PI3KCA Mutations in Uterine Cervix Carcinoma

Journal of Clinical Medicine

Article PI3KCA Mutations in Uterine Cervix Carcinoma

Ioannis A. Voutsadakis 1,2

1 Algoma District Cancer Program, Sault Area Hospital, Sault Ste. Marie, ON P6B 0A8, Canada; [email protected] 2 Section of Internal Medicine, Division of Clinical Sciences, Northern Ontario School of Medicine, Sudbury, ON P3E 2C6, Canada

Abstract: Background: Squamous cervical carcinoma represents an infection-associated malignancy that produces a high mortality when metastatic or recurrent after primary local treatment. There is an urgent need for new therapies for this cancer. Molecular lesions in cervical cancer may provide opportunities for targeted therapies development. Methods: Publicly available data from the Cancer Genome Atlas (TCGA) were analyzed to deﬁne the molecular landscape of squamous cervical carcinomas with and without mutations of PIK3CA, the gene encoding the alpha catalytic subunit of phosphatidylinositol 3 kinase (PI3K). Associations with alterations in other critical genes and pathways of cancer and the total mutation burden and copy number alteration burden of cervical cancers were examined. Results: Mutations in PIK3CA are observed in 27.1% of squamous cervical cancers. PIK3CA represents the most frequently mutated gene in these cancers. Mutations in PIK3CA are associated with higher rates of mutations in other genes of important cancer-associated pathways such as the tyrosine kinase receptors/K-Ras/BRAF/MAPK and the Wnt/β catenin pathway. In addition, PIK3CA mutated cervical cancers display a higher tumor mutation burden (TMB) than non- mutated cancers. Conclusion: Frequent mutations of PIK3CA gene in squamous cervical carcinomas may represent an opportunity for targeted therapies development both inhibiting the PI3K kinase and associated pathway defects. Increased TMB may additionally confer immunotherapy sensitivity.

Keywords: cervical cancer; squamous; mutations; PI3K kinase; catalytic subunit; PIK3CA mutations

Citation: Voutsadakis, I.A. PI3KCA Mutations in Uterine Cervix Carcinoma. J. Clin. Med. 2021, 10, 220. 1. Introduction https://doi.org/10.3390/jcm Squamous carcinoma of the uterine cervix is an infectious cancer associated with 10020220 high risk sub-types of the human papilloma virus (HPV) and represents a major public health burden worldwide. Uterine cervical cancer represents the fourth most common Received: 31 October 2020 cancer in both incidence and mortality in women and led to more than 300,000 deaths Accepted: 7 January 2021 worldwide in 2018 [1]. The introduction of HPV vaccines a few years ago is expected to Published: 10 January 2021 improve these grim statistics in the coming decades [2]. However, cervical cancer will

Publisher’s Note: MDPI stays neu- remain a signiﬁcant health problem for the coming years with an unmet need for effective tral with regard to jurisdictional clai- therapies, especially in the metastatic setting. Molecular characterization of cervical cancer ms in published maps and institutio- pathogenesis holds the promise for advancing therapeutics through the development of nal afﬁliations. rational targeted therapies. PIK3CA, the gene encoding for the alpha catalytic subunit of phosphatidylinositol- 4,5-bisphosphate 3-kinase, is one of the most frequently mutated oncogenes in cancer [3]. The gene is located at the long arm of human chromosome 3 at 3q26.32. The catalytic Copyright: © 2021 by the author. Li- unit encoded by PIK3CA consists of 1068 aminoacids, has a molecular weight of 110 kDa, censee MDPI, Basel, Switzerland. and is regulated by a regulatory subunit of 85 kDa, encoded by PIK3R1. The kinase This article is an open access article receives activating signals from receptor tyrosine kinases and functions to phosphorylate distributed under the terms and con- phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol-3,4,5-triphosphate, which ditions of the Creative Commons At- binds kinase PDK1, the kinase complex mTORC2 and the serine/threonine kinase AKT. The tribution (CC BY) license (https:// proximity of AKT with the two other kinases facilitates phosphorylation of AKT [4]. This creativecommons.org/licenses/by/ phosphorylation results in activation of kinase AKT. AKT phosphorylates and activates 4.0/).

