{"paper_id":"bee91b49-b32c-42de-96e6-4ab0ae499876","body_text":"Ovarian cancer (OC) is the eighth leading cause of cancer death among women worldwide, with 324,398 new cases and 206,839 deaths in 2022 [ 1 ]. When it is diagnosed at the early stage of disease, the 5-year survival rate is very high (~90%). However, due to the lack of specific symptoms, many OCs are diagnosed in advanced stages, where the 5-year survival declines to ~30% [ 2 ].\nMost OCs are of epithelial origin [ 3 ]. Histologically, epithelial OC is divided into five major subtypes: high-grade serous, endometrioid, clear-cell, mucinous, and low-grade serous [ 2 ]. Based on genetic alterations, disease development, and prognoses, epithelial OC can be further grouped into type I and type II carcinomas [ 4 ]. Type I tumors include slow-growing, low-grade serous, endometrioid, mucinous, and clear carcinomas characterized by mutations in  KRAS ,  PIK3CA ,  PTEN ,  BRAF ,  CTNNB1 , and  ARID1A , mostly arising from endometriosis or borderline serous tumors. Type II tumors, which occur more frequently, are aggressive, high-grade serous carcinomas, carcinosarcomas, and undifferentiated carcinomas with a high prevalence of  TP53  and  BRCA1/2  mutations, distal fallopian tube origin, and poor prognoses compared to type I OC [ 3 , 5 , 6 ].\nDespite the molecular diversity, most OCs are treated in the same manner, which includes radical surgery followed by carboplatinum and paclitaxel chemotherapy, with or without bevacizumab, an angiogenesis inhibitor. Depending on genetic test results on  BRCA1/2  mutations and homologous recombination deficiency (HRD), maintenance therapy may include poly (ADP-ribose) polymerase (PARP) inhibitors, bevacizumab, or a combination of both [ 7 ]. Unfortunately, despite achieving initial remission, up to 70% of patients will experience recurrence because of platinum resistance [ 8 ]. To improve therapeutic efficacy and patient survival, extensive research is underway to examine aberration in cancer signaling pathways, which would help to develop aberration-specific inhibitors.\nAlterations in the PI3K pathway are common in cancers, making this pathway one of the most important for therapeutic intervention [ 9 ]. These alterations involve  PIK3CA  (p110α catalytic subunit of PI3K),  PIK3R1  (p85α regulatory subunit of PI3K), and  PTEN , the negative regulator of PI3K. Upon activation, PI3K generates phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), which activates critical downstream targets, such as AKT [ 10 ]. The induction of PI3K can be further enhanced by direct binding with activated RAS protein [ 11 ]. In physiological conditions, PI3K is mainly in the autoinhibited state. p85α inhibits and stabilizes the catalytic subunit, and it may also negatively regulate the PI3K pathway by forming homodimers that stabilize PTEN [ 12 ]. In addition to p85α’s canonical role in regulating the PI3K catalytic subunit, free p85α has a function in oxidative stress response, mediating apoptosis regulated by p53 [ 13 , 14 ].\nSomatic mutations in the  PIK3R1  gene have been reported in 4.4% of all tumors, with the highest frequency in endometrial carcinoma (31%), glioblastoma multiforme (11%), and colorectal cancer (8%) [ 15 , 16 ]. The 5q13.1 region containing  PIK3R1  is frequently lost in breast (23%) and prostate (24%) cancers [ 17 , 18 ].  PIK3R1  mRNA expression is also markedly lower in tumors than in normal tissues across multiple cancer types, including breast, lung, kidney, prostate, and endometrial carcinomas [ 17 ]. Low  PIK3R1  mRNA expression is associated with unfavorable prognoses in breast cancer patients, increased PI3K signaling, and the tumorigenic transformation of breast cancer models [ 17 , 18 , 19 ].\nPIK3R1  mutations are infrequent in ovarian cancers, except for the endometrioid and clear-cell types, in which they have been detected with a frequency ranging from 7% to 50% [ 20 , 21 , 22 ]. In contrast to mutations,  PIK3R1  copy number loss is frequently observed in high-grade serous ovarian cancers (HGSOCs) (68%) and is correlated with reduced  PIK3R1  mRNA expression [ 23 ].  PIK3R1  loss favors ovarian tumorigenesis through the co-activation of AKT and STAT3 signaling and confers sensitivity to these pathway inhibitors [ 23 ].\nThe present study aimed to evaluate  PIK3R1  status in ovarian carcinomas by analyzing mutations, copy number alterations, and mRNA expression, in the context of the clinicopathological features of these tumors. We also related  PIK3R1  gene status to changes in other PI3K pathway components, like  PIK3CA  and  PTEN , as well as to mutations in  TP53  and  KRAS . Finally, we evaluated the clinical importance of  PIK3R1  alterations in ovarian cancer patients. Our data provide insight into the clinicopathological and molecular characteristics of tumors with  PIK3R1  dysfunction and may have important implications in the future planning of ovarian cancer therapy with PI3K pathway inhibitors.