a hopeful DCE-MRI angiogenic biomarker to assess preoperative treatment response after SCR with delayed surgery, and it permits to discriminate pCR allowing to direct surgery for tailored and conservative treatment.