- Email: [email protected]
Breast cancer and Epstein–Barr virus infection
S38
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
nephropathy (AAN), often complicated by development of multiple urothelial carcinomas of sequential onset. We used deep sequencing of multiple urinary tract tumours of AAN cases from a unique, well-characterized women population in Belgium, to characterize the relationships of the patients’ lateonset second cancers to the exposure to AA as well as to the first cancers. Materials and Methods: Aristolactam-DNA adduct-positive AAN patients (n = 4) who developed cancer within 8 years following the initial exposure to AA were chosen for analysis of their first cancers (upper tract urothelial carcinomas, UTUC) and second cancers with delayed onset (1−9 years after first-cancer diagnosis, involving the bladder or ureteral meatus). One case developed a third malignancy in the bladder, 3 years after the second cancer. All patients had received a kidney transplant before developing the second cancers and had a functional renal graft prior to prophylactic nephroureterectomy. Genomic DNAs were isolated from FFPE sections of the renal cortex and from the upper and lower tract tumours of each patient using laser capture microdissection or macrodissection of the tumour areas. Lowcoverage (~15x) exome 100-bp paired-end sequencing was performed using Illumina HiSeq2500. Customized variant calling identified somatic variants absent in non-tumour tissues. Non-negative matrix factorisation was applied to extract exome-wide mutational signatures in the tumour tissues. Results and Discussion: In all cases, we established the mutational signature of AA (the COSMIC signature 22) in the first UTUC as well as second cancers involving the bladder or lower ureter (meatus). Additionally, the first and second cancers of all cases harboured considerable overlaps in exposure-specific (A>T) somatic mutations. This finding provides evidence that the delayed onset of bladder urothelial carcinomas in AAN patients is likely due to distal seeding of cancer cells originating from the primary UTUC tumours. Conclusions: Our first-of-its-kind study addresses the risk as well as mechanistic factors leading to the second, late-onset bladder urothelial carcinomas following kidney transplantation and primary UTUC development. Our results underline the importance of long-term bladder follow-up in high-risk populations with established or suspected AA exposure. Acknowledgments: Funding from IARC; CMMI is supported by the European Regional Development Fund and the Walloon Region. We thank Ms. C. Carreira and Dr Y. Song for expert assistance with histopathology and Dr S. Villar and O. Aminova for sample processing and sequencing. The NYU Genome Technology Center is partially supported by the NIH/NCI (P30CA016087). No conflict of interest. 210 Reactivation of thyroid hormone receptors in fully developed rat hepatocellular carcinomas causes their regression and prevents lung metastasis A. Perra1 , M.A. Kowalik1 , L. Cabras1 , E. Puliga1 , G.M. Ledda-Columbano1 , A. Columbano1 . 1 University of Cagliari, Department of Biomedical Sciences, Cagliari, Italy Introduction: Hepatocellular carcinoma (HCC) is the second cause of cancer related deaths Worldwide. To date, the only curative treatment available is surgery. Our previous data demonstrated that a short treatment with T3 causes a rapid regression of rat preneoplastic nodules, suggesting that T3 induces a differentiation program towards a normal phenotype. Moreover, repeated administrations of T3 to rats bearing preneoplastic nodules, resulted in reduction of HCC incidence by 50%. Based on these results we evaluated whether fully developed HCCs are still responsive to the anti-tumorigenic and pro-differentiating effects of T3. Materials and Methods: F-344 rats were subjected to Resistant-Hepatocyte rat model of hepatocarcinogenesis, considting of a single dose of the carcinogen diethylnitrosamine (DENA) and a 2 week exposure to 2-acetylaminofluorene coupled with partial hepatectomy (PH). Ten month after DENA all rats developed early HCC (eHCC) and were divided in 2 groups. Group 1 was fed a T3-supplemented diet a week every three weeks and rats were sacrificed after 1 or 5 cycles; group 2 was maintained on a basal diet and rats were sacrificed at the same time points as T3 treated animals. After sacrifice liver was either snap frozen or formalin fixed. Histopathological analysis and immunohistochemical staining for the prognostic marker KRT-19 were done. Frozen samples were cryosectioned and HCC were lasermicrodissected (LMD) after histo-pathological classification. Total RNA was extracted from LMD samples and used for gene expression analysis by qPCR. Results and Discussion: Results showed that eHCCs are still able to respond to T3, as shown by increased expression of the TRs target genes Dio1 and Spot14 after 1 week. Reactivation of the T3/TRs axis was associated with a significant reduction of the more aggressive KRT19+ HCCs. Fourteen months after initiation, while 100% of the rats not treated with T3 showed well- or poorly-differentiated HCCs, the liver of rats treated with 5 cycles of T3 did not show macroscopic tumors and, microscopic analysis confirmed the absence of HCCs, with only a few adenomas being visible. Moreover, T3 treatment totally abolished the occurrence lung metastases.
