Tiziana Latiano

Heterogeneity of KRAS, NRAS, BRAF and PIK3CA mutations in metastatic colorectal cancer and potential effects on therapy in the CAPRI GOIM trial

BACKGROUND Evidence suggests that metastatic colorectal carcinoma (mCRC) has a high level of intratumor heterogeneity. We carried out a quantitative assessment of tumor heterogeneity for KRAS, NRAS, BRAF and PIK3CA mutations, in order to assess potential clinical implications. PATIENTS AND METHODS Tumor samples (n = 182) from the CAPRI-GOIM trial of first-line cetuximab + FOLFIRI in KRAS exon-2 wild-type mCRC patients were assessed by next-generation sequencing that allows quantitative assessment of mutant genes. Mutant allelic frequency was normalized for the neoplastic cell content and, assuming that somatic mutations usually affect one allele, the Heterogeneity Score (HS) was calculated by multiplying by 2 the frequency of mutant alleles in neoplastic cells. Therefore, HS virtually corresponds to the fraction of neoplastic cells carrying a specific mutation. RESULTS The KRAS HS ranged between 12 and 260 with mean value of 87.1 and median value of 84.4, suggesting that in most CRC, the majority of neoplastic cells carry mutant KRAS. Similar findings were observed for NRAS (HS range 35.5-146.7; mean 102.8; median 117.1). In contrast, in BRAF (HS range 17.1-120; mean 54.8; median 54.3) and PIK3CA (HS range 14.3-120; mean 59.5; median 47.3) mutant cases, only a fraction of neoplastic cells seem to carry the mutant allele. The response rate was 70% in KRAS mutant patients with an HS <33 (low KRAS; n = 10) and 45.7% in KRAS HS >33 patients (high KRAS; n = 35); median progression-free survival were 7.97 and 8.37 months, respectively. Low-KRAS tumors had a higher frequency of additional mutations in PIK3CA when compared with high-KRAS (6/10 versus 8/35). CONCLUSIONS KRAS and NRAS mutations are usually present in the majority of neoplastic cells, whereas BRAF and PIK3CA mutations often affect a limited fraction of transformed cells. Resistance to cetuximab in low-KRAS patients might be driven by the complex mutational profile rather than KRAS mutation load.

Phase II study of panitumumab, oxaliplatin, 5-fluorouracil, and concurrent radiotherapy as preoperative treatment in high-risk locally advanced rectal cancer patients (StarPan/STAR-02 Study)

BACKGROUND The aim of this phase II study was to assess the activity of panitumumab in combination with oxaliplatin, 5-fluorouracil, and external radiotherapy (RT) as preoperative treatment in locally advanced rectal cancer patients. PATIENTS AND METHODS Patients had rectal adenocarcinoma, cT3N+ or cT4N-/+ stage, located <12 cm from the anal margin. Panitumumab was administered before the start of chemo-RT, and every 2 weeks in combination with 5-fluorouracil-oxaliplatin with concurrent RT. Rectal surgery was carried out 7-8 weeks after the end of neoadjuvant treatment. The primary end point was a pathological complete response rate of 25%. RESULTS Sixty patients were enrolled from February 2007 to October 2009. Fifty-five (91.7%) patients underwent surgery. Rate of pathological complete response was 21.1% (95% confidence interval 10.4% to 31.6%). Pathological downstaging occurred in 33 of 57 (57.9%) patients. Grade 3-4 toxicity during neoadjuvant treatment was diarrhea (38.9%), cutaneous reactions (18.6%), nausea (5.1%), asthenia (3.4%), anorexia (3.4%), and neutropenia (1.7%). One toxic death was observed for diarrhea. CONCLUSIONS In our study, the primary end point is not reached and panitumumab combination treatment was associated with high incidence of grade 3-4 diarrhea. The higher pathological complete response rate in comparison with the results of previous neoadjuvant rectal cancer trials with anti-epidermal growth factor receptor monoclonal antibodies supports further studies necessary to understand the possibility of optimal regimens and sequences with chemo-RT.

