Nlr & Brca Mutational Status In Patients With High Grade Serous Advanced Ovarian Cancer: A Large Population Study

Laboratory-markers of the systemic inammatory-response, such as neutrophil/lymphocyte-ratio (NLR) have been studied as prognostic factors in several tumors but in OC-patients their role is still controversial and no data about the possible correlation with the BRCA-status has been ever reported. We consecutively enrolled a series of 397 newly diagnosed high-grade serous-advanced OC-patients. All patients were tested for BRCA-mutational-status and blood-parameters have been collected 48 hours before staging-surgery. A signicant correlation of NLR with disease distribution (p<0.005) was found and patients with NLR<4 underwent primary-debulking-surgery more frequently (p-value 0.001), with a lower surgical-complexity-score (p-value 0.002). Regarding survival-data, patients with NLR< 4 had a signicant 7-month increase in mPFS (26 vs 19 months, p=0.009); focusing on the BRCA-status, among both BRCA-mutated and BRCA-wild type patients, those with lower NLR had a signicantly prolonged mPFS compared to patients with NLR> 4 (BRCA-mutated: 35 vs 23 months, p= 0.03; BRCA-wt: 19 vs 16 months, p=0.05). At multivariate-analysis, independent factors of prolonged PFS were BRCA mutational status, having received complete cytoreduction and NLR< 4. Also, the strongest predictors of longer OS were BRCA-mutational status, having received complete, NLR< 4 and age. NLR is conrmed to be a prognostic marker in OC-patients and it seems unrelated with BRCA-mutational status.


Introduction
Ovarian cancer remains the most lethal gynecological malignancy in developed countries [1].
Nearly 75% of OC affected women present with advanced disease (stage III or IV), and most of these will die from their disease, with 5-year overall survival rates around 30% [2].
Several prognostic factors have been identi ed to predict the outcome and guide personalized treatment of patients with advanced OC, including histological type, FIGO-stage, residual tumor after surgery, response to chemotherapy, and BRCA1/2-mutation status [3]. In addition to clinical and molecular features, recent data suggest the in uence of the host-driven in ammatory response on tumors' behavior and treatments outcome [4,5]. In fact, tumor growth and metastatic spread result from several interactions between tumoral and stromal factors, including blood vessels, in ammatory cells and the immune system, leading to a chronic in ammation status [6,7].
Laboratory markers of systemic in ammatory response, such as white blood cell count, have been studied as prognostic and predictive factors in several tumors [8,9]. High NLR, de ned as the absolute neutrophils count divided by the absolute lymphocytes count [9][10][11] has shown a negative prognostic impact in stomach, colorectal cancer and other cancers [12][13][14]. The mechanism underlying the association between high NLR and worse outcome has not been clari ed yet. Probably, neutrophilia can inhibit the immune system, by blocking the cytolytic activity of immune cells [15][16], and promotes tumour growth, by producing vascular endothelial growth factor [17,18]. On the other hand, lymphocytopenia is frequently found in patients with advanced disease, indicating a lower immune activity against tumor antigens released by cancer cells [19,20,23]. Moreover, lymphocytes T seem keener to apoptosis [21,22].
In OC, in ammation markers have shown interesting but controversial results [24]. In particular, recent trials have suggested that a high NLR is correlated with an immunosuppressive pro le [25], and with poorer overall survival and could be a predictive marker for treatment e cacy [26][27][28].
Albeit OCs harboring a BRCA mutation are considered more immunoreactive and with a higher mutational and neoantigen loads compared with BRCA wild type tumors, no study has investigated the possible correlation between NLR values and BRCA mutational status. In light of these aspects, we investigated the relationship between BRCA status and systemic in ammatory factors in a large high-grade serous OC population.