Conclusion: These data demonstrate a strog anti-tumorigenic effect of T3 and suggest that thyroid hormone may induce a reprogramming of neoplastic cells towards a more benign phenotype. The evidence of a strong anti-neoplastic effect associated to the T3/TRs axis activation in already developed HCC could lead to new therapeutic strategies aimed to reactivate TRs signaling. No conflict of interest. 211 Breast cancer and Epstein–Barr virus infection Y. Shlyakhtunou1 , A. Savchenko1 . 1 Vitebsk State Order of Peoples’ Friendship Medical University, oncology, Vitebsk, Belarus At the present time we do not stop looking for new predictors of tumor flow in breast cancer. Association of viruses and neoplastic processes in one body continues to interest and excite many of the scientific community worldwide. Objective: To investigate persistence of Epstein–Barr virus in the tumor tissue of breast carcinoma as a possible new prognostic factor in tumor progression. Materials and Methods: The study included all 178 tumor tissue samples of breast carcinomas. Of these, 40 tumor samples for retrospective analysis, and 138 samples for prospective study. Conducted research presence core antigen of Epstein–Barr virus (EBNA-1) in breast carcinoma tissue immunohistochemistry (IHC) method (n = 40 retrospectively, n = 70 prospectively) and (n = 68) by the polymerase chain reaction (PCR) you searched the DNA of the virus. Results of the study: It is found that in 53.5% of tumor tissue samples occurs presence of viral nuclear antigen confirmed by IHC and in 42.6% of samples − confirmed by PCR. Most often the presence of EBNA-1 and DNA of Epstein–Barr virus was determined in patients materials having metastasized to regional lymph (N +) components, as well as tumor-high and medium grade (G2-G3), having lymphovenous invasion (LVSI «+») as well as Her2-neooverexpressing cancers and high proliferative activity index (Ki-67 >50%). The risk of progression of tumor after completion of special treatment in terms of up to 21 months increased in 69.2–96.6% of women who have the persistence of Epstein–Barr virus in the tumor tissue. In 87.5% of patients with the presence of the viral genome (EBNA-1), confirmed by IHC method has a risk of dying from breast cancer within 5 years after diagnosis. Conclusion: Association of Epstein–Barr virus can be considered as a negative prognostic factor contributing to the progression of the tumor process, increasing the risk of death from breast cancer. So far, not established pathogenetic mechanism of influence of Epstein–Barr virus in the tumor tissue of breast carcinoma and the ability of the virus to influence the development of drug resistance to different groups of chemotherapeutic agents used to treat cancer of this localization. Further investigation of the mechanisms of tumor cells using the culture will optimize approaches to breast cancer treatment. No conflict of interest. 212 New approach to analyzing gene expression in neuroendocrine tumors M.V. Comanescu1 , M. Dobre2 , F. Vasilescu3 . 