J. Clin. Med. 2021, 10, 220. https://doi.org/10.3390/jcm10020220 https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 220 2 of 13

or inhibits, in its turn, several downstream effectors with pro-carcinogenic actions. An important down-stream effector is the mTORC1 complex, whose activation results in cell growth and protein translation promotion through inhibition of kinase S6K1 and of translation negative regulator EIF4EBP1, respectively [5]. In addition, through the action of multiple downstream effectors, PI3K regulates other cancer associated pathways. For example, mTORC1 executes an inhibitory phosphorylation on kinase GSK3 (Glycogen Synthase Kinase 3), an inhibitor of the Wnt/β-catenin pathway [6]. AKT phosphorylation also leads to inhibitory phosphorylation of tumor suppressors including the transcription factor FOXO1 [7]. PIK3CA mutations are observed frequently in a broad spectrum of cancers, including the most prevalent cancers such as breast, endometrial, and colorectal cancers [8]. In breast cancer, PIK3CA mutations are present in about a third of patients and are more common in ER-positive, HER2-negative cancers [9]. The great majority of mutations are observed in hotspot codons H1047, E542, E545, and N345. PIK3CA mutations have come to the therapeutic forefront in metastatic ER-positive, HER2-negative breast cancers, as they predict response to PI3K inhibitors [10]. In this sub-type of breast cancer, the alpha-specific PI3K inhibitor alpelicib has been approved in combination with hormonal therapy in the metastatic setting after progression on CDK inhibitors combinations [11]. In colorectal adenocarcinomas, PIK3CA mutations occur in 20% to 25% of cases [12–14]. Their presence has been suggested to confer resistance to standard colorectal cancer FOLFOX (5-FU, Folinic acid, Oxaliplatin) chemotherapy [15]. This article examines the landscape of PIK3CA mutated cervical cancers and compares it with that of PIK3CA wild-type carcinomas. In the current therapeutic environment of targeted and personalized therapies, identification and characterization of specific sub-sets of cancers that used to be thought and treated as homogeneous is increasingly important. Currently, the VEGF inhibitor bevacizumab and the PD-1 inhibitor pembrolizumab are the only targeted therapies approved for metastatic cervical carcinomas. Bevacizumab indication is not biomarker based and pembrolizumab indication in the molecule PD-1 positive population provides an imperfect targeting, as the response rate is less than 15%. Additional targeted drugs with associated biomarkers would be more than welcome in metastatic cervical carcinomas. PI3K inhibitors are already in the clinic for other cancers and, given the high prevalence of PIK3CA mutations in the disease, as ascertained in the current study, they could be prioritized for further development in cervical carcinomas.

2. Materials and Methods The Cancer Genome Atlas (TCGA) study of cervical cancer was used to compare patients with or without PIK3CA mutations regarding clinical and genomic characteristics of interest [16]. The study includes a total of 297 patients, among whom 46 patients have adenocarcinoma and were excluded from the current analysis. The remaining 251 patients with squamous cervical carcinoma were included in the analysis. The analysis was performed in the cBioPortal for Cancer Genomics Portal (cBiopor- tal, http://www.cbioportal.org), a genomics site initially developed by Memorial Sloan Kettering Cancer Center (MSKCC) and currently maintained by MSKCC in collaboration with others, which allows for a user friendly interrogation of any gene of interest and includes data on mutations, copy number alterations (CNAs), mRNA expression, as well as correlative clinical data [17]. For the analysis of CNAs in TCGA, the GISTIC (Genomic Identification of Significant Targets in Cancer) algorithm is used. A score of 2 or above in GISTIC denotes putative amplification of a gene, while a score of -2 and below denotes putative deep deletion. The Aneuploidy Score (AS) is derived by summing the number of chromosome arms in each sample that have copy number alterations (gains or losses). A chromosome arm is considered copy number altered, gained, or lost, if there is a somatic copy number alteration in more than 80% of the length of the arm, as calculated by the ABSOLUTE algorithm from Affymetrix 6.0 SNP arrays [18]. Chromosomal arms with somatic copy number alterations in 20% to 80% of the arm length are not called, and chro- J. Clin. Med. 2021, 10, x FOR PEER REVIEW 3 of 13