Investigation of Multiple Susceptibility Loci for Inflammatory Bowel Disease in an Italian Cohort of Patients

Background Recent GWAs and meta-analyses have outlined about 100 susceptibility genes/loci for inflammatory bowel diseases (IBD). In this study we aimed to investigate the influence of SNPs tagging the genes/loci PTGER4, TNFSF15, NKX2-3, ZNF365, IFNG, PTPN2, PSMG1, and HLA in a large pediatric- and adult-onset IBD Italian cohort. Methods Eight SNPs were assessed in 1,070 Crohns disease (CD), 1,213 ulcerative colitis (UC), 557 of whom being diagnosed at the age of ≤16 years, and 789 healthy controls. Correlations with sub-phenotypes and major variants of NOD2 gene were investigated. Results The SNPs tagging the TNFSF15, NKX2-3, ZNF365, and PTPN2 genes were associated with CD (P values ranging from 0.037 to 7×10−6). The SNPs tagging the PTGER4, NKX2-3, ZNF365, IFNG, PSMG1, and HLA area were associated with UC (P values 0.047 to 4×10−5). In the pediatric cohort the associations of TNFSF15, NKX2-3 with CD, and PTGER4, NKX2-3, ZNF365, IFNG, PSMG1 with UC, were confirmed. Association with TNFSF15 and pediatric UC was also reported. A correlation with NKX2-3 and need for surgery (P  =  0.038), and with HLA and steroid-responsiveness (P  =  0.024) in UC patients was observed. Moreover, significant association in our CD cohort with TNFSF15 SNP and colonic involvement (P  =  0.021), and with ZNF365 and ileal location (P  =  0.024) was demonstrated. Conclusions We confirmed in a large Italian cohort the associations with CD and UC of newly identified genes, both in adult and pediatric cohort of patients, with some influence on sub-phenotypes.

Associations between Genetic Polymorphisms in IL-33, IL1R1 and Risk for Inflammatory Bowel Disease

Background Recent evidence suggests that the IL-33/IL1RL1 axis plays a critical role in several autoimmune and inflammatory disorders; however, its mechanistic role in inflammatory bowel disease (IBD) has not been clearly defined. We investigated the contribution of IL-33 and IL1RL1 polymorphisms to IBD risk, and possible correlations with phenotype in an Italian cohort of adult and pediatric patients. Methods We evaluated the association of six SNPs in IL-33 and IL1RL1 genes, in 805 Crohn’s disease (CD), 816 ulcerative colitis (UC), and 752 controls, using Taqman. IL-33 and IL1RL1 mRNA expression was also analyzed. Results Significant allele and genotype associations with IL-33 rs3939286 were found in CD (P = 0.004; P = 0.035) and UC patients (P = 0.002; P = 0.038). After stratifying the cohort for age at diagnosis, the differences remained significant only in the IBD adult-onset. Significant associations were also obtained in CD patients with two IL1RL1 polymorphisms (rs13015714 and rs2058660, P<0.015). By combining homo- and heterozygous carriers of the rs13015714 risk allele, differences were still significant for both CD adult- and pediatric-onset. Upon genotype-phenotype evaluation, an increased frequency of extensive colitis in adult UC (P = 0.019) and in steroid-responsive pediatric patients (P = 0.024) carrying the IL-33 rs3939286 risk genotype, was observed. mRNA expression of IL-33 and IL1RL1 in inflamed IBD biopsy samples was significantly increased. Conclusions Common IL-33 and IL1RL1 polymorphisms contribute to the risk of IBD in an Italian cohort of adult and pediatric patients, with some influence on sub-phenotypes.

Sequential evaluation of thiopurine methyltransferase, inosine triphosphate pyrophosphatase, and HPRT1 genes polymorphisms to explain thiopurines' toxicity and efficacy

To evaluate the polymorphisms of several genes involved in the azathioprine and mercaptopurine metabolism, in an attempt to explain their toxicity and efficacy in Crohn’s disease and ulcerative colitis.

Changes in miR-143 and miR-21 expression and clinicopathological correlations in pancreatic cancers.

