Emanuele Damiano Luca Urso

Complete Pathologic Response Following Preoperative Chemoradiation Therapy for Middle to Lower Rectal Cancer Is Not a Prognostic Factor for a Better Outcome

PURPOSEThe aim of this study was to evaluate factors associated with pathologic tumor response following preoperative chemoradiation therapy, and the prognostic impact of pathologic response on overall and disease-free survival.METHODSBetween 1994 and 2002, 132 patients underwent chemoradiation therapy followed by surgery for middle to lower rectal cancer. After excluding 26 cases (metastatic cancer, n = 13; nonradical surgery, n = 6; local excision procedure, n = 4; non-5-fluorouracil-based chemotherapy, n = 2; incomplete data on preoperative chemoradiation therapy regimen used, n = 1), the remaining 106 patients were included in the study. Variables considered were the following: age, gender, tumor location, pretreatment T and N stage, modality of 5-fluorouracil administration, total radiotherapy dose delivered, chemoradiation therapy regimen used (Regimen A: chemotherapy (bolus of 5-fluorouracil and leucovorin, days 1–5 and 29–33) + radiotherapy (45 Gy/25 F/1.8 Gy/F); Regimen B: chemotherapy (5-fluorouracil continuous venous infusion ± weekly bolus of carboplatin or oxaliplatin) + radiotherapy (50.4 Gy/28 F/1.8 Gy/F)), time interval between completion of chemoradiation therapy and surgery, postoperative chemotherapy administration, surgical procedures, pT, pN, and pTNM stage, and response to chemoradiation therapy defined as tumor regression grade, scored from 1 (no tumor on surgical specimen) to 5 (absence of regressive changes). Statistical analysis was performed by means of logistic regression analysis (Cox’s model for overall and disease-free survival).RESULTSMedian age of the 106 patients was 60 (range, 31–79) years and the male:female ratio, 66:40. Median distance of tumor from the anal verge was 6 (range, 1–11) cm. Pretreatment TNM stage, available in 104 patients, was cT3–T4N0, n = 41; cT2N1, n = 9; cT3N1, n = 39; and cT4N1, n = 17. The median radiotherapy dose delivered was 50.4 (range, 40–56) Gy; 58 patients received 5-fluorouracil by continuous venous infusion, and carboplatin with oxaliplatin was added to the chemotherapy schedule in 71 cases. Patients were given Regimen A in 47 cases and Regimen B in 59. The median interval between chemoradiation therapy and surgery was 42.5 (range, 19–136) days, and 94 patients underwent a sphincter-saving procedure. Tumor regression grade, available in 104 cases, was 1, n = 19; 2, n = 18; 3, n = 15; 4, n = 13; and 5, n = 39. At a median follow-up of 42 (range, 1–110) months, 11 patients had died, and 95 were alive. None of the patients had local recurrences, but 13 had distant recurrences. At logistic regression analysis, the chemoradiation therapy regimen used was the only independent predictor of tumor response following preoperative chemoradiation therapy (odds ratio = 0.29, 95% confidence interval = 0.13–0.67, P = 0.003). At Cox’s regression analysis, pretreatment T stage was the only independent prognostic factor for both disease-free survival (relative risk = 7.13, 95% confidence interval = 2.3–21.8, P = 0.001) and overall survival (relative risk = 4.83, 95% confidence interval = 1.1–19.9, P = 0.029).CONCLUSIONSTumor response following preoperative chemoradiation therapy is mainly related to the preoperative regimen used. For patients receiving preoperative chemoradiation therapy, pretreatment T stage, but not tumor response to preoperative chemoradiation therapy, is prognostic for outcome (both disease-free and overall survival).