J. Clin. Med. 2021, 10, 220 3 of 13 ABSOLUTE algorithm from Affymetrix 6.0 SNP arrays [18]. Chromosomal arms with somatic copy number alterations in 20% to 80% of the arm length are not called, and chromosomal arms with somatic copy number alterations in less than 20% of the arm length mosomal arms with somatic copy number alterations in less than 20% of the arm length are are considered not altered. The fraction of genome altered (FGA) in TCGA is derived by considered not altered. The fraction of genome altered (FGA) in TCGA is derived by source source segment files and is calculated by summing the length of segments with log2 segment ﬁles and is calculated by summing the length of segments with log2 greater than greater than 0.2 divided by the total length of all segments measured in the sample. FGA 0.2 divided by the total length of all segments measured in the sample. FGA represents anotherrepresents measure another of themeasure burden of ofthe copy burden number of copy alterations number in alterations a sample. Informationin a sample. ofInfor- the pathogenicmation of the implications pathogenic of implications mutations inof genesmutations of interest in genes was of derived interest fromwas derived the OncoKB from knowledgebasethe OncoKB knowledgebase [19]. [19]. PrognosisPrognosis of of squamous squamous cervical cervical cancer canc patientser patients with with or without or withoutPIK3CA PIK3CAmutations mutations was evaluatedwas evaluated by construction by construction of Kaplan of Kaplan Meier Meier curves. curves. Statistical Statistical comparisons comparisons of categorical of categor- andical continuousand continuous data data were were carried carried with with the Fisher’s the Fisher’s exact exact test ortest the orx the2 test x2 andtest theand t the test. t Thetest. LogThe RankLog Rank test test was was used use tod compareto compare Kaplan–Meier Kaplan–Meier survival survival curves. curves. All All statistical statistical comparisonscomparisons werewere consideredconsidered signiﬁcantsignificant ififp p< < 0.05.0.05.

3.3. ResultsResults PIK3CAPIK3CA mutationsmutations prevalenceprevalence inin TCGATCGA cervicalcervical squamoussquamous cellcell carcinomacarcinoma studystudy isis 27.1%27.1% (67(67 ofof 251251 cases,cases, FigureFigure1 ).1). Thirty-nine Thirty-nine cases cases (15.5%) (15.5%) display display ampliﬁcations amplifications of of the the genegene locatedlocated atat 3q26,3q26, andand aa totaltotal ofof 39%39% ofof casescases havehave eithereither mutationsmutations oror ampliﬁcationsamplifications oror both.both. This This makes makes PIK3CAPIK3CA thethe most most prevalent prevalent lesions lesions in insquamous squamous cervical cervical carcinomas carcino- masin TCGA. in TCGA. PIK3CAPIK3CA mutationsmutations observed observed are misse are missensense mutations mutations,, and andtwo twothirds thirds are con- are concentratedcentrated in aminoacid in aminoacid positions positions E542 E542 and andE545 E545 located located in the in helical the helical PI3Kα PI3K familyα family acces- accessorysory domain domain (aminoacids (aminoacids 520 to 520 703) to of 703) the ofprotein. the protein. These Thesehot spot hot mutations spot mutations are known are knownto be oncogenic to be oncogenic,, as listed as in listed the inOncoKB the OncoKB cancer cancer mutations mutations knowledgebase. knowledgebase. Most muta- Most mutationstions in other in other aminoacid aminoacid positions positions observed observed in inPIK3CAPIK3CA areare also also listed listed as as oncogenic oror likelylikely oncogeniconcogenic inin OncoKB.OncoKB. ConsistentConsistent withwith theirtheir prevalenceprevalence inin cervicalcervical squamoussquamous cellcell carcinomas,carcinomas, PIK3CAPIK3CA mutationsmutations areare observedobserved inin 27.8%27.8% andand 28.1%28.1% ofof casescases inin HPV+ HPV+ headhead andand neckneck cancerscancers andand analanal carcinomascarcinomas fromfrom thethe headhead andand neckneck TCGATCGA studystudy andand thethe analanal carcinomacarcinoma subsetsubset of of the the MSK-IMPACT MSK-IMPACT cohort cohort (Figure (Figure1)[ 81),20 [8,20]. In]. contrast, In contrast, HPV– HPV head– head and neckand neck cancers cancers display displa a lowery a lowerPIK3CA PIK3CAmutations mutations prevalence prevalence of 15.9%. of 15.9%.

