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IntroductionBladder cancer (urothelial carcinoma) is a common malignancy characterized by high recurrence rates and intense clinical follow-up, indicating the necessity for more effective therapies. Current treatment regimens include intra-vesical administration of mitomycin C (MMC) for non-muscle invasive disease and systemic cisplatin for muscle-invasive or metastatic disease. Hyperthermia, heating a tumor to 40-44 degrees C, enhances the efficacy of these chemotherapeutics by various modes of action, one of which is inhibition of DNA repair via homologous recombination. Here, we explore whether ex vivo assays on freshly obtained bladder tumors can be applied to predict the response towards hyperthermia.Material and methodsThe cytochrome C release assay (apoptosis) and the RAD51 focus formation assay (DNA repair) were first established in the bladder cancer cell lines RT112 and T24 as measurements for hyperthermia efficiency, and subsequently tested in freshly obtained bladder tumors (n = 59).ResultsHyperthermia significantly increased the fraction of apoptotic cells after cisplatin or MMC treatment in both RT112 and T24 cells and in most of the bladder tumors (8/10). The RAD51 focus formation assay detected both morphological and numerical changes of RAD51 foci upon hyperthermia in the RT112 and T24 cell lines. In 64% of 37 analyzed primary bladder tumor samples, hyperthermia induced similar morphological changes in RAD51 foci.ConclusionThe cytochrome C assay and the RAD51 focus formation assay are both feasible on freshly obtained bladder tumors, and could serve to predict the efficacy of hyperthermia together with cytotoxic agents, such as MMC or cisplatin.

Cancer puts an increasing burden on our healthcare system and is a major cause of death. Therefore, novel approaches are required to improve cancer treatment. Cancer cells have several hallmarks that could be therapeutically targeted. Importantly, every tumor has a different combination of aberrations affecting the different hallmarks. This review focuses on targeting one of these hallmarks, the DNA damage response (DDR). DDR defects can not only cause cancer, but they can also be exploited therapeutically. This plays an important role even in ‘classical’ (DNA damaging) chemotherapy and radiotherapy, but more precise targeting of specific defects is expected to increase treatment efficacy and decrease normal tissue toxicity. Poly-(ADP-ribose) polymerase (PARP) inhibitors are the first clinical example of such synthetic lethality in tumors having specific DDR defects. They are currently under investigation as DDR-targeting anticancer drugs and they progress quickly in clinical trials.

Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors are promising targeted treatment options for hereditary breast tumors with a homologous recombination (HR) deficiency caused by BRCA1 or BRCA2 mutations. However, the functional consequence of BRCA gene mutations is not always known and tumors can be HR deficient for other reasons than BRCA gene mutations. Therefore, we aimed to develop a functional test to determine HR activity in tumor samples to facilitate selection of patients eligible for PARP inhibitor treatment. Experimental design: We obtained 54 fresh primary breast tumor samples from patients undergoing surgery. We determined their HR capacity by studying the formation of ionizing radiation induced foci (IRIF) of the HR protein RAD51 after ex vivo irradiation of these organotypic breast tumor samples. Tumors showing impaired RAD51 IRIF formation were subjected to genetic and epigenetic analysis. Results: Five of 45 primary breast tumors with sufficient numbers of proliferating tumor cells were RAD51 IRIF formation deficient (11%, 95% CI, 5%–24%). This HR defect was significantly associated with triple-negative breast cancer (OR, 57; 95% CI, 3.9–825; P = 0.003). Two of five HR-deficient tumors were not caused by mutations in the BRCA genes, but by BRCA1 promoter hypermethylation. Conclusion: The functional RAD51 IRIF assay faithfully identifies HR-deficient tumors and has clear advantages over gene sequencing. It is a relatively easy assay that can be performed on biopsy material, making it a powerful tool to select patients with an HR-deficient cancer for PARP inhibitor treatment in the clinic. Clin Cancer Res; 20(18); 4816–26. ©2014 AACR.

Bladder cancer has a high incidence with significant morbidity and mortality. Attenuated expression of the DNA damage response protein Xeroderma Pigmentosum complementation group C (XPC) has been described in bladder cancer. XPC plays an essential role as the main initiator and damage-detector in global genome nucleotide excision repair (NER) of UV-induced lesions, bulky DNA adducts and intrastrand crosslinks, such as those made by the chemotherapeutic agent Cisplatin. Hence, XPC protein might be an informative biomarker to guide personalized therapy strategies in a subset of bladder cancer cases. Therefore, we measured the XPC protein expression level and functional NER activity of 36 bladder tumors in a standardized manner. We optimized conditions for dissociation and in vitro culture of primary bladder cancer cells and confirmed attenuated XPC expression in approximately 40% of the tumors. However, NER activity was similar to co-cultured wild type cells in all but one of 36 bladder tumors. We conclude, that (i) functional NER deficiency is a relatively rare phenomenon in bladder cancer and (ii) XPC protein levels are not useful as biomarker for NER activity in these tumors.