1 INCD Victor Babes, Pathology, Bucuresti, Romania, 2 INCD Victor Babes, Pathology, Bucharest, Romania, 3 Military Central Hospital, Pathology, Bucharest, Romania Neuroendocrine tumors (NETs) are epithelial neoplasms arising mainly from the gastrointestinal tract, pancreas and the bronchial tree that have different behavior, from indolent tumors to clinically aggressive, poorly differentiated carcinomas. The objective of the study was to investigate NETs in order to identify an alternative method for the immunohistochemical testing by performing a qRT-PCR reaction and to create a PCR array that will be used as a virtual device with direct application in molecular diagnosis, prognosis and therapy of NETs. Materials and Methods: We studied tumoral and peritumoral fragments fixed in formalin and embedded in paraffin from 26 patients diagnosed with NET (pulmonary, gastrointestinal and pancreatic) by histopathological and immunohistochemical techniques. In order to assess gene expression a PCRarray method was used (Qiagen) which identified relative expression of 18 genes of interest by comparing the tumour with the peritumoral tissue, the normalization being carried out by means of two reference genes. The quality control test was performed using primers for the control of genomic DNA contamination, reverse transcription control and control of the efficiency of amplification. Results: In cases of pulmonary NETs, VGF, MGMT and SSTR1 gene were strongly over-expressed, GAST, SYP, and CCND1 genes were moderately overexpressed while mTOR, INS, CHGA, MKI67, SSTR5 and SLIT2 genes had a low overexpression. In gastrointestinal NETs, CHGA and SSTR1 genes were moderately overexpressed, whileGRPR, mTOR, SSTR3 and SSTR5 genes had a low overexpression. sStr2 and M KI67 genes were downregulated. In pancreatic tumors, GAST, VGF and TYMS genes were moderately overexpressed, SSTR3 and SSTR1 genes had a low overexpression and MGMT and SLIT2 genes were downregulated. We identified different gene profiles for the three anatomical locations of NET studied. Somatostatin receptor (SSTR) overexpression represents a predictive marker for the therapy
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
nephropathy (AAN), often complicated by development of multiple urothelial carcinomas of sequential onset. We used deep sequencing of multiple urinary tract tumours of AAN cases from a unique, well-characterized women population in Belgium, to characterize the relationships of the patients’ lateonset second cancers to the exposure to AA as well as to the first cancers. Materials and Methods: Aristolactam-DNA adduct-positive AAN patients (n = 4) who developed cancer within 8 years following the initial exposure to AA were chosen for analysis of their first cancers (upper tract urothelial carcinomas, UTUC) and second cancers with delayed onset (1−9 years after first-cancer diagnosis, involving the bladder or ureteral meatus). One case developed a third malignancy in the bladder, 3 years after the second cancer. All patients had received a kidney transplant before developing the second cancers and had a functional renal graft prior to prophylactic nephroureterectomy. Genomic DNAs were isolated from FFPE sections of the renal cortex and from the upper and lower tract tumours of each patient using laser capture microdissection or macrodissection of the tumour areas. Lowcoverage (~15x) exome 100-bp paired-end sequencing was performed using Illumina HiSeq2500. Customized variant calling identified somatic variants absent in non-tumour tissues. Non-negative matrix factorisation was applied to extract exome-wide mutational signatures in the tumour tissues. Results and Discussion: In all cases, we established the mutational signature of AA (the COSMIC signature 22) in the first UTUC as well as second cancers involving the bladder or lower ureter (meatus). Additionally, the first and second cancers of all cases harboured considerable overlaps in