\n\nThe study group comprised 197 patients with ovarian carcinomas (age range: 17–88 years, median 53 years). The patients were treated in the Maria Sklodowska-Curie National Research Institute of Oncology between 1995 and 2009. The tumors were uniformly reviewed histopathologically and classified according to the World Health Organization (WHO) criteria [ 24 ]. There were 126 (64%) serous—including 109 (55%) high-grade serous ovarian cancers (HGSOCs) and 17 (9%) low-grade serous ovarian cancers (LGSOCs)—25 (13%) endometrioid, 19 (10%) clear-cell, 10 (5%) mucinous, 9 (5%) undifferentiated, and 8 (4%) mixed-type or unclassified carcinomas. Of the tumors, 12 (6%) were well differentiated (G1), 46 (23%) showed moderate differentiation (G2), and 139 (71%) were poorly differentiated (G3). Tumors were staged according to the criteria of the International Federation of Gynecologists and Obstetricians (FIGO) [ 25 ]. Of the tumors, 35 (18%) were in FIGO stage I, 10 (5%) in stage II, 140 (71%) in stage III, and 12 (6%) in stage IV.\nPIK3R1  mutations were analyzed in 141 cases including 74 serous, 25 endometrioid, 19 clear-cell, 10 mucinous, 7 mixed, and 6 undifferentiated ovarian cancers. The somatic copy number alteration (CNA) in the  PIK3R1  gene was analyzed for 197 ovarian cancers.  PIK3R1  mRNA expression analysis was carried out for 144 cases that met the criteria: at least 85% tumor cell content and sufficient quality of RNA.\nBlood samples from healthy women were used to assess the normal copy number of the  PIK3R1  gene in CNA analysis. Five specimens of noncancerous fallopian tubes constituted a control group to compare the  PIK3R1  mRNA expression levels between normal and tumor tissues.\nIn a group of 151 patients treated with chemotherapy, 111 patients were cured with standard taxane–platinum (TP: paclitaxel with cisplatin or carboplatin), and 40 patients were cured with a platinum-based regimen (PC: cisplatin–cyclophosphamide or carboplatin–cyclophosphamide). Only patients with FIGO stage IIB-IV disease treated with standard protocols of chemotherapy were accepted for this study. The first-line chemotherapy consisted of 6 cycles in the TP-treated group and 6–8 cycles in the PC-treated group. Taxol was given in a 24 h (135 mg/m 2 ) or 3 h infusion (175 mg/m 2 ) and was followed by cisplatin (75 mg/m 2 ) or carboplatin (AUC6). For the PC regimen, it was 75 mg of cisplatin/m 2  or carboplatin (350 mg/m 2  or AUC6) and 750 mg of cyclophosphamide/m 2 . Patients’ follow-up time ranged from 296 to 4062 days (median 1125 days) for the TP group and from 104 to 4080 days (median 887 days) for the PC group. Overall survival (OS) was defined as the interval from the date of initial surgical resection to the date of death or last contact. Disease-free survival (DFS) time was defined for patients who reached complete response (CR) as the interval from the date of the last chemotherapy course to the date of recurrence or last contact. Response to chemotherapy was evaluated retrospectively according to WHO response evaluation criteria based on data from medical records describing patient’s clinical condition and CA125 levels in 3–4 week intervals [ 26 ]. Clinical complete response (CR) was defined as the disappearance of all clinical and biochemical symptoms of ovarian cancers, evaluated after the completion of first-line chemotherapy and confirmed four weeks later. The platinum-sensitive group (PS) had a DFS time longer than six months.\nTumors and non-tumor tissues obtained during the surgical procedure (before chemotherapy) were snap-frozen in liquid nitrogen and stored at −68 °C. All tumor specimens were reviewed by a pathologist to ensure the maximal amount of tumor cells (mostly >70% for mutation and CNA analyses and at least 85% for mRNA analysis). Genomic DNA and total RNA were isolated using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) and the RNeasy Plus Mini Kit (QIAGEN), respectively. DNA and RNA quantity and quality were measured with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and additionally, RNA quality was assessed on an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). One microgram of RNA was transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Vilnius, Lithuania) according to the manufacturer’s protocol.