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PO-468 LAMININ332 (α3; ß3;γ2) genes and protein expression in cervical carcinomas

Introduction Laminin332 (α3; ß3; γ2 chains) has been identified as an important macromolecule in cancer invasion and metastasis, influencing cell differentiation, migration, adhesion, cell proliferation and survival. The α3, β3, and γ2 chains of LAM-332 are encoded by three distinct genes, LAMA3, LAMB3, and LAMC2, respectively. LAMC2 is associated to the invasive and metastatic abilities of several tumour types such as colon and pancreas. However, the molecular role of LAMC2 in cervical carcinomas has not yet been fully elucidated. We investigated the functional significance of LAMC2 in cervical carcinomas: squamous cell carcinomas (SCC) and adenocarcinoma (ADC) as well as its role in SCC of the cervix. Material and methods SiHa (SCC) and HeLa (ADC) cell lines were used. The expression of LAMA3, LAMB3, LAMC2 and EGFR was evaluated by qPCR in the presence/absence of EGF. Lamγ2 levels were evaluated by immunofluorescense. Lamγ2 role in migration and invasion was evaluated by wound healing and trans-well assays in control, EGF stimulated and LAMC2 silenced cells (shRNA). Immunohistochemical study evaluated lamα3, β3, and γ2 chains and EGFR, in formalin-fixed, paraffin-embedded in 122 cases, both in situ (n=15) and invasive cervical carcinomas: SCC (n=106) and ADC (n=41). Fisher’s exact test was performed for statistical analysis. Results and discussions EGF stimulates the migration and invasion in SiHa cells. However knocking-down Lamγ2 decreases cell migration and invasion capacities in SiHa cells. Also, the knockdown of LAMC2 lead to an increased transcription of LAMA3 and LAMB3 genes only in SiHa. The immunohistochemical study demonstrated that lamγ2 chain in SCC cases was significantly associated with invasion (p=0.0001) and with that histological type (p=0.0183). In SCC cases, there was also a significant association of lamß3 and γ2 chains overexpression (p=0.008). Lamα3 was associate with ADC type (p<0.0001). EGFR immuno-overexpression was also associated with SCC (p<0.0001). Conclusion Laminin332 and EGFR are differently expressed in the two most common histological types of cervical carcinomas (SCC and ADC). Lamγ2 expression is associated with SCC in both in vitro and clinical context and Lamg2 -dependent invasion is probably mediated by EGFR dependent signalling pathways. Together, results demonstrate a role of extracellular matrix proteins in the progression of cervix carcinoma. Additionally, Siha and HeLa can be an effective in-vitro models for an overview of the processes involved in cervical cancer progression.

Introduction Lynch syndrome (LS) is an autosomal dominantly inherited form of colorectal cancer (CRC) and is implicated in 2%-4% of CRC cases. LS develops from a mutation in one allele of one of the DNA mismatch repair (MMR) genes, most commonly MLH1 and MSH2, or less frequently MSH6 and PMS2. Loss of functional MMR proteins leads to defects in DNA repair and, subsequently, high DNA microsatellite instability (MSI-High). LS diagnostics currently consists of an analysis of MMR protein expression by means of Immunohistochemistry (IHC) and a molecular analysis to detect MSI. During our 10 year experience in performing LS diagnostics we have encountered few cases where IHC and MSI analyses do not match. This study is to clarify these inconsistent molecular alterations which will contribute to our understanding of LS diagnostics. Material and methods Of 2335 LS cases 27 (1.1%) showed discrepant IHC and MSI results. To clarify this, IHC and MSI analyses were repeated if possible with different antibodies and different MSI markers (mononucleotide instead of dinucleotide markers). Using MS-MLPA MLH1 hypermethylation, BRAF V600E mutation status and LOH of the MMR genes was analysed. Finally, germline and somatic mutations in MMR genes were analysed by using NGS. Protein modelling was also performed to visualise structural protein changes. Results and discussions Among these 27 cases, due to the decline of patients for further testing, 4 cases were excluded from further analyses. Of the remaining cases in 2 cases using a different antibody explained the unexpected results and in 2 cases switching from dinucleotide to mononucleotide MSI markers. In respectively 8 and 4 cases somatic and germline mutations explained the IHC and MSI results. Finally, in 2 cases protein modelling of identified mutations explained the presence of protein staining in MSI-high cases. The rest cases cannot be further studied due to the lack of tissues. Conclusion Based on these results, we have concluded: 1. Mononucleotide markers are more sensitive to detect microsatellite instability. 2. Protein modelling can explain presence of protein staining for mutations that do not cause a major conformational change. 3. Somatic sequencing is a valuable addition to Lynch syndrome diagnostics. Introduction Laminin332 (a3; ß3; g2 chains) has been identified as an important macromolecule in cancer invasion and metastasis, influencing cell differentiation, migration, adhesion, cell proliferation and survival.

