banda transcan 2 2016ul

Title: Clinical utility of tumour heterogeneitiy in triple negative breast cancer and high-grade serous ovarian carcinoma for prediction of therapy response

Project Coordinator:
Manfred DIETEL (Germany) Institute of Pathology, Charité University Hospital Berlin

Project Partners:
Violeta SERRA (Spain) Vall d' Hebron Institute of Oncology, Experimental Therapeutics Group, Barcelona
Giuseppe VIALE (Italy) European Institute of Oncology, Department of Pathology, Milan
Alexandra LEARY (France) Institut Gustave Roussy Gynecology Unit, Translational Research Laboratory INSERM U981, Villejuif

Project Abstract:
Triple negative breast cancer (TNBC) and ovarian high grade serous carcinoma (HGSOC) are two poor-prognosis tumour subtypes with many molecular similarities. Recurrence and therapy resistance are related to the selection of resistant sub-clones during disease progression. Therefore, tumour heterogeneity (TH) is a major underlying mechanism for the dismal disease course of TNBC and HGSOC.
The TH4-RESPONSE project is based on the hypotheses that TH is a major mechanism of recurrence and therapy resistance of HGSOC and TNBC, and that mechanisms of TH will become evident during treatment, as part of an evolutionary adaptation of tumour cells. The impact of TH on patient outcome can be defined in large and well-characterized patient cohorts, preferably from clinical trials.
Aims of our project are (1) to define molecular features of TH as drivers of chemotherapy resistance in TNBC and HGSOC, (2) to profile TH and tumour evolution in tissue samples and liquid biopsies of ovarian cancer, (3) to assess the clinical utility of differences in molecular alterations in breast cancer between primary tumour and metastases, to (4) identify resistance mechanisms against targeted therapy in PDX models, and (5) to integrate the results for further validation including prospective validation studies as well as development of concepts for future validation trials.
This multidisciplinary project including pre-clinical, clinical, pathological, and bioinformatics expertise will assess the various aspects of tumour heterogeneity of TNBC and HGSOC. In a discovery/validation approach we will define molecular biomarkers of TH in clinical samples from primary, recurrent, and metastatic TNBC and HGSOC using multi-omics techniques. Pre-clinical investigation of PDX models will render biomarkers of PARP inhibitor resistance. Based on large biobanking resources including sample cohorts from multicenter clinical trials as well as in prospective studies, the defined biomarkers will be validated the biomarkers in terms of their clinical utility.
TH4-RESPONSE will lead to integration of results into new biomarker assays to assess and monitor tumoural heterogeneity, which will be translated into the routine diagnostic workup of cancer patients. Identification of actionable oncogenic drivers will help to design novel therapeutic strategies, and new clinical trial concepts will result in diagnostic and therapeutic options, transferrable to the clinic, with a high level of evidence. Identification of clinically relevant features of tumour heterogeneity will help to understand the biology of the two most aggressive tumour types in women. In the long run, the results of the TH4-RESPONSE project will contribute to reduce mortality from breast and ovarian cancer.

Final Summary:
Aims: Triple negative breast cancer (TNBC) and high-grade serous ovarian carcinoma (HGSOC) share many molecular and morphological similarities. The aim of this project was to analyse both tumour entities according to molecular and proteomic heterogeneity focusing on primary and recurrent tumor samples and the clinical impact.
Methods: In order to analyse this question 128 breast cancer samples were analysed by large multi-gene NGS panels detecting different types of genetic alterations such as single nucleotide variants (SNVs), insertion/deletions (InDels), copy number variants (CNVs), and fusion genes. HGSOC samples were analyzed by HTG Edge Seq platform with the Oncology Biomarker Panel that captures the expression of 2500 transcripts with Ion Torrent S5 semiconductor sequencing. Furthermore HGSOC were evaluated according to genomic instability by SNParray (N=80) or CGH (N=60) on paired samples focusing on DNA repair genes. PDX models of ovarian and breast cancer patients were established and RAD51 was evaluated both on patient samples and PDX models to discover mechanisms of PARPi resistance.
Results: The molecular analysis of the breast cancer samples showed molecular subtype changes between primary tumors and relapses in 10 of 128 (7.8%) cases. Most driver genomic alterations (55.8%) were shared between primary tumors and matched recurrences. However, in 39 of 61 cases (63.9%), additional private alterations were detected in the relapse samples only, including 12 patients with potentially actionable aberrations. The HTG analysis of the ovarian tumors resulted in 233 DEGs (adj.p ≤0,05, log2FC>0,8) after evaluation of the RNA. After further analysis of publicly available datasets 41 genes remained significant and out of these eight markers (HMGCS2, FABP4, AHRR, GREM1, ITGA5, COL5A2, WNT9B and SFRP2) were chosen for IHC validation. For three markers, differential expression within the tumor compartment could be validated on the protein level. The expression of AHRR was higher in the primary tumors compared to the recurrences (p<0,001, n=49). ITGA5 (p=0,048, n=51) and GREM1 (Z = -4,124, p<0,001, n=51) were found to be expressed higher in the recurrent tumors.
The genomic instability analysis showed that at baseline a significant proportion of tumors lacked expression in DDR biomarkers such as RAD51, PAR or FANCD2, however marked changes were noted after platinum NACT. Post-NACT re-expression of DDR markers was associated with poor outcome especially for tumors co-expressing RAD51 and PARP or FANCD2.
The search for PARPi resistance via PDX models showed a frame-restoring secondary mutation in BRCA2 in PDX405OR, an olaparib-resistant counterpart from PDX405. None of the BRCA1/2 mutant models harbored alterations in 53BP1. All PARPi-resistant models exhibited high RAD51 score, and most of them produced BRCA1 hypomorphs. Overall, 14 out of 18 BRCA1-mutant PDX models (78%) harbored BRCA1 and RAD51 foci, and were resistant to PARPi. Out of the 4 BRCA1 foci-deficient PDXs, three were PARPi resistant and one was PARPi sensitive.
Conclusion: A rather stable molecular situation between primary and recurrent breast cancer might be stated, but private actionable alterations in recurrence exist. The results of the ovarian cancers point to a relevance of proteins engaged in the processes of extracellular matrix organization (GREM1; ITGA5) and metabolic pathways (AHRR). The combination of only few but specific newly discovered signaling and survival pathways for recurrence in high-grade serous ovarian carcinoma and breast cancer make these purposeful targets for therapy and further research about these pathways very fascinating. Furthermore homolougos recombination proved to be essentially involved in ovarian tumor relapse and resistance mechanisms to PARPi therapy could be detected with PDX models and RAD51 analysis. In passing 12 papers have been published from this consortium in the TH4Respons project and an ERA PerMed grant has been obtained for the continuation of the RAD51 clinical validations (ERAPERMED2019-215).


(Project funded under JTC 2014)


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