Objectives Despite advances in clinical management of pancreatic cancer (PC), there is still room for improvement in early detection, diagnosis, and treatment strategies. The role of microRNAs (miRNAs) in tumor biology might pinpoint an alteration in expression of miRNAs as new diagnostic/prognostic biomarkers. Methods Expression levels of miR-143 and miR-21 and correlations with clinicopathological features were analyzed in 26 matched pairs of tumor and adjacent noncancerous tissue samples collected from patients with PCs, including 18 pancreatic ductal adenocarcinomas (PDACs) and 8 adenocarcinomas of Vater’s papilla (PVACs). Results Compared to normal tissues, miR-143 was up-regulated in both PDAC and PVAC tumor samples (P = 0.0028 and P = 0.039, respectively). Conversely, alterations in miR-21 expression were significantly different in PDAC versus PVAC samples (P = 0.0049). Tumor levels of miR-21 were associated with preoperative serum levels of CA 19-9 (r = 0.63, P = 0.0022), whereas miR-143 expression was negatively correlated to lymph node spreading (r = −0.64; P = 0.0004). Correlation between miR-143 and miR-21 expression levels in patients with PDAC was observed (r = 0.53, P = 0.023). Conclusions Deregulation of miR-143 and miR-21 may reflect histological features and biological behavior of different PCs. Association data with clinical parameters might indicate a prognostic significance for miR-143 and miR-21 in PCs.

Evaluating the role of the genetic variations of PTPN22, NFKB1, and FcGRIIIA genes in inflammatory bowel disease: A meta-analysis

Background: We tested several polymorphisms of genes involved in the mucosal immune system in a population of inflammatory bowel disease (IBD) patients to investigate their possible implication in disease predisposition. Methods: Polymorphisms of 3 candidate genes (PTPN22, NFkB1, and FcGRIIIA) were investigated in 649 IBD patients (343 with Crohns disease [CD] and 306 with ulcerative colitis [UC]), 176 unaffected relatives, and 256 healthy controls. Allele and genotype frequencies were correlated with clinical characteristics and major variants of the CARD15 gene. Our findings were pooled in a meta‐analysis with the available studies in the literature. Results: No significant difference for the PTPN22 and NFkB1 variants was found. In contrast, allele and genotype frequencies of the G559T allele of the FcGRIIIA gene were significantly different in CD patients compared to controls (allele T 12% versus 8%, odds ratio [OR] = 1.58, 95% confidence interval [CI] 1.06–2.35; GT genotype 23% versus 16%, OR = 1.64, 95% CI = 1.08–2.5). However, no significant overtransmission of the T allele was confirmed at the family‐based analysis. For all genes, neither an interaction with CARD15 gene, nor a significant difference at genotype/phenotype analysis was demonstrated, included response to medical therapy. Conclusions: Although involved in autoimmune diseases, the PTPN22 and NFkB1 genes do not seem involved in the IBD predisposition, also according to meta‐analysis results. The association with the G559T polymorphism of the FcGRIIIA gene in CD patients deserves further investigation. (Inflamm Bowel Dis 2007)

High RAD51 mRNA expression characterize estrogen receptor‐positive/progesteron receptor‐negative breast cancer and is associated with patient's outcome

Mutations in DNA double‐strand breaks (DSB) repair genes are involved in the pathogenesis of hereditary mammary tumors, it is, however, still unclear whether defects in this pathway may play a role in sporadic breast cancer. In this study, we initially determined mRNA expression of 15 DSB related genes by reverse transcription quantitative polymerase chain reaction in paired normal tissue and cancer specimen from 20 breast cancer cases to classify them into homogeneous clusters. G22P1/ku70, ATR and RAD51 genes were differentially expressed in the three branches recognized by clustering analysis. In particular, a breast cancer subgroup characterized by high RAD51 mRNA levels and estrogen receptor (ER)‐positive/progesteron receptor (PR)‐negative phenotype was identified. This result was confirmed by the analysis of G22P1/ku70, ATR and RAD51 mRNA levels on paired normal and tumor specimens from an extended breast cancer cohort (n = 75). RAD51 mRNA levels were inversely associated with PR status (p = 0.02) and the highest levels were, indeed, detected in ER‐positive/PR‐negative tumors (p = 0.03). RAD51 immunostaining of a tissue microarray confirmed the inverse relationship between high RAD51 expression and negative PR status (p = 0.002), as well as, the association with ER‐positive/PR‐negative phenotype (p = 0.003). Interestingly, the analysis of microarray expression data from 295 breast cancers indicate that RAD51 increased mRNA expression is associated with higher risk of tumor relapse, distant metastases and worst overall survival (p = 0.015, p = 0.009 and p = 0.013 respectively). Our results suggest that RAD51 expression determination could contribute to a better molecular classification of mammary tumors and may represent a novel tool for evaluating postoperative adjuvant therapy for breast cancer patients.

