Genomic profiling in ovarian cancer retreated with platinum based chemotherapy presented homologous recombination deficiency and copy number imbalances of CCNE1 and RB1 genes

Ovarian cancer, the most lethal gynecologic cancer, is expected to account for 14,700 deaths in the USA in 2018 [1]. High grade serous carcinoma (HGSC), the most frequent histological type [2], is molecularly characterized by a few recurrent mutations, including in TP53 (almost all tumors) and BRCA1 or BRC2 genes. HGSCs present high genomic instability with copy number alterations (CNA) affecting a large fraction of the genome [3]. Approximately 50% of these tumors are characterized by homologous recombination (HR) deficiency, which has been associated with BRCA1 or BRCA2 germline or somatic mutations (20 and 5% of cases, respectively), BRCA1 promoter methylation (10% of cases), additional mutations in HR repair pathway genes and CNA in their regulators (PTEN and EMSY) [3].

Patients carrying tumors with BRCA mutation tend to have elevated sensitivity to platinum-based chemotherapy [4] and PARP inhibitors [5‐8]. These cases have shown a better medium term prognosis [9] even if the cure rate and long term prognosis is unaltered [10]. Secondary mutations have been associated with resistance to PARP inhibitors [11, 12].

Two strategies have been used to identify tumors with HR deficiency or alterations in genes involved in the DNA repair system other than BRCA1 and BRCA2 mutations. The first approach is to identify mutations in genes related to HR pathway [13], based on next generation sequencing (NGS), which has become feasible with the fast development of sequencing technologies. The second is the identification of “genomic scars”, which are supposed to be a functional consequence of HR deficiency independently of its cause [14‐17]. Clinical trials revealed that even patients with no HR deficiency, evaluated by two different HR deficiency scores, can achieve response to PARP inhibitors [8, 13]. These outcomes could be explained by other mechanisms of action than synthetic lethality of the HR pathway deficiency or failure in identifying the HR defect, or both.

In addition to the genomic scars of HR deficiency, gains or losses involving specific genes have also been associated with response to therapy in ovarian cancer [18]. Cyclin E1 and RB1 are cell-cycle proteins associated with the G1-S phase cell-cycle transition. CCNE1 copy number gain is described in about 20% of HGSC and is seemingly rare in BRCA mutated tumors [3]. Tumors with CCNE1 copy number gains are more resistant to platinum therapy [19] while RB1 loss are associated with high sensitivity to platinum therapy [20, 21].

Platinum resistant disease has a low chance to be responsive to platinum-based chemotherapy. The standard treatment is monotherapy using different drugs than platinum salts [2]. In daily clinical practice and despite of the resistant profile, a set of patients is retreated with platinum therapy and some of them are responsive to platinum retreatment [22].

In this study, we sought to evaluate the association of HR pathway mutations, HR deficiency scores and CCNE1 and RB1 CNA with response to platinum retreatment in ovarian cancer patients in the platinum-resistant setting.

A total of 31 patients with platinum resistant ovarian cancer was retreated with platinum therapy during a period of 10 years (2005 to 2014) at AC Camargo Cancer Center, SP, Brazil. High grade serous ovarian cancer was found in 68% of all cases, half of the patients received four or more prior treatment lines and 61.3% of the cases presented primary platinum resistant disease. All patients except three, were treated with taxane or gemcitabine in combination with platinum. Table 1 summarizes the clinical and pathological features of these 31 patients.

In this study, we evaluated markers of response to platinum retreatment in a selected group of patients with heavily pretreated platinum resistant ovarian cancer. The rationale was to identify patients that are still sensitive even after a platinum free interval shorter than 6 months and numerous previous treatment lines.

Nine of 15 cases (60%) of our cohort presented HR deficiency, which is in accordance with the 50% described in the TCGA dataset [3]. Patients with CS score higher than 42 had an ORR of 55.6% versus 33.3% observed in those with lower scores. In addition, the HRD median values of each score were higher among responders than in non-responders. However, the differences were not statistically significant probably due to the small number of cases evaluated.

Two scores based on the patterns of CNA and LOH were used in the phase III clinical trials of PARP inhibitors showing their ability to identify patients with BRCA mutation [7, 8, 13]. These studies also described a second group of patients negative for BRCA mutations but with high sensitivity to PARP inhibitors. Our findings give additional evidence that the scores are able to identify patients with high sensitivity to platinum agents. The absence of statistical significance in our study may be due to the small number of cases or the accuracy of these scores. Interestingly, the BRCA non-mutated cases and those with low HRD (based on the scores) benefited from treatment with PARP inhibitors [7, 8, 13]. The definition of all scores, except tAI [14], were based on the BRCA mutation as a gold standard to define HRD [15‐17]. Therefore, the CNA and LOH pattern calculated by the scores are similar to the ones promoted by BRCA mutations, which may be true for most but not necessarily all causes of the HRD.