exposure-specific (A>T) somatic mutations. This finding provides evidence that the delayed onset of bladder urothelial carcinomas in AAN patients is likely due to distal seeding of cancer cells originating from the primary UTUC tumours. Conclusions: Our first-of-its-kind study addresses the risk as well as mechanistic factors leading to the second, late-onset bladder urothelial carcinomas following kidney transplantation and primary UTUC development. Our results underline the importance of long-term bladder follow-up in high-risk populations with established or suspected AA exposure. Acknowledgments: Funding from IARC; CMMI is supported by the European Regional Development Fund and the Walloon Region. We thank Ms. C. Carreira and Dr Y. Song for expert assistance with histopathology and Dr S. Villar and O. Aminova for sample processing and sequencing. The NYU Genome Technology Center is partially supported by the NIH/NCI (P30CA016087). No conflict of interest. 210 Reactivation of thyroid hormone receptors in fully developed rat hepatocellular carcinomas causes their regression and prevents lung metastasis A. Perra1 , M.A. Kowalik1 , L. Cabras1 , E. Puliga1 , G.M. Ledda-Columbano1 , A. Columbano1 . 1 University of Cagliari, Department of Biomedical Sciences, Cagliari, Italy Introduction: Hepatocellular carcinoma (HCC) is the second cause of cancer related deaths Worldwide. To date, the only curative treatment available is surgery. Our previous data demonstrated that a short treatment with T3 causes a rapid regression of rat preneoplastic nodules, suggesting that T3 induces a differentiation program towards a normal phenotype. Moreover, repeated administrations of T3 to rats bearing preneoplastic nodules, resulted in reduction of HCC incidence by 50%. Based on these results we evaluated whether fully developed HCCs are still responsive to the anti-tumorigenic and pro-differentiating effects of T3. Materials and Methods: F-344 rats were subjected to Resistant-Hepatocyte rat model of hepatocarcinogenesis, considting of a single dose of the carcinogen diethylnitrosamine (DENA) and a 2 week exposure to 2-acetylaminofluorene coupled with partial hepatectomy (PH). Ten month after DENA all rats developed early HCC (eHCC) and were divided in 2 groups. Group 1 was fed a T3-supplemented diet a week every three weeks and rats were sacrificed after 1 or 5 cycles; group 2 was maintained on a basal diet and rats were sacrificed at the same time points as T3 treated animals. After sacrifice liver was either snap frozen or formalin fixed. Histopathological analysis and immunohistochemical staining for the prognostic marker KRT-19 were done. Frozen samples were cryosectioned and HCC were lasermicrodissected (LMD) after histo-pathological classification. Total RNA was extracted from LMD samples and used for gene expression analysis by qPCR. Results and Discussion: Results showed that eHCCs are still able to respond to T3, as shown by increased expression of the TRs target genes Dio1 and Spot14 after 1 week. Reactivation of the T3/TRs axis was associated with a significant reduction of the more aggressive KRT19+ HCCs. Fourteen months after initiation, while 100% of the rats not treated with T3 showed well- or poorly-differentiated HCCs, the liver of rats treated with 5 cycles of T3 did not show macroscopic tumors and, microscopic analysis confirmed the absence of HCCs, with only a few adenomas being visible. Moreover, T3 treatment totally abolished the occurrence lung metastases.