\nThe  PIK3R1  gene (GeneBank:  NG_012849.2 ) consists of 16 exons with 15 exons encoding protein (from 2 to 16). All 15 protein-coding exons were searched for mutations with the use of Sanger sequencing. Primers were designed using Primer3web v4.1.0 ( https://primer3.ut.ee ) and checked for their specificity with Primer-BLAST ( www.ncbi.nlm.nih.gov/tools/primer-blast , accessed on 20 November 2024) software. PCR mixtures were prepared according to the standard procedure (Applied Biosystems, Austin, TX, USA). Reactions were carried out on an Eppendorf thermocycler (Eppendorf, Hamburg, Germany) with an initial denaturation step at 95 °C for 5 min, followed by 36 cycles consisting of denaturation at 94 °C, annealing at 60 °C, and extension at 72 °C, each for 30 s. PCR products were further purified with exonuclease I and alkaline phosphatase (EURx Ltd., Gdansk, Poland) treatment and sequenced with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Austin, TX, USA) on a 3500 Genetic Analyzer (Applied Biosystems).\nThe  PIK3R1  somatic copy number alteration (CNA) was assessed with quantitative real-time PCR (qPCR) using TaqMan Copy Number Assays (Applied Biosystems) on a 7500 Fast Real-Time PCR system (Applied Biosystems). Reactions were carried out as a duplex in a 10 µL mixture containing 10 ng genomic DNA, TaqMan Universal Master Mix II, primers, and probes for  PIK3R1  (TaqMan Copy Number Assay Hs06008293_cn) and for the reference gene (TaqMan Copy Number Reference Assay RNase P). The conditions of the qPCR were as follows: 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min. Samples were analyzed in four replicates. Each 96-well plate included a negative control without a DNA template (NTC) and a calibrator sample with a known copy number for the target of interest. The number of copies of the  PIK3R1  sequence was determined by relative quantitation using the comparative C T  (ΔΔC T ) method and CopyCaller Software v2.0 (Applied Biosystems). This method measures the C T  difference (ΔC T ) between the target and reference sequence and then compares the ΔC T  values of the test samples to a calibrator sample known to have two copies of the target sequence. The copy number of the target (Q) is calculated to be two times the relative quantity. Copy number gain and loss were assigned as Q > 2.5 and Q < 1.5, respectively.\nPIK3R1  expression at the mRNA level was evaluated with the use of the quantitative real-time PCR (qPCR) method and TaqMan Gene Expression Assays (Applied Biosystems) on a 7500 Fast Real-Time PCR system. qPCRs were run as separate reactions for the target and each of the reference genes,  HPRT1 ,  PPIA,  and  GUSB . Reactions were carried out in three replicates in a 10 µL mixture containing 11 ng cDNA, TaqMan Universal Master Mix II with UNG, and the TaqMan Gene Expression Assay for  PIK3R1  (Hs00933163_m1) or for the reference gene ( HPRT1 , 4326321E;  PPIA , 4326316E; or  GUSB , 4326320E). Each 96-well plate included negative controls (NTC) for the analyzed genes. The thermal profile of the qPCR was as follows: 50 °C for 2 min (Uracil-N-Glycosylase activity), 95 °C for 10 min, followed by 40 cycles consisting of two steps: 95 °C for 15 s and 60 °C for 1 min. The comparative C T  (ΔΔC T ) method was used to analyze changes in the  PIK3R1  expression normalized to the mean of the reference gene expression in a given sample relative to a calibrator sample. The sample exhibiting the highest expression of the  PIK3R1  gene was used as a calibrator.  PIK3R1  expression was analyzed as a categorical variable with the median value of the expression used as a cut-off point.\nComparison between groups was performed using the Mann–Whitney U test for continuous variables and the Chi-squared or Fisher’s exact test for categorical variables. Analyses of prognoses were performed using the univariate and multivariate Cox proportional hazards models and further confirmed with the log-rank test and Kaplan–Meier survival estimates. The univariate and multivariate logistic regression models were used to analyze predictions. In all multivariate models, the  PIK3R1  alterations were correlated with clinicopathological tumor characteristics, including patient age (categorized by median split), histological type (categorization: HGSOCs vs. other types), grade (categorization: grade III vs. I-II), clinical stage (FIGO IIIC-IV vs. I-IIIB), residual tumor size (categorization: Rt < 2 cm vs. 0 and Rt ≥ 2 cm vs. 0), and type of chemotherapy regimen (TP vs. PC). All multivariate statistical models were simplified by backward stepwise elimination of variables if their  p -values were higher or equal to 0.1. The level of significance was set as  p  < 0.05. All calculations were performed using STATA v11 software.\n\nFive  PIK3R1  mutations were identified in 141 (3.5%) ovarian carcinomas ( Table 1 ,  Figure 1 A). All mutations were deletion or deletion–insertion type and were localized within the nSH2 and iSH2 domains. Two truncating mutations were found in exon 9, two in-frame changes were detected in exons 10 and 12, and one deletion was in the intron 12 splice acceptor site. cDNA sequencing revealed that the intronic mutation leads to the deletion of 69 bp encoded by exon 13 and probably results in the exon 13 skipping during transcription and the insertion of a single residue Ile.