Patients' characteristics.
397 patients ful lled the inclusion criteria and were evaluable for the biomarkers of interest.
Characteristics of evaluated patients are described in Table 1 The association between the preoperative NLR score and clinicopathologic characteristics of EOC patients are also shown in Table 1.
Regarding the BRCA status, no signi cant differences were found with regard of NLR values (p-value: 0.97). The majority of patients presented as stage III of disease (282, 71.8%), without differences related to NLR value. We found a signi cant correlation of NLR with disease distribution, with more patients with low tumor load in Group 1 (NLR <4) versus Group 2 (NLR > 4) (44.6% vs 67.3%) (p<0.0001).

Discussion
In this study, we found for the rst time that high NLR (>4) has a negative prognostic role in patients with primary advanced OC, in terms of both PFS and OS, regardless of BRCA status.
Several evidences suggest that BRCA-mutated OC disease exhibits statistically signi cantly higher mutational and neoantigen loads and may be more immunologically "hot"/T -cell in amed compared to BRCAwt and HR pro cient ovarian cancers. Therefore, we wanted to explore whether or not, this could be predicted by a simple blood biomarker. Nevertheless, we weren't able to nd such correlation, as NLR values seem unrelated to the presence/absence of the BRCA mutation. In other words, if we assume that NLR is correlated, at least indirectly, with the immune status, based on our results, individual immunoreactivity to cancer is independent from BRCA status. As a matter of fact, we shouldn't be surprised that BRCA, as well as HRD status, do not linearly predict response to immune checkpoint inhibitors (ICIs) [34,35] and we should consider NLR as a more reliable predictor of immunotherapy response even in OC, as it has been recently demonstrated in other cancers [36][37][38].
More importantly, we came up with the evidence that OC BRCA-mutated disease is not "one disease" with peculiar and good survival outcomes and that, even in presence of a BRCA mutation, the prognosis can be determined by other factors, of which NLR is one easily identi able. Indeed, among BRCAmut patients, those with low NLR had 7 months advantage of mPFS with respect to women with high NLR.
Moreover, it is now recognized that PARPi, which are known to achieve their greatest e cacy in BRCA mutated and HRD cancer cells, can also get a response in HR pro cient cancer [39]. This can be explained by both a cytotoxic activity (depending on HR de ciency), and an antitumor immune activity, that might be more relevant on HR pro cient cells [40][41][42][43][44]. This hypothesis should be further investigated in the HR pro cient population, to provide NLR as a marker able to identify those patients who can rely on their reactive antitumor-immune response to bene t from PARPi.
We also found that high NLR in patients with primary advanced OC is predictive of larger tumor burden (expressed as LPS-PIV score) and those with higher NLR have higher chances of receiving NACT instead of PDS, compared with those with lower NLR. These observations are in line with the negative prognostic value of high NLR reported in other retrospective series of different OC settings [45,46]. Finally, our data con rm that NLR at baseline has an independent prognostic impact for both PFS and OS.
The strength of our cohort relies in the collection of data from a large single-centre population consecutively enrolled in a prospective study for tissue-BRCA status investigation. Furthermore, no data about NLR according to BRCA status in OC have been published before. However, it should be recognized that neutrophils and lymphocytes counts are non-speci c parameters, because they could be in uenced by concomitant conditions, such as infections or in ammation. The cut-off value to discriminate between the high or low group using NLR is not clearly established. We decided to use the cut-off closer to the median value, which was 4 in our population, as it has been previously proposed [47] and considering that the more often used values are 3 or 5 [19,48]. This might be a critical limitation in the general application of our assessment, though it would reasonably not change the nal ndings of our analysis.
In conclusion, NLR is con rmed to be a prognostic marker in OC patients. The information obtained from our study has revealed a potential new biologic subtype of BRCA patients, correlated with in ammation status and easily detectable. Next research should be focused on the role of NLR with regard of PARPi and ICIs response, regardless of BRCA/HRD status, underlining others less common but not less effective mechanisms of action of these drugs and allowing further personalization of treatment.