FigureFigure 1.1. PrevalencePrevalence ofof PIK3CAPIK3CA mutationsmutations inin cervicalcervical carcinomas, HPV+ and HPV HPV–– head and neck carcinomascarcinomas and and anal anal carcinomas. carcinomas. Fisher’s Fisher’s exact exact test testp p= = 0.01 0.01 for for the the comparison comparison of ofPIK3CA PIK3CAmutations mutations in HPV+ and HPV– head and neck carcinomas. Data are from TCGA and MSK-IMPACT. in HPV+ and HPV– head and neck carcinomas. Data are from TCGA and MSK-IMPACT.

PatientsPatients withwith cervical cervical squamous squamous carcinomas carcinomas and andPIK3CA PIK3CAmutations mutations tend tend to presentto present in ain more a more advanced advanced age age than than the the cervical cervical cancer cancer patients patients without withoutPIK3CA PIK3CAmutations. mutations. More More than 20% of cases in the group with PIK3CA mutations were older than 65, while the percentage of older than 65 years-old patients in the group without PIK3CA mutations

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than 20% of cases in the group with PIK3CA mutations were older than 65, while the percentage of older than 65 years-old patients in the group without PIK3CA mutations was wasless lessthan than 10% 10% (Figure (Figure 2). Conversely,2). Conversely, half half of the of thepatients patients without without PIK3CAPIK3CA mutationsmutations pre- presentsent at age at age 45 45and and younger. younger.

PIK3CA mut % % 30 PIK3CA wt 20

0 <45 45-65 >65

FigureFigure 2.2. AgeAge distribution distribution of of squamous squamous cervical cervical carcinoma carcinoma patients patients with with and and without without PIK3CAPIK3CA 2 mutationsmutations (x(x2 pp == 0.006).