\nAmong tumors harboring  PIK3R1  mutations, we observed the loss of the  PIK3R1  allele in one tumor, amplification in two tumors, and the retainment of both alleles in another two tumors. In the case of amplification, we did not determine which allele, with or without mutation, was amplified.\nMutations were detected in three endometrioid (12%, 3/25), two clear-cell (10.5%, 2/19), and none of the 74 serous or 10 mucinous cancer cases. Most tumors with mutations were FIGO I or II, moderately differentiated carcinomas. Women with  PIK3R1 -mutated and -unmutated tumors had a mean age at diagnosis of 58.4 and 54.3 years, respectively.\nWe compared  PIK3R1  mutations with  PIK3CA ,  PTEN ,  KRAS,  and  TP53  status previously determined in these tumors [ 27 , 28 , 29 ].  PIK3R1  mutations were mutually exclusive with  PIK3CA  mutations. They were associated with  PTEN  mutations ( p  = 0.041) but not with  KRAS  mutations and tended to coexist with  TP53  wild-type tumors ( p  = 0.076,  Table 2 ). In detail, two endometrioid tumors with a  PIK3R1  mutation had a coexisting  PTEN  mutation, and one of them also had a mutation in the  KRAS  gene. Another endometrioid tumor harbored a concurrent mutation in  TP53 . In contrast, in clear-cell cancers,  PIK3R1  mutations occurred alone.\nThe prevalence of PI3K pathway mutations, including  PIK3R1 ,  PIK3CA , and  PTEN  was 13.5% (19/141) in ovarian cancers. In the endometrioid histological type, this frequency was 52% (13/25,  Figure 1 B) and was significantly higher than in clear-cell type cancers (15.8%, 3/19,  p  = 0.025), LGSOCs (6.7%, 1/15,  p  = 0.005), and HGSOCs (1.7%, 1/59,  p  < 0.001). Because  KRAS  mutations can stimulate PI3K activity and were observed in 16% (4/25) of endometrioid tumors, the frequency of the PI3K pathway aberration due to  PIK3R1 ,  PIK3CA ,  PTEN , and  KRAS  mutations may increase to 60% (15/25) in this histotype.\nThe number of  PIK3R1  copies ranged from 0.25 to 3.7 (mean value 1.93). The copy number alteration was observed in 70 of 197 (35.5%) ovarian cancers. Specifically, there were 28.4% (56/197) allelic losses and 7.1% (14/197) amplifications at the  PIK3R1  locus.\nThe gene allele loss was detected in HGSOCs (36.7%, 40/109) and LGSOCs (11.8%, 2/17) and endometrioid (16%, 4/25), clear-cell (26.3%, 5/19), mixed-type (25%, 2/8), mucinous (10%, 1/10), and undifferentiated (22.2%, 2/9) ovarian carcinomas. The allelic loss was more common in HGSOCs than in other tumors (36.7%, 40/109 vs. 18.2%, 16/88,  p  = 0.004,  Table 3 ). This is in contrast to  PIK3R1  mutations, which were observed exclusively in endometrioid and clear-cell types. Moreover,  PIK3R1  copy loss was associated with adverse clinicopathological features. The loss of  PIK3R1  was more frequently observed in advanced FIGO IIIC-IV stages (34.8%, 47/135) than in FIGO stage I-IIIB cancers (14.5%, 9/62,  p  = 0.003). Similarly, high-grade cancers had a higher frequency of allelic losses (34.5%, 48/139) than low-grade carcinomas (13.8%, 8/58,  p  = 0.003). There was no relationship between allele loss and patient age.\nWe investigated the potential relationship between  PIK3R1  and  PIK3CA ,  PTEN ,  KRAS , or  TP53  alterations described in our previous studies [ 27 , 28 , 29 ].  PIK3R1  copy loss was associated with both  PIK3CA  amplification ( p  = 0.038) and  PTEN  allele loss ( p  = 0.006,  Table 2 ). Interestingly, there seemed to be an association between  PIK3R1  copy loss and decreased or absent PTEN protein expression. The frequency of tumors that showed  PIK3R1  copy loss steadily increased from tumors with strong PTEN protein expression (14%, 6/41), to those with moderate expression (38%, 14/37), to those with decreased or absent PTEN protein expression (41%, 14/34,  p  = 0.019). The loss of the  PIK3R1  allele was more common in  TP53 -mutated cancers than in  TP53  wild-type tumors (35.8%, 48/134 vs. 12.7%, 8/63,  p  = 0.001).  PIK3CA ,  PTEN,  and  KRAS  mutations were not associated with  PIK3R1  allele loss.\nPIK3R1  amplification was found in HGSOC (8.3%, 9/109), LGSOC (5.9%, 1/17), endometrioid (8%, 2/25), and clear-cell (10.5%, 2/19) histological types. There were no significant associations between this alteration and clinicopathological or molecular factors, except for  PIK3R1  mutations. Tumors with  PIK3R1  amplifications harbored  PIK3R1  mutations more frequently than tumors without amplification (20%, 2/10 vs. 2.3%, 3/131,  p  = 0.040).\nPIK3R1  mRNA expression was significantly decreased in ovarian cancers compared with control tissues ( p  = 0.003, Mann–Whitney U test). The difference in mean expressions between these groups was 69% (0.116 ± 0.11 and 0.376 ± 0.31 in ovarian cancers and control tissues, respectively).  