Chemokine receptor CXCR4: Role in gastrointestinal cancer

Chemokines (CK)s, small proinflammatory chemoattractant cytokines that bind to specific G-protein coupled seven-span transmembrane receptors, are major regulators of cell trafficking and adhesion. The CXCL12 [stromal cell-derived factor-1 (SDF-1)] binds primarily to CXC receptor 4 (CXCR4; CD184). The binding of CXCL12 to CXCR4 induces intracellular signaling through several divergent pathways initiating signals related to chemotaxis, cell survival and/or proliferation, increase in intracellular calcium, and gene transcription. CXCR4 is expressed on multiple cell types including lymphocytes, hematopoietic stem cells, endothelial and epithelial cells, and cancer cells. One of the most intriguing and perhaps important roles that CKs and the CK receptors have is in regulating metastasis. Here, CK receptors may potentially facilitate tumor dissemination at each of the key steps of metastasis, including adherence of tumor cells to endothelium, extravasation from blood vessels, metastatic colonization, angiogenesis, proliferation, and protection from the host response via activation of key survival pathways such as ERK/MAPK, PI-3K/Akt/mTOR, or Jak/STAT, etc. In addition, it is increasingly recognized that CKs play an important role in facilitating communication between cancer cells and non-neoplatic cells in the tumor microenvironment (TME), including endothelial cells and fibroblasts, promoting the infiltration, activation of neutrophils, and tumor-associated macrophages within the TME. In this review, we mainly focus on the roles of chemokines CXCL12 and its cognate receptors CXCR4 as they pertain to cancer progression. In particular, we summarizes our current understanding regarding the contribution of CXCR4 and SDF-1 to gastrointestinal tumor behavior and its role in local progression, dissemination, and immune evasion of tumor cells. Also, describes recent therapeutic approaches that target these receptors or their ligands.

Association Study of a Polymorphism in Clock Gene PERIOD3 and Risk of Inflammatory Bowel Disease

Altered body rhythmicity and deregulated clock gene expression may cause circadian disruption, which can lead to immune dysregulation and chronic inflammatory diseases. PERIOD3 (PER3) polymorphisms have been associated with circadian disruption and changed secretion of cytokines involved in chronic inflammation. Crohns disease (CD) and ulcerative colitis (UC) are multifactorial diseases resulting from complex interaction among environmental/microbial factors and the intestinal immune system, triggering an abnormal immune response in genetically susceptible individuals. We evaluated the influence of a polymorphism of the clock gene PER3 on susceptibility and behavior of these inflammatory bowel diseases. The rs2797685 variant of the PER3 gene was assessed in 1082 CD and 972 UC patients, 754 of whom had been diagnosed <18 yrs of age, and 1311 unrelated healthy controls. Allele and genotype frequencies of rs2797685 were significantly increased in both CD (p = 1.6 × 10−4, odds ratio [OR] = 1.38, 95% confidence interval [CI]: 1.17–1.63) and UC (p = .012, OR = 1.25, 95% CI: 1.05–1.48) patients. Difference between frequency distributions remained statistically significant after stratifying the cohort according to age at diagnosis for CD, but not for UC. Statistically significant association was found between PER3 polymorphism and use of immunosuppressive drugs in pediatric CD patients (p < .001) and with stricturing and fistulizing disease behavior in adult CD patients (p = .031). In conclusion, results of this association study suggest a possible role of PER3 polymorphism in determining susceptibility to CD and UC and phenotypic characteristics of CD. In particular, the rs2797685 variant of the PER3 gene is associated with a more aggressive form of CD, highlighted by higher use of immunosuppressants and more frequent stricturing and fistulizing disease behaviors, as well as early onset of CD. This is a descriptive study, and functional data are needed to prove a causal relationship; nonetheless, involvement of the clock gene machinery in the susceptibility and the behavior of inflammatory bowel diseases may suggest new pathophysiological mechanisms and new therapeutic approaches. (Author correspondence: g.mazzoccoli@ operapadrepio.it)