TheThe signalingsignaling pathway pathway transducing transducing signals signals from from cell cell surface surface tyrosine tyrosine kinase kinase receptors recep- (TKRs)tors (TKRs) through through K-Ras K-Ras to MAPK to MAPK kinases kinases is commonlyis commonly dysregulated dysregulated in in cancer. cancer. Several Several componentscomponents of of this this pathway pathway show show low low numbers numbers of of mutations mutations in in squamous squamous cervical cervical cancer. can- Thecer. prevalenceThe prevalence of mutations of mutations of the of backbone the backbone component component genes of genes the pathway of the pathway is higher is inhigher cervical in cervical cancers withcancersPIK3CA with PIK3CAmutations mutations compared compared with the with group the without group PIK3CA without mutationsPIK3CA mutations (Figure3 ).(Figure Overall, 3). theOverall,PIK3CA the PIK3CAmutant mutant group hasgroup mutations has mutations in one orin one more or genesmore ofgenes the TKRs/K-Ras/MAPKof the TKRs/K-Ras/MAPK pathway pathway in 38.8% in 38.8% of cases, of cases while, while the PIK3CA the PIK3CAwild-type wild- grouptype group has mutations has mutations in one in or one more or more genes genes of the of TRK/K-Ras/MAPK the TRK/K-Ras/MAPK pathway pathway in 19% in 19% of casesof cases (Fisher’s (Fisher’s exact exact test testp = p 0.002).= 0.002). The The most most prevalent prevalent mutations mutations are are at at the the gene gene for for the the neurofibrominneurofibromin 11 (NF1),(NF1),a a negativenegative regulatorregulator ofof K-RAS,K-RAS, KRASKRAS itself,itself, andand thethe EGFREGFR familyfamily TKRsTKRs ERBB3ERBB3 andand ERBB2,ERBB2,which which areare mutated mutated in in 9%, 9%, 6%, 6%, 7.5%, 7.5%, and and 6% 6% of ofPIK3CA PIK3CAmutated mutated cases,cases, respectively respectively (Figure(Figure3 ). 3). All All but but one one KRAS KRAS mutations mutations are are hotspot hotspot mutations mutations and and knownknown toto bebe oncogenic.oncogenic. AboutAbout halfhalf ofof thethe mutationsmutations inin thethe threethree otherother genesgenes areare known known oncogenic,oncogenic, whilewhile thethe restrest areare currently currently variants variants of of unknown unknown significance. significance. TheThe signalingsignaling cascade cascade in in which which PI3K PI3K kinase kinase belongs belongs receives receives also also input input from from TKRs TKRs in parallelin parallel with with K-RAS-MAPK, K-RAS-MAPK, through through the th PI3Ke PI3K and and AKT AKT kinases kinases and and presents presents increased increased prevalenceprevalence of of mutationsmutations in inPIK3CA PIK3CAmutated mutated cervicalcervical cancerscancers comparedcompared with with non-mutatednon-mutated counterpartscounterparts (Figure(Figure4 ).4). In In the the PIK3CAPIK3CA mutatedmutated group,group, oneone oror moremore keykey proteinsproteins ofof thethe pathwaypathway areare mutated in in 34.3% 34.3% of of cases cases,, and and in inthe the PIK3CAPIK3CA wildwild-type-type group group,, they they are mu- are mutatedtated in in22.3% 22.3% of ofcases. cases. The The most most frequently frequently mutated mutated genes genes are the tumortumor suppressorssuppressors PTENPTEN and and FBXW7, FBXW7 which, which are are both both mutated mutated in 14.9%in 14.9% of cases of cases in PIK3CA in PIK3CAmutated mutated cancers cancers and PIK3CA inand 5% in and 5% 10.6% and 10.6% of cases, of respectively,cases, respectively in , in PIK3CAwild-type wild cancers-type (Figure cancers4). (FigureIn addition, 4). In small numbers of recurrent deep deletions are present in both PTEN and FBXW7, which addition, small numbers of recurrent deep deletions are present in both PTEN and increases the total prevalence of molecular lesions in these genes in cervical squamous FBXW7, which increases the total prevalence of molecular lesions in these genes in cervi- carcinomas to 13% and 14%, respectively. Most PTEN and FBXW7 cervical carcinoma muta- cal squamous carcinomas to 13% and 14%, respectively. Most PTEN and FBXW7 cervical tions are considered likely oncogenic in OncoKB database, while for a few the significance carcinoma mutations are considered likely oncogenic in OncoKB database, while for a few is unknown. the significance is unknown.

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10 10 9 9 8 8 7 7 6 6 5 5 PIK3CA mut PIK3CA mut % mutant % 4