Cancer risk associated with STK11/LKB1 germline mutations in Peutz-Jeghers syndrome patients: Results of an Italian multicenter study

BACKGROUND Germline mutations in the STK11/LKB1 gene cause Peutz-Jeghers syndrome, an autosomal-dominantly inherited condition characterized by mucocutaneous pigmentation, hamartomatous gastrointestinal polyposis, and an increased risk for various malignancies. We here report the results of the first Italian collaborative study on Peutz-Jeghers syndrome. AIMS To assess cancer risks in a large homogenous cohort of patients with Peutz-Jeghers syndrome, carrying, in large majority, an identified STK11/LKB1 mutation. METHODS One-hundred and nineteen patients with Peutz-Jeghers syndrome, ascertained in sixteen different Italian centres, were enrolled in a retrospective cohort study. Relative and cumulative cancer risks and genotype-phenotype correlations were evaluated. RESULTS 36 malignant tumours were found in 31/119 (29 STK11/LKB1 mutation carriers) patients. The mean age at first cancer diagnosis was 41 years. The relative overall cancer risk was 15.1 with a significantly higher risk (p < 0.001) in females (22.0) than in males (8.6). Highly increased relative risks were present for gastrointestinal (126.2) and gynaecological cancers (27.7), in particular for pancreatic (139.7) and cervical cancer (55.6). The Kaplan-Meier estimates for overall cumulative cancer risks were 20%, 43%, 71%, and 89%, at age 40, 50, 60 and 65 years, respectively. CONCLUSION Peutz-Jeghers syndrome entails markedly elevated cancer risks, mainly for pancreatic and cervical cancers. This study provides a helpful reference for improving current surveillance protocols.

Integrated analysis of unclassified variants in mismatch repair genes

Purpose: Lynch syndrome is a genetic disease that predisposes to colorectal tumors, caused by mutation in mismatch repair genes. The use of genetic tests to identify mutation carriers does not always give perfectly clear results, as happens when an unclassified variant is found. This study aimed to define the pathogenic role of 35 variants present in MSH2, MLH1, MSH6, and PMS2 genes identified in our 15-year case study.Methods: We collected clinical and molecular data of all carriers, and then we analyzed the variants pathogenic role with web tools and molecular analyses. Using a Bayesian approach, we derived a posterior probability of pathogenicity and classified each variant according to a standardized five-class system.Results: The MSH2 p.Pro349Arg, p.Met688Arg, the MLH1 p.Gly67Arg, p.Thr82Ala, p.Lys618Ala, the MSH6 p.Ala1236Pro, and the PMS2 p.Arg20Gln were classified as pathogenic, and the MSH2 p.Cys697Arg and the PMS2 p.Ser46Ile were classified as likely pathogenic. Seven variants were likely nonpathogenic, 3 were nonpathogenic, and 16 remained uncertain.Conclusion: Quantitative assessment of several parameters and their integration in a multifactorial likelihood model is the method of choice for classifying the variants. As such classifications can be associated with surveillance and testing recommendations, the results and the method developed in our study can be useful for helping laboratory geneticists in evaluation of genetic tests and clinicians in the management of carriers.

The role of MYH gene in genetic predisposition to colorectal cancer: Another piece of the puzzle

Biallelic germline mutations in the MYH gene cause MYH-Associated Polyposis but patients with a single mutation possibly have an increased colorectal cancer (CRC) risk. Using DNA from consecutive CRC patients we carried out a case-control study, with the aim to contribute data on the Italian population. Genotyping of four MYH mutations found two biallelic and two monoallelic carriers among 439 cases, and only one heterozygous individual among 247 age-matched controls. The frequencies of the mutant alleles were 0.68% (6/878) and 0.20% (1/494), respectively. These differences were not statistically significant. Results on the monoallelic carriers were combined with those from 11 studies on other populations, and the risk of developing a CRC was estimated with an OR=1.11 (95% CI=0.90; 1.36), yet not reaching a significant evidence of increased CRC risk.

A ten markers panel provides a more accurate and complete microsatellite instability analysis in mismatch repair-deficient colorectal tumors

UNLABELLED Tumour microsatellite instability (MSI) is useful in identifying patients with hereditary non-polyposis colorectal cancer (HNPCC) with defective DNA mismatch repair (MMR) genes. A reference Bethesda panel has limitations resulting from the inclusion of dinucleotide markers, which are less sensitive and specific for detection of tumours with MMR deficiencies. We developed a multiplex PCR assay with additional four mononucleotide markers and one dinucleotide marker (NR-21, NR-24, BAT-40, TGF-BetaR and D18S58) for a rapid and proper classification of MSI-H, MSI-L and MSS colorectal cancers. Two tetranucleotide markers were added to identify sample mix-ups and/or contamination. RESULTS all the 44 cases test cases were in agreement with previous classification except for three cases: one case MSI-H-Bethesda unstable only for dinucleotides markers shifted to MSI-L category and two cases MSI-L-Bethesda unstable for mononucleotide markers shifted to MSI-H category. Immunohistochemistry analysis revealed that these two MSI-H cases did not expressed hMLH1 and they were found to be methylated at the MLH1 promoter, while the first one that shifted to MSI-L showed MMR protein expression. CONCLUSION a complete panel of ten markers including four dinucleotide and six mononucleotide microsatellites allows accurate evaluation of tumor MSI status.