PIK3R1  mRNA was downregulated in 95.8% (138/144) of ovarian cancers relative to the control group’s mean, indicating the relevance of  PIK3R1  in ovarian tumorigenesis.\nWe examined  PIK3R1  expression in relation to DNA copy number loss. The samples were divided into those with low and high  PIK3R1  mRNA levels according to the median expression in the cancer group (0.0989) (n = 72 in each group). Low expression may be a consequence of allelic loss of the  PIK3R1  gene because there is an association between mRNA level and the copy number alteration of the gene ( p  = 0.009,  Figure 1 C). DNA copy number loss was more frequently observed in tumors with low mRNA expression (45.8%, 33/72) than in tumors with high expression (25%, 18/72,  p  = 0.009). There was no significant relationship between  PIK3R1  mutations and mRNA levels.\nWe assessed  PIK3R1  expression levels in ovarian cancer subtypes. Low expression was present in HGSOCs (49.5%, 50/101) and endometrioid (75%, 6/8), clear-cell (83%, 5/6), mixed-type (71%, 5/7), and undifferentiated (75%, 6/8) cancers but not in LGSOCs (0%, 0/13) or mucinous (0%, 0/1) ovarian cancers. The difference in the expression levels between HGSOCs and LGSOCs was statistically significant ( p  <0.001). Interestingly, diminished expression was more common in the endometrioid and clear-cell types than in serous type (75.6%, 11/14 vs. 43.9%, 50/114,  p  = 0.021).\nNext, we examined  PIK3R1  expression in relation to clinicopathological and molecular factors. Lower levels of  PIK3R1  mRNA were more common in poorly differentiated than in well and moderately differentiated tumors (54%, 65/120 vs. 29%, 7/24,  p  = 0.025). There was no relationship between expression and clinical stage or patients’ age ( Table 3 ). We found an association between expression and  PIK3CA  amplification or  TP53  mutations. Low  PIK3R1  expression was more frequent in cancers with  PIK3CA  amplifications than those with  PIK3CA  WT tumors (64%, 14/22 vs. 38%, 24/64,  p  = 0.033). Similarly, tumors with diminished  PIK3R1  expression tended to harbor  TP53  mutations more often than  TP53  WT tumors (53%, 62/116 vs. 36%, 10/28,  p  = 0.092,  Table 3 ). There was no association between  PIK3R1  expression and  PTEN  or  KRAS  changes ( Table 2 ).\nUnivariate and multivariate statistical analyses were performed to assess the associations of  PIK3R1  copy number alteration and expression with patients’ outcomes in the entire group of patients and subgroups treated with either the standard taxane–platinum (TP) or the platinum-based (PC) regimen. In the group analyzed for mRNA expression, 103 patients were treated with TP regimens and 34 with PC.\nLow  PIK3R1  expression diminished the probability of complete response (CR) in the PC-treated patients. Low expression was rarely observed in patients who reached CR (29%, 7/24) compared to those with other responses (70%, 7/10,  p  = 0.028). In both univariate and multivariate analyses, low expression negatively influenced the probability of CR (OR 0.18,  p  = 0.035 and OR 0.07,  p  = 0.030, respectively,  Table 4 ). There were no associations between  PIK3R1  expression and CR in the TP-treated and the entire group of patients.  PIK3R1  expression was not associated with platinum sensitivity in any of the analyzed groups.\nLow  PIK3R1  expression tended to diminish the risk of recurrence in the entire group of patients. The mean disease-free survival (DFS) time of patients with low and high expression was 569 and 470 days, respectively. Univariate and multivariate analyses showed a trend toward better prognoses for patients with low  PIK3R1  expression compared to those with high expression in terms of recurrence (HR = 0.68,  p  = 0.081, log-rank,  p  = 0.079, and HR = 0.064,  p  = 0.054, respectively;  Figure 1 D). DFS also showed associations with grade and debulking status. These correlations were not confirmed in the smaller TP and PC-treated patient subgroups. Patients’ overall survival was not influenced by the  PIK3R1  mRNA level in either group.\nThere were no associations between clinical endpoints and the  PIK3R1  DNA copy number.\n\nThe current study showed that the vast majority of ovarian cancers carry  PIK3R1  alterations (>90%). We identified mutations, gene deletions, and a very high frequency of mRNA underexpression. We also found that tumors with  PIK3R1  mutations had other clinicopathological and molecular characteristics than tumors with gene loss or reduced mRNA expression.