Cyclin E1 overexpression promotes cell cycle progression abrogating DNA repair during the G1 phase. In addition, CCNE1 amplification has been associated with an apparent synthetic lethality in cases with HR deficiency [28]. Nine of our 15 tumors (60%) showed CCNE1 copy number gains while data from TCGA (509 high grade serous ovarian carcinomas) presented CCNE1 gains in 32.4% or focal amplification in 20.8% [3]. We observed that cases showing CCNE1 gains had lower ORR, and shorter PFS and OS compared with those not presenting gains. Previous studies in ovarian cancer patients described increased expression levels of cyclin E1 and an association with worse survival [29]. At the genomic level, two studies described high frequency of CCNE1 gains in patients with primary platinum resistant disease [19, 30]. The worse survival observed in the TCGA cohort for patients with CCNE1 amplification was attributed to its negative association with BRCA mutations [3]. In our study, two of four patients harboring BRCA mutations also presented CCNE1 gains. This finding suggests that the correlation between CCNE1 gain and outcome is not exclusively due to its negative association with BRCA mutations. Ten of our 15 patients had primary platinum resistant disease and 7 of 9 patients with CCNE1 gains presented primary resistant disease. This finding supports the association of CCNE1 aberrations and resistance to platinum therapy and may explain the higher than expected frequency of CCNE1 gains in our study. Previous studies showed CCNE1 gains and BRCA mutation or homologous recombination deficiency as mutually exclusive [29]. However, the authors showed that complete mutually exclusive alterations were not observed between low levels of CCNE1 gains and BRCA mutations. Our two patients with co-occurrence of BRCA mutation and CCNE1 gain presented low CCNE1 copy number gains (2.2 and 2.3).

The RB1 protein is involved in the S-phase checkpoint to repair DNA breaks and in the regulation of DNA replication. The loss of the tumor suppressor RB1 leads to cell cycle progression and replication fork progression leading to replication stress and DNA damage, which could be repaired by HR machinery [31]. We found an association of RB1 loss with PFS and OS. To our knowledge, two previous studies addressed the impact of RB1 loss in ovarian carcinoma. In 2013, Milea et al. showed loss of RB1 protein expression associated with longer OS [20]. Recently, Garsed et al. reported the association of RB1 loss and HR gene mutations with extremely long PFS and OS [21]. Taken together, these findings suggest that RB1 loss is a biomarker with the potential to identify sensitive patients to platinum treatment. In addition, this alteration could be used in the clinical practice and potentially select BRCA mutated patients with higher chance to be PARP inhibitors responsive.

Four of 15 tumors (26%) presented BRCA mutations, an expected frequency in high grade serous carcinomas [32]. No differences in the response of the treatment were found between mutated and non-mutated tumors. Although, BRCA mutations are well known markers of response to platinum and PARP inhibitors therapy, other studies also failed to show higher frequency of BRCA mutations in long term responders [21]. In addition to BRCA, two non-responder patients presented tumors with low HR deficiency scores and XRCC2 mutations. XRCC2 is involved in the repair of DNA double-strand breaks by HR pathway. This finding highlights the value of an investigation using a panel of genes in ovarian cancer. Furthermore, conclusions regarding HR deficiency based solely on the presence of certain mutations may not be precise, as was found in the ARIEL2 trial [13].

Our study has several limitations, mostly due to the small sample size and its retrospective nature. For example, we found the ORR higher than expected for platinum resistant patients. High ORR may be due to patient over-selection including those who have received several previous treatment lines. Despite the limitation of the ORR evaluation, CCNE1 gain and RB1 loss were both associated to OS, which is an objective endpoint even in retrospective studies. No association between HR deficiency scores and response to therapy was found. The small number of patients limits conclusions regarding the low accuracy of these scores, even if previous literature data also pointed out to the limitation of the scores. However, the strength of our study was the evaluation of well selected individuals for whom it would be expected a low response rate with platinum retreatment. Unexpectedly, a high response rate to platinum retreatment was found suggesting that this selected cohort might be enriched for extremely sensitive tumors. HR deficiency scores were not able to show a strong association with therapy response. Interestingly, CCNE1 copy number gain was a negative prediction marker of platinum sensitivity, and RB1 copy number loss identified patients with sensitive disease.

In this study we explored the mutational profile and HR deficiency score in ovarian cancer patients to better understand the platinum resistant recurrence as defined by the platinum free interval. We demonstrated that HR deficiency scores, CCNE1 gains and RB1 losses could be used to distinguish patients who are still sensitive to platinum retreatment from those resistant to platinum therapy. Considering similar mechanisms of sensitivity to platinum salts and PARP inhibitors, these markers could be useful to better select the patients for PARP inhibitors therapy in the platinum resistant relapse.

The prediction of response to platinum retreatment goes beyond HR deficiency. CCNE1 copy number gains revealed a different subtype of ovarian carcinoma and could be used as a negative selection marker for platinum therapy and PARP inhibitors. Furthermore, RB1 losses identified patients with higher chance to be responsive to the treatment. Theses markers add information to precision therapy in the context of the cost-effectivity for PARP inhibitors therapy.