Prediction of rectal lymph node metastasis by pelvic computed tomography measurement

AIM Rectal cancer staging represents a crucial step to select the best treatment for this tumour. Particularly after neo-adjuvant chemoradiotherapy (CRT), it may influence the surgical procedure (e.g. radical resection vs. local excision). The aim of this study was to determine the best lymph node size cut-off at computed tomography (CT) to predict nodal metastasis in rectal cancer patients with and without preoperative CRT. METHODS A consecutive series of patients operated on for primary mid-low rectal adenocarcinoma, all staged with pelvic CT scan, were subdivided as follows: those who underwent surgery alone treatment without CRT (Group A) and those who underwent preoperative CRT (Group B). All CT scans were re-viewed by a single radiologist and, based on the lymph node size, findings were compared with pathologic lymph node status (pN). At each lymph node size cut-off value, the following were calculated: accuracy, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). The best cut-off value was defined as having an accuracy >or=70% with the highest NPV. RESULTS The study population consisted of 162 patients: Group A (n=52) and Group B (n=110). Patients classified as pN-positive (n=45) had a higher number of and larger sized lymph nodes by CT scan than patients classified as pN-negative (n=117). The cut-off values with an accuracy >or=70% ranged between 7 and 11 mm in Group A and between 9 and 14 mm in Group B. The cut-off with the best NPV was 7 mm for Group A and 10mm for Group B. CONCLUSIONS Acknowledging the limitations of the dimensional criterion, lymph node size cut-off values found in our study may be useful for planning rectal cancer treatment using CT scan.

A Specific Mutational Signature Associated with DNA 8-Oxoguanine Persistence in MUTYH-defective Colorectal Cancer

8-Oxoguanine, a common mutagenic DNA lesion, generates G:C > T:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:C > T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strong sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. The occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs.

Rectum-sparing surgery may be appropriate for biallelic MutYH-associated polyposis.

PURPOSE: The risk of cancer or severe polyposis of the rectal stump after total colectomy for MutYH-associated polyposis is scarcely defined. To evaluate this risk, we describe the findings of endoscopic surveillance of the rectal stump in a series of patients with biallelic MutYH mutations and polyposis. METHODS: This is a retrospective, observational, multicenter case series derived from 2 familial cancer registries. Biallelic, germ-line MutYH mutations were found in 14 patients with no adenomatous polyposis coli gene mutations. Eleven of them underwent total colectomy with ileorectal anastomosis and yearly proctoscopic surveillance thereafter. Phenotype and histology of rectal polyps were recorded at diagnosis and during follow-up. Development of adenomas and carcinomas during endoscopic surveillance of the rectal stump was observed. RESULTS: At diagnosis, 6 patients had attenuated polyposis (10–100 adenomas), 5 patients had classical polyposis, 8 patients had colon carcinoma, and no patient had rectal carcinoma. The mean number of rectal polyps at diagnosis was 2.64 ± 2.11 (range, 0–6). No patients had rectal cancer. The most frequent MutYH mutations were Y165C/Y165C and G382D/G382D in 6 and 2 patients, respectively. During surveillance of the rectal stump after surgery (median duration, 5 y; range, 2–23 y), no patient developed rectal cancer. The mean number of adenomas per proctoscopy was 1.23 ± 2.19 (range, 0–10 adenomas per proctoscopy). This study was limited by the small size and retrospective nature of the case series. CONCLUSION: Total colectomy with ileorectal anastomosis may be appropriate for patients with MutYH-associated polyposis, provided that they have no rectal cancer or severe rectal polyposis at presentation and that they undergo yearly endoscopic surveillance thereafter.