PO-468
The a3, b3, and g2 chains of LAM-332 are encoded by three distinct genes, LAMA3, LAMB3, and LAMC2, respectively. LAMC2 is associated to the invasive and metastatic abilities of several tumour types such as colon and pancreas. However, the molecular role of LAMC2 in cervical carcinomas has not yet been fully elucidated.
We investigated the functional significance of LAMC2 in cervical carcinomas: squamous cell carcinomas (SCC) and adenocarcinoma (ADC) as well as its role in SCC of the cervix. Material and methods SiHa (SCC) and HeLa (ADC) cell lines were used. The expression of LAMA3, LAMB3, LAMC2 and EGFR was evaluated by qPCR in the presence/absence of EGF. Lamg2 levels were evaluated by immunofluorescense. Lamg2 role in migration and invasion was evaluated by wound healing and trans-well assays in control, EGF stimulated and LAMC2 silenced cells (shRNA). Immunohistochemical study evaluated lama3, b3, and g2 chains and EGFR, in formalin-fixed, paraffin-embedded in 122 cases, both in situ (n=15) and invasive cervical carcinomas: SCC (n=106) and ADC (n=41). Fisher's exact test was performed for statistical analysis. Results and discussions EGF stimulates the migration and invasion in SiHa cells. However knocking-down Lamg2 decreases cell migration and invasion capacities in SiHa cells. Also, the knockdown of LAMC2 lead to an increased transcription of LAMA3 and LAMB3 genes only in SiHa. The immunohistochemical study demonstrated that lamg2 chain in SCC cases was significantly associated with invasion (p=0.0001) and with that histological type (p=0.0183). In SCC cases, there was also a significant association of lamß3 and g2 chains overexpression (p=0.008). Lama3 was associate with ADC type (p<0.0001). EGFR immuno-overexpression was also associated with SCC (p<0.0001). Conclusion Laminin332 and EGFR are differently expressed in the two most common histological types of cervical carcinomas (SCC and ADC). Lamg2 expression is associated with SCC in both in vitro and clinical context and Lamg2 -dependent invasion is probably mediated by EGFR dependent signalling pathways.
Together, results demonstrate a role of extracellular matrix proteins in the progression of cervix carcinoma. Additionally, Siha and HeLa can be an effective in-vitro models for an overview of the processes involved in cervical cancer progression. Introduction Melanoma is an aggressive disease, curable by surgical resection if caught at an early stage (>95% 5 year survival) but with a poor prognostic outcome when diagnosed at advanced metastatic stage (<5% 5 year survival). Therefore, there is an urgent need for biomarkers of early diagnosis. In this respect, liquid biopsies hold great promise as they are fast, non-invasive and allow repeated sampling and monitorisation of patient response. Circular RNAs (circRNAs) are covalently closed RNA molecules that much more stable than linear RNAs to the high level of RNase activity in blood, and therefore have great potential as circulating biomarkers for cancer patient management. We therefore set out to identify and validate potential biomarker circRNAs from the plasma of melanoma patients' representing differing stages of the disease, along with healthy controls. Material and methods Next generation sequencing (RNAseq) was performed on pools of plasma from melanoma patients with stages 0, I/II, III or IV disease, as well as healthy individuals. Back-spliced junction reads were mapped using a combination of TopHat and TopHat-Fusion, and differential expression analysis carried out with the DEseq algorithm. Three circRNA (CR-3320,-2465 and À1445) were selected for validation in 106 plasma samples from 84 melanoma patients and 22 healthy controls by qRT-PCR. Results and discussions We obtained 20-30 million reads by RNAseq and identified between 120-200 circRNA species (after QC) in each sample. Of these 37 were differentially (p<0.05) expressed. Of the three circRNAs measured in an independent validation cohort, all were expressed at higher levels in melanoma patient samples than in controls. The highest levels were observed in stage 0 and I patients, decreasing in stage II and stage III/IV patients, probably due to treatment differences in the cohorts. The AUC values of CR-3320 and CR-2465 were 0.72 and 0.66 with a sensitivity of 53.33% and 30.77% and a specificity of 82.53% and 100%, respectively.
Conclusion This study suggests that circRNAs could represent a novel source of biomarkers for liquid biopsies for melanoma, and in all probability other cancers. Introduction Regorafenib demonstrated efficacy in pre-treated metastatic colorectal cancer (mCRC) patients. Limited clinical benefit in unselected patient populations highlights the unmet need for better patient selection and identification of mechanisms of action. MicroRNAs (miRs) are small non-coding RNAs involved in cell homeostasis, carcinogenesis and control multiple oncogenic pathways. Numerous miRs deregulation in mCRC are associated with clinical outcome and cancer progression. Material and methods We ran a translational phase II trial of regorafenib in chemo-refractory mCRC patients with biopsiable metastases. Tissue biopsies were obtained at baseline (BL), after 2 months of treatment, and at disease progression (PD). Patient Derived Organoids (PDOs) and PDO-xenotransplants were generated to study primary and acquired resistance to regorafenib. MiR profiling was performed in BL serum of all patients by NanoString nCounter platform and validated with digital droplet (dd)PCR in serum, plasma and exosomes and by In Situ Hybridization (ISH) in matching tissue biopsies. Fisher's exact test investigated potential associations between patient groups and categorical variables whilst t-test or nonparametric equivalent tests were used for continuous variables. Progression Free Survival (PFS) was measured from date of registration to date of first progression/relapse or death from cancer progression. Overall Survival (OS) was measured from date of randomisation to death from cancer. The Kaplan-Meier method summarised the survival estimates while the Cox proportional hazards model used to compare the survival rates between patient groups with and without adjustment for the effect of covariates. Results and discussions MiR expression was tested in 43 BL sera. Dysregulation in 28 miRs was associated with PFS and/ or OS. Among these miRs, up-regulation of miR-652-3 p and down-regulation of miR-3614-3 p was associated with worse PFS and OS. Results were validated by ddPCR on the same serum samples, and matching plasmas. ISH confirmed dysregulation of two miRs in sequential tissues biopsies, PDOs and PDO-xenotransplants of patients with primary and acquired resistance. Validation in an independent patient's cohort (n=70) is ongoing. Functional experiments to define miRmediated resistance are ongoing. Conclusion Circulating miR-652-3 p and miR-3614-3 p might be exploited as biomarkers for the upfront selection of patients' candidate to regorafenib treatment and might be used to track and forecast acquired resistance to treatment.