Patients
Between January 2017 and December 2019, newly diagnosed high grade serous ovarian cancer (HGSOC) patients with FIGO Stage IIIC-IV, admitted at the Gynaecologic Oncology Unit, Fondazione Policlinico A. Gemelli IRCCS in Rome, were consecutively tested for the tissue/blood BRCA mutation within a prospective study [29].
All women received gynecologic oncologist counseling before BRCA testing and a signed written informed consent. BRCA-mutations were classi ed according to the ENIGMA BRCA1/2 Gene Variant Classi cation Criteria (http://www.enigmaconsortium.org/) and women with variants of uncertain signi cance (VUS) were considered wild-type. Tissue samples for somatic testing were obtained during surgery. Patients were included if they had a primary diagnosis of high grade serous ovarian cancer (HGSOC), they received 3 weekly carboplatin-paclitaxel as rst line treatment, with or without maintenance therapy, if their BRCA mutational status was available. Their blood parameters should have been collected in the local laboratory at Fondazione Policlinico A. Gemelli IRCCS 48 hours before staging laparoscopy/laparotomy. All women gave written informed consent for their data to be collected and analyzed for scienti c purposes. The Institutional Review Board of the Catholic University of the Sacred Hear approved the study (CICOG-01-07-19/35).

Clinical data and follow-up
According to our Institutional model, patients were initially submitted to clinical evaluation, CT-scan and staging laparoscopy (S-LPS) [30] to be triaged to primary debulking surgery (PDS) or neoadjuvant chemotherapy (NACT). Intraperitoneal tumor burden was evaluated at diagnosis using a laparoscopic predictive-index value (LPS-PIV) [30], classifying women as having: low tumor load in presence of LPS-PIV < 8, and high tumor load when a LPS-PIV≥ 8 was observed. In all patients selected for PDS, maximal surgical effort was attempted, and residual tumor recorded. The Complexity of surgical procedures in patients receiving PDS was graded according to the surgical complexity score (SCS) by Aletti et al. [31]. Regardless upfront treatment strategy, all women received six cycle of carboplatin (area under the curve [AUC] 5 or 6) plus paclitaxel (175 mg/mq) every 21 day (Q3W); maintenance therapy was also administrated as indicated according to internal protocol and included Bevacizumab (15 mg/kg) in combination with chemotherapy and maintenance Q3W for 22 cycles or Olaparib 300 mg tablets orally two times per day until disease progression or toxicity for a maximum of 2 year, in BRCA mutated patients (since April 2019).
After treatment administration, patients were entered into routine follow-up program including gynecological examination, CA125 assessment and CT-scan every 6 months.
Data from medical records were consecutively collected including medical history, surgery results, treatment approach, and genetic counseling.

Statistical analysis
NLR was de ned as the absolute neutrophils count divided by the absolute lymphocytes count. Neutrophils and lymphocytes count collected within 48 hours before staging laparoscopy or laparotomy were taken into consideration. A cut-off value of 4 was adopted to discriminate patients with low (NLR <4) (Group 1) versus high (NLR > 4) (Group 2) as primary analysis, according with previous published data and median value in the present series [27,32,33].
Chi-square or Fisher's exact test were used for comparison of categorical variables.
Regarding survival analysis, PFS was de ned as the time relapsed between the date of diagnosis (staging laparoscopy/laparotomy) and recurrence; patients without evidence of progression disease at the time of the analysis were censored on the date of their last tumor evaluation. Overall survival (OS) was de ned as the time interval between the diagnosis and death of any cause. Patients who were no longer alive at the time of the analysis or had been lost to follow-up were censored on the date of their last follow-up visit. PFS and OS were estimated by the Kaplan-Meier method, and curves were compared by the log-rank or Breslow (Generalized Wilcoxon) tests (at a signi cance level of 5%), as appropriate. Estimated hazard ratios (HRs) and their two-sided 95% con dence intervals (95% CIs) were calculated using the Cox proportional-hazard model. All statistical calculations were carried out using SPSS 26.0 for Mac (SPSS Inc., Chicago, IL, USA).

Ethical approval.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.     Kaplan-Meyer plots for overall survival (OS) according to NLR-value and BRCA status, overall population