APC I1307K mutations and forkhead box gene (FOXO1A): another piece of an interesting correlation.

Purpose Germline nonsense and frameshift mutations in the adenomatous polyposis coli (APC) gene are found in approximately 90% of individuals affected by familial adenomatous polyposis (FAP) and a genotype-phenotype relationship has been observed. Missense mutations have also been found in a few cases, even if their role in FAP is still unknown. An association between a missense mutation, APC I1307K, and the risk of sporadic colorectal cancer (CRC) has been reported. In order to improve the knowledge about the genetic effect of APC I1307K on the phenotype, we tried a new approach using matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). Experimental design An APC mutation (I1307K) was found in an index case of a non-Jewish woman and her son with attenuated familial adenomatous polyposis (A-FAP) and no family history of cancer. In order to evaluate whether the presence and abundance of the ionic species are related to the presence of cancer or the presence of mutation, comparative analyses of 11 healthy clean-colon subjects, 59 patients with CRC (stage II n=19, stage III n=23, stage IV n=17) without polyps, and 9 FAP patients, carriers of a nonsense mutation in the APC gene, were evaluated. Results Comparative analysis of serum protein profiles of the index patient and her healthy son, FAP and sporadic CRC patients, and subjects with preneoplastic lesions showed a characteristic abundance of ionic species at m/z 905, which was not present in healthy controls. Two peptides were identified from MALDI/MS/MS spectra of m/z 905 belonging to the kininogen-1 precursor and the human forkhead box protein 01A (FOXO1A). FOXO1A was present in only two subjects carrying I1307K, but not in other patients. Conclusions Our findings seem to suggest a relationship between m/z 905, FOXO1A and the development and growth of colorectal cancer. FOXO1A fragment determination in serum with MALDI/MS might be a promising approach for early detection of colon carcinoma or for the development of targeted therapies.

Glutathione S-Transferase P1 Ile105Val Polymorphism is Associated with Haematological Toxicity in Elderly Rectal Cancer Patients Receiving Preoperative Chemoradiotherapy

AbstractBackground: Increasing evidence suggests that common gene polymorphisms may influence the toxicity of various cytotoxic agents used in the treatment of cancer. Objective: To evaluate the predictive value of acute toxicity of methylenetetrahydrofolate reductase 677T polymorphism, glutathione S-transferase P1 (GSTP1) substitution of isoleucine with valine at codon 105 (Ile105Val) polymorphism and the tandem repeat polymorphism in the thymidylate synthase gene promoter in elderly patients with rectal cancer receiving preoperative chemoradiotherapy (CRT). Method: From 1994 to 2002, 166 Caucasian patients underwent surgery following CRT for mid-low rectal cancer at a single institution, 42 (male-to-female ratio, 25:17) of whom were aged ≥65 years (median age 70 years, range 65–79). The pre-treatment clinical stage was tumour (T) stage 3–4 in 38 patients and node (N)-positive in 29 patients. Patients received external-beam radiotherapy with conventional fractionation and fluorouracil-based chemotherapy. Blood samples were used to extract and amplify DNA. Gene polymorphisms were determined by polymerase chain reaction and restriction enzyme digestion. Acute toxicity to preoperative therapy was reported according to the National Cancer Institute Common Toxicity Criteria, version 2. Univariate and multivariate analyses were performed using one-way analysis of variance and linear regression, respectively. Results: Haematological toxicity (grade 1–2) was observed in 15 of 40 patients for whom toxicity data were available and gastrointestinal toxicity (grade 1–4) in 24 of these same 40 patients. At univariate analysis, female sex (p = 0.036) and GSTP1 Ile105Val (p = 0.0376) were associated with haematological toxicity. At multivariate analysis, GSTP1 Ile105Val polymorphism (p = 0.041) was the only factor found to be associated with haematological toxicity. Patients carrying the Val/Val genotype in the GSTP1 gene had a lower risk of haematological toxicity (odds ratio = 0.322, 95% CI 0.101, 0.957) than patients with the Ile/Ile genotype. Conclusion: GSTP1 Ile105Val polymorphism is a promising marker of potential haematological toxicity in elderly patients with rectal cancer receiving preoperative CRT.