Conclusion: These data demonstrate a strog anti-tumorigenic effect of T3 and suggest that thyroid hormone may induce a reprogramming of neoplastic cells towards a more benign phenotype. The evidence of a strong anti-neoplastic effect associated to the T3/TRs axis activation in already developed HCC could lead to new therapeutic strategies aimed to reactivate TRs signaling. No conflict of interest. 211 Breast cancer and Epstein–Barr virus infection Y. Shlyakhtunou1 , A. Savchenko1 . 1 Vitebsk State Order of Peoples’ Friendship Medical University, oncology, Vitebsk, Belarus At the present time we do not stop looking for new predictors of tumor flow in breast cancer. Association of viruses and neoplastic processes in one body continues to interest and excite many of the scientific community worldwide. Objective: To investigate persistence of Epstein–Barr virus in the tumor tissue of breast carcinoma as a possible new prognostic factor in tumor progression. Materials and Methods: The study included all 178 tumor tissue samples of breast carcinomas. Of these, 40 tumor samples for retrospective analysis, and 138 samples for prospective study. Conducted research presence core antigen of Epstein–Barr virus (EBNA-1) in breast carcinoma tissue immunohistochemistry (IHC) method (n = 40 retrospectively, n = 70 prospectively) and (n = 68) by the polymerase chain reaction (PCR) you searched the DNA of the virus. Results of the study: It is found that in 53.5% of tumor tissue samples occurs presence of viral nuclear antigen confirmed by IHC and in 42.6% of samples − confirmed by PCR. Most often the presence of EBNA-1 and DNA of Epstein–Barr virus was determined in patients materials having metastasized to regional lymph (N +) components, as well as tumor-high and medium grade (G2-G3), having lymphovenous invasion (LVSI «+») as well as Her2-neooverexpressing cancers and high proliferative activity index (Ki-67 >50%). The risk of progression of tumor after completion of special treatment in terms of up to 21 months increased in 69.2–96.6% of women who have the persistence of Epstein–Barr virus in the tumor tissue. In 87.5% of patients with the presence of the viral genome (EBNA-1), confirmed by IHC method has a risk of dying from breast cancer within 5 years after diagnosis. Conclusion: Association of Epstein–Barr virus can be considered as a negative prognostic factor contributing to the progression of the tumor process, increasing the risk of death from breast cancer. So far, not established pathogenetic mechanism of influence of Epstein–Barr virus in the tumor tissue of breast carcinoma and the ability of the virus to influence the development of drug resistance to different groups of chemotherapeutic agents used to treat cancer of this localization. Further investigation of the mechanisms of tumor cells using the culture will optimize approaches to breast cancer treatment. No conflict of interest. 212 New approach to analyzing gene expression in neuroendocrine tumors M.V. Comanescu1 , M. Dobre2 , F. Vasilescu3 . 1 INCD Victor Babes, Pathology, Bucuresti, Romania, 2 INCD Victor Babes, Pathology, Bucharest, Romania, 3 Military Central Hospital, Pathology, Bucharest, Romania Neuroendocrine tumors (NETs) are epithelial neoplasms arising mainly from the gastrointestinal tract, pancreas and the bronchial tree that have different behavior, from indolent tumors to clinically aggressive, poorly differentiated carcinomas. The objective of the study was to investigate NETs in order to identify an alternative method for the immunohistochemical testing by performing a qRT-PCR reaction and to create a PCR array that will be used as a virtual device with direct application in molecular diagnosis, prognosis and therapy of NETs. Materials and Methods: We studied tumoral and peritumoral fragments fixed in formalin and embedded in paraffin from 26 patients diagnosed with NET (pulmonary, gastrointestinal and pancreatic) by histopathological and immunohistochemical techniques. In order to assess gene expression a PCRarray method was used (Qiagen) which identified relative expression of 18 genes of interest by comparing the tumour with the peritumoral tissue, the normalization being carried out by means of two reference genes. The quality control test was performed using primers for the control of genomic DNA contamination, reverse transcription control and control of the efficiency of amplification. Results: In cases of pulmonary NETs, VGF, MGMT and SSTR1 gene were strongly over-expressed, GAST, SYP, and CCND1 genes were moderately overexpressed while mTOR, INS, CHGA, MKI67, SSTR5 and SLIT2 genes had a low overexpression. In gastrointestinal NETs, CHGA and SSTR1 genes were moderately overexpressed, whileGRPR, mTOR, SSTR3 and SSTR5 genes had a low overexpression. sStr2 and M KI67 genes were downregulated. In pancreatic tumors, GAST, VGF and TYMS genes were moderately overexpressed, SSTR3 and SSTR1 genes had a low overexpression and MGMT and SLIT2 genes were downregulated. We identified different gene profiles for the three anatomical locations of NET studied. Somatostatin receptor (SSTR) overexpression represents a predictive marker for the therapy