\nPIK3R1  mutations are rare in ovarian cancer. In our study, this alteration was observed in 3.5% of tumors, which is similar to the rate (0–3.8%) reported by other groups [ 15 , 21 , 30 , 31 ]. All mutations were clustered within the iSH2 and nSH2 domains involved in PI3K inhibition and stabilization. It has been shown that mutations in these domains disrupt the inhibitory interface and retain the stabilizing interconnection between the regulatory and catalytic subunits, resulting in PI3K activation [ 15 , 32 ]. In contrast, p85α with truncating mutations N-terminal to iSH2 cannot bind the p110α subunit and probably acts independently of PI3K via the activation of JNK signaling [ 33 , 34 ]. In our series, two truncating mutations were identified, which may represent this PI3K-independent course of action. The distribution of mutations we observed was similar to that found in endometrial cancers, in which the vast majority of mutations (93.3%) clustered within the iSH2 and nSH2 domains [ 35 ]. In contrast, in prostate cancers, the truncating mutations were found mainly in the cSH2 domain, suggesting that the localization of  PIK3R1  mutations may vary between tumor types and may have different functional consequences [ 17 ].\nWe found mutations mostly in low-stage, moderately differentiated endometrioid carcinomas (12%) and in clear-cell (10.5%) carcinomas, which is in agreement with the scarce literature on this subject. To date, Cybulska et al. has reported mutations in 11.1% of analyzed endometrioid ovarian cancers, mostly low-stage, and in 0.5% of HGSOCs from The Cancer Genome Atlas [ 20 ]. Fieuws detected  PIK3R1  mutations in 6.7% of clear-cell carcinomas and 2.4% of HGSOCs with no clinical characteristics given [ 21 ]. Teer et al. found  PIK3R1  mutations in 50% of endometrioid ovarian cancers [ 22 ].\nThis study revealed  PIK3R1  copy number alterations, including allelic losses (28.4%) and amplifications (7.1%). We also observed that these allele losses were associated with an advanced FIGO stage, high tumor grade, and HGSOC histotype. The association between the loss of a gene allele and clinicopathological parameters of ovarian cancers has not been previously reported. The chromosome region with  PIK3R1  was frequently deleted in HGSOCs according to TCGA data [ 36 ]. Similarly, Huang et al. reported significantly frequent copy deletions in the  PIK3R1  region in serous histotypes [ 37 ]. A detailed analysis of the  PIK3R1  copy number in serous ovarian cancer across TCGA revealed 68.4% heterozygous and 3.5% homozygous loss, respectively [ 23 ]. However, a small fraction of  PIK3R1  allele amplification (3.3%) was also observed in TCGA ovarian cancer patients [ 23 ]. Moreover, Teer et al. found one endometrioid ovarian cancer with both  PIK3R1  mutation and amplification [ 22 ]. Investigations performed on other cancer types demonstrated  PIK3R1  copy number loss in 23% of breast cancers, as well as in 24% and 36% of primary and metastatic prostate cancers, respectively [ 17 , 18 ]. Data from murine models revealed that, for both  Pik3r1  and  Pten , heterozygous mice display increased AKT activity and increased intestine neoplasia compared with  Pten  heterozygotes alone [ 38 ]. In addition, single-copy ablation of  Pik3r1  accelerated a mouse model of HER2-/neu-driven breast cancers [ 18 ]. Collectively, these data suggest that  PIK3R1  copy number losses are frequent in ovarian cancers and other cancer types and may have tumorigenic potential. However, the clinicopathological characteristics and clinical significance of this alteration has not yet been fully explored.\nOur study revealed that  PIK3R1  is lowly expressed in ovarian cancers. Similarly, low  PIK3R1  mRNA expression was found in ovarian cancer and many other human cancers in the OncoMine microarray database [ 39 ]. Decreased expression of  PIK3R1  was also observed in serous ovarian cancers in TCGA cohorts [ 23 , 40 ] and, in agreement with our results, was correlated with the gene copy numbers, suggesting that reduced expression may be related to gene allele loss [ 23 ]. However, opposite results of  PIK3R1  and p85α overexpression have also been reported in ovarian tumors [ 41 ]. In our study, we found diminished mRNA expression in all the histological types analyzed. Still, it was significantly more frequent in endometrioid and clear-cell types than in serous types and was inversely associated with the grade of malignancy. Low  PIK3R1  expression has previously been linked to advanced histological grade and/or clinical stage in other carcinomas, like in breast cancer [ 19 ], neuroblastoma [ 42 ], hepatocellular cancer [ 39 ], and others [ 17 , 43 ].