Purpose. BRCA1 and BRCA2 related breast and ovarian tumors respond very well to treatment with PARP inhibitors (PARPi) because of their deficiency in homologous recombination (HR). We have indications that a substantial number of ovarian tumors might have deficiencies in HR unrelated to germline mutations in BRCA1 or BRCA2. A functional assay to assess HR efficacy might therefore allow identification of additional ovarian cancer patients that could benefit from PARPi treatment. Experimental procedure. Unselected fresh ovarian tumor samples were collected from patients undergoing surgery or ascites/pleural fluid drainage. Samples were irradiated with ionizing radiation ex vivo, fixed after 2 hours and embedded in paraffin. Sections were stained with hematoxylin and eosin and examined by pathologist. The presence of RAD51 foci in replicating cells as visualized by immunofluorescence was used as a functional read out for HR proficiency. Results. We determined the ability to induce RAD51 foci following radiation in ex vivo cultured ovarian tumor samples that contained sufficient numbers of replicating cells (n = 13). RAD51 foci formation was absent (A). Genetic and epigenetic analyses of the other samples are ongoing. Conclusion. The ex vivo analysis of RAD51 foci induction in fresh tumor tissue is a promising new tool to identify HR deficient tumors. Not only is it an important time saving strategy compared to BRCA1/BRCA2 mutation analysis, it may also identify a much broader patient population that will be eligible for the treatment with PARP inhibitors. Citation Format: Maaike Vreeswijk, M. Meijers, K.A.T. Naipal, N.S. Verkaik, R. Kanaar, J.H.J. Hoeijmakers, A. Jager, D.C. van Gent, K.N. Gaarenstroom, V.T.H.B.M. Smit, H. Vrieling. Ex vivo analysis of RAD51 foci induction in fresh tumor tissue: a promising new tool for the identification of homologous recombination-deficient tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3392. doi:10.1158/1538-7445.AM2015-3392

One of the hallmarks of cancer is genetic instability. Many chemotherapeutic strategies make use of DNA damaging agents, that preferentially target dividing cells. Recently, DNA repair defects have been identified in many different types of tumors, suggesting that the effectiveness of such treatments may be modulated by the precise genetic make-up of the tumor; defects in specific DNA repair pathways can be used as the Achilles' Heel of the tumor. An important new concept is synthetic lethality, the observation that combinations of mutations can be lethal, while both single mutations are viable. In selected tumors, the specific DNA repair defect can be targeted in such a way that the tumor cells can be killed without causing severe side effect on normal tissues. The combination of PARP inhibitors and homologous recombination (HR) deficient hereditary breast cancer is on its way to the clinic and more combinations are currently in the preclinical phase. Selection of breast cancer patients for targeted treatment with PARP inhibitors requires a validated test to identify HR deficient tumors. For this purpose, we have assessed ionizing radiation induced RAD51 foci (IRIF) after ex vivo irradiation of individual breast tumor slices as a marker for HR defects. We validated this approach in known HR- deficient tumors, where we found a severe defect in RAD51 IRIF formation. Subsequently, we used this assay to identify a subgroup of HR deficient primary breast cancers. Out of 42 tumors, we found five HR deficient breast cancers, caused by two distinct modes of BRCA gene inactivation, mutations and promoter hypermethylation. HR deficiency was significantly associated with high grade, triple-negative breast cancer (TNBC). A large fraction of TNBC showed HR defects, suggesting that PARP inhibitors may be particularly promising for this group of tumors that currently have a poor prognosis. We conclude that the RAD51 IRIF assay is a powerful tool to select patients with HR-deficient primary breast cancers eligible for PARP-inhibitor treatment in the clinic. These approaches may change cancer medicine in a fundamental way, from a one-size-fits-all concept to an individualized treatment strategy based on the functional molecular make-up of the tumor for each patient. Citation Format: Kishan A.T. Naipal, Nicole S. Verkaik, Petra ter Brugge, Najim Ameziane, Carolien H.M. van Deurzen, John W. Martens, Johan P. de Winter, Jos Jonkers, Maaike P. Vreeswijk, Agnes Jager, Jan Hoeijmakers, Roland Kanaar, Dik C. van Gent. Exploiting DNA repair defects in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2425. doi:10.1158/1538-7445.AM2014-2425