\np85α can inhibit PI3K activity not only by binding and inhibiting the catalytic subunit but also by competing with PI3K to bind to receptors. This is because p85 is in excess of the catalytic subunit in cells [ 44 ]. Thus, the binding of p85 monomers to receptors prevents interactions between the p85-p110 complex and RTKs, resulting in reduced PI3K signaling. This mechanism of PI3K inhibition is particularly sensitive to free p85α levels. In fact, decreased  PIK3R1  expression mainly affects free p85 monomers, resulting in a significant increase in PI3K signaling. Indeed, even transient knockdown of  PIK3R1  (leading to a partial loss of  PIK3R1  mRNA expression) alone was sufficient to induce AKT activation and the proliferation of prostate cancer cells [ 17 ]. Additionally, it was shown that serous ovarian cancer cell lines with  PIK3R1  loss demonstrated multiple tumorigenic properties like increased proliferation, migration, and invasion [ 23 ]. Consequently, in mice, reduced  PIK3R1  expression promoted oncogenic transformation and metastatic dissemination of ovarian cancer [ 23 ]. Together, these data support the function of  PIK3R1  as a tumor suppressor, the loss of which may enhance tumorigenesis in ovarian cancer and other cancer types.\nRegarding the changes in other genes of the PI3K pathway, we found that  PIK3R1  mutations are mutually exclusive with  PIK3CA  and coexist with  PTEN  mutations. This is in agreement with a study of a pan-cancer cohort across 20 different cancer types, including ovarian cancer, in which only 2 patients out of the 1200 analyzed harbored both a  PIK3R1  and a  PIK3CA  mutation [ 45 ]. Similarly, a study on breast cancers did not reveal the coexistence of mutations in the regulatory and catalytic PI3K subunits [ 19 ]. However,  PIK3R1  and  PIK3CA  mutations co-occur in endometrial [ 35 ] and colorectal cancers [ 15 ], suggesting that these double events may contribute to pathogenesis in particular tumor types.\nSeveral studies have shown that  PIK3R1  frequently coexists with  PTEN  and  KRAS  mutations in endometrial tumors [ 35 , 46 ]. According to Cheung and colleagues,  PIK3R1  mutations may co-occur with heterozygous  PTEN  mutations to compensate for the incomplete loss of PTEN protein [ 46 ]. It was revealed that the sole  PIK3R1  mutations present in normal uterine endometrium are not sufficient to initiate malignant transformation. Still, they are associated with a higher tendency towards endometriosis development, while endometriotic epithelia carrying additional mutations in  PTEN  are more likely to transform into endometriosis-associated ovarian cancer [ 47 ]. These data, together with our findings, indicate that mutations in  PIK3R1  and  PTEN  or  KRAS  may work together for efficient transformation into endometrioid ovarian cancer that may represent type I tumors in ovarian cancer classification.\nIn contrast to  PIK3R1  mutations,  PIK3R1  allele loss and decreased mRNA expression are associated with aggressive clinicopathological features and unfavorable molecular changes, indicating that they may contribute to type II tumor development. These molecular alterations include  PIK3CA  amplification,  PTEN  loss, and  TP53  mutations. Similar to low  PIK3R1  expression,  PIK3CA  amplification can disrupt the levels of free p85α leading to PI3K pathway activation. This is because an excess of p110α, due to  PIK3CA  amplification, can sequester a surplus of free p85α monomers into the p85α-p110α complex, increasing the amount of PI3K enzymes [ 46 ]. Moreover, an excess of p110α may disturb free p85α homodimerization. p85α homodimers are able to bind and stabilize PTEN protein, protecting PTEN from WWP2-mediated proteasomal degradation [ 13 ]. Thus, apart from competing with PI3K to bind to RTKs, p110α-free p85α can negatively regulate the PI3K pathway through PTEN stabilization, whose function may be attenuated by  PIK3CA  amplification.\nTP53  is the most frequently mutated gene in HGSOCs [ 16 ]. In our series consisting of different histological types,  TP53  mutations were present in the majority of tumors. TP53 activates the transcription of numerous genes directing cell cycles, DNA repair, or apoptosis in response to DNA damage [ 48 ]. The role of free p85α in cellular stress pathways has also been confirmed [ 13 ]. In response to oxidative stress, apoptotic cell death mediated by p53 was blocked in cells with little to no p85α expression [ 14 , 49 ]. Moreover, free p85α is a positive regulator of p53 acetylation and a critical upstream proapoptotic mediator in the UV-B response [ 50 ]. In addition, p85α is upregulated by p53 at the transcription level, further indicating proapoptotic roles for p85α in response to cellular damage [ 13 , 14 ].\nIn this study, we observed a decreased probability of complete response in PC-treated patients with low  PIK3R1  mRNA expression. Our finding is consistent with the proapoptotic function of free p85α via the positive regulation of p53, which may be weakened in cells with low p85α amounts. Similarly, a study on serous ovarian cancer cell lines demonstrated that  PIK3R1  downregulation induced the expression of antiapoptotic  BCL2 , as well as genes involved in cell cycle progression ( CCNB1 ) and metastasis ( MMP9  and  VEGFA ) [ 23 ]. However, an opposite effect of enhanced apoptotic response to platinum treatment in platinum-resistant cells was also observed in HGSOC cell lines with  PIK3R1  knockdown [ 51 ]. Nevertheless, our findings imply that  PIK3R1  loss and  TP53  mutations may have a negative, additive influence on the response to platinum-based therapy. On the other hand, low  PIK3R1  expression tended to diminish the risk of recurrence in the entire group of patients. This observation is surprising, since in our study low expression was associated with other pathological factors linked to poor outcomes, like high nuclear grade. We propose that the decreased risk of recurrence may be attributed to a significantly higher proportion of endometrioid and clear-cell types than HGSOCs in the group with low expression. These two histological types of ovarian cancer develop more slowly and are usually diagnosed at a lower clinical stage than HGSOCs even with comparable high nuclear grades. Contrary to this conclusion, an ovarian serous cystadenocarcinoma analysis from TCGA revealed an association between high  PIK3R1  expression and worse prognoses [ 40 ]. This discrepancy in these study results indicate that the prognostic significance of  PIK3R1  expression warrants further investigations in ovarian cancer patients. Nevertheless, it should be underlined that low  PIK3R1  expression was a negative prognostic factor in multiple cancer types, including prostate, gastric, liver, and non-small-cell lung cancers as well as breast and uterine endometrial carcinomas [ 17 , 40 ].\nAlterations in the PI3K pathway found in a wide variety of human cancers provided the rationale for the development of PI3K/AKT inhibitors, and some of them have been clinically approved [ 9 ]. Unfortunately, no PI3K-pathway-targeted drugs have been approved for ovarian cancer treatment so far. Several preclinical studies suggest the possibility that PI3K pathway inhibitors might be effective in  PIK3R1 -deficient tumors. D’Ambrosio et al. demonstrated that an ovarian cancer patient-derived xenograft (PDX) with the  PIK3R1  mutation W624R was sensitive to the pan-class I PI3K inhibitor buparlisib, and also to the p110α specific inhibitor alpelisib, as well as to the dual PI3K and mTOR inhibitors dactolisib [ 52 ]. In another study,  PIK3R1  copy number loss or reduced  PIK3R1  expression rendered ovarian cancer cells vulnerable to the inhibition of AKT or JAK2/STAT3 inhibitors [ 23 ]. In a mice model of breast cancer, pan-PI3K and p110α-selective pharmacological inhibition effectively blocks transformation driven by partial p85α loss [ 18 ]. These data suggest that PI3K pathway therapeutics may be effective in the treatment of ovarian cancer patients with  PIK3R1  loss. Recently, Passarelli et al. reported the outcomes of patients with  PIK3CA -mutated recurrent gynecological tumors, including 10 ovarian cancers, prospectively treated with alpelisib within a controlled program ( NCT04085653 ) [ 53 ]. In the ovarian cancer group, the disease control rate (DCR) was 50% (4: stable disease; 1: partial response), and the median progression-free survival (PFS) was 5.0 months. Our results and those of preclinical studies suggest that  PIK3R1  alterations like  PIK3R1  gene deletion—common in type II cancers—may serve as an alternative to  PIK3CA  markers for therapy with these pathway inhibitors and should be taken into consideration while planning future clinical trials.\n\nIn summary, we showed that the  PIK3R1  gene is frequently altered in ovarian cancers due to mutations, DNA copy number alterations, and decreased  PIK3R1  mRNA expression. We found that  PIK3R1  mutations mainly contribute to the development of low-stage endometrioid and clear-cell carcinomas with coexisting  PTEN  mutations. In contrast, copy number loss and decreased mRNA expression are features of aggressive high-grade ovarian cancers representing different histological types harboring  PIK3CA  amplifications and  TP53  mutations. Moreover, we found that low  PIK3R1  expression significantly diminished the probability of complete response in patients treated with platinum-based regimens. To our knowledge, this is the first ovarian cancer study comprising the analysis of  PIK3R1  mutations, copy number alterations, mRNA expression with respect to clinicopathological and molecular factors, and patients’ endpoints. This study may be important since we found that there is a significant group of ovarian cancer patients with different  PIK3R1  alterations that may potentially benefit from treatment with PI3K and AKT inhibitors.","source_license":"CC-BY-4.0","license_restricted":false}