% mutant % 4 PIK3CA wt 3 PIK3CA wt 3 2 2 1 1 0 0

FigureFigure 3.3. PercentagePercentage ofof mutations mutations of of genes genes of of the the receptor receptor tyrosine tyrosine kinase-RAS-RAF-MEK kinase-RAS-RAF-MEK cascade cascade in Figure 3. Percentage of mutations of genes of the receptor tyrosine kinase-RAS-RAF-MEK cascade PIK3CAin PIK3CAmutant mutant and and wild-type wild-type cervical cervical cancers. cancers. Fisher’s Fisher’s exact exact test testp =p = 0.002 0.002 for for the the comparison comparison of ofin PIK3CAcases with mutant mutations and wild in any-type of thesecervical genes cancers. versus Fisher’s cases withexact none test pof = these0.002 genesfor the mutated comparison in the cases with mutations in any of these genes versus cases with none of these genes mutated in the two twoof cases groups with (PIK3CA mutations mutant in any and of thesePIK3CA genes wild versus-type). cases Data with are fr noneom TCGA. of these genes mutated in the groupstwo groups (PIK3CA (PIK3CAmutant mutant and PIK3CA and PIK3CAwild-type). wild-type). Data Data are from are fr TCGA.om TCGA.

16 16 14 14 12 12 10 10 8 PIK3CA mut 8 PIK3CA mut PIK3CA wt % mutant % PIK3CA wt % mutant % 6 6 4 4 2 2 0 0 PTEN PIK3CG PIK3R1 MTOR FBXW7 PTEN PIK3CG PIK3R1 MTOR FBXW7

Figure 4. Percentage of mutations of genes of the PI3K-AKT-PTEN cascade in PIK3CA mutant and FigurewildFigure-type 4.4. PercentagePercentage cervical cancers. ofof mutationsmutations Fisher’s ofof exact genesgenes test ofof pthethe = 0.01 PI3K-AKT-PTENPI3K for-AKT the -comparisonPTEN cascadecascade of incasesin PIK3CAPIK3CA with mutationsmutantmutant and wild-typeinwild any-type of thesecervical cervical genes cancers. cancers. versusFisher’s Fisher’s cases with exactexact none testtest p ofp= =thes 0.010.01e forforgenes thethe mutated comparisoncomparison in the ofof casestwocases groups withwith mutationsmutations (PIK3CA in anymutantin any ofthese of and these genesPIK3CA genes versus wild versus cases-type). cases with Data with none are none offrom these of TCGA. thes genese genes mutated mutated in the in two the groupstwo groups (PIK3CA (PIK3CAmutant mutant and PIK3CA wild-type). Data are from TCGA. and PIK3CA wild-type). Data are from TCGA. The Wnt/β catenin pathway also presents an increased number of mutations in key TheThe Wnt/Wnt/β catenin pathway also presents an increasedincreased numbernumber ofof mutationsmutations inin keykey component genes in PIK3CA mutated cervical cancers compared with PIK3CA wild-type componentcomponent genesgenes ininPIK3CA PIK3CAmutated mutated cervicalcervical cancerscancers comparedcompared withwithPIK3CA PIK3CAwild-type wild-type counterparts. Most frequently mutated genes of the pathway are the co-receptor LRP1B counterparts.counterparts. MostMost frequentlyfrequently mutatedmutated genesgenes ofof thethe pathwaypathway areare thethe co-receptorco-receptor LRP1BLRP1B (LDL receptor related protein 1B) and the FAT type cadherins FAT4 and FAT1, which (LDL(LDL receptorreceptor related related protein protein 1B) 1B) and and the the FAT FAT type type cadherins cadherins FAT4 FAT4 and FAT1,and FAT1 which, which have have all a prevalence of mutations in PIK3CA mutated cancers of 13.4%, while in the non- allhave a prevalence all a prevalence of mutations of mutations in PIK3CA in PIK3CAmutated mutated cancers ofcancers 13.4%, of while 13.4%, in while the non-mutated in the non- mutated group they are mutated in 10%, 3.3%, and 7.8% of cases, respectively (Figure 5). groupmutated they group are mutated they are inmutated 10%, 3.3%, in 10%, and 3.3% 7.8%, and of cases, 7.8% respectively of cases, respectively (Figure5). (FigureThe core 5). The core component genes of the pathway CTNNB1, encoding for β catenin, APC and componentThe core component genes of thegenes pathway of the pathway CTNNB1, CTNNB1, encoding encoding for β catenin, for β catenin, APC and APC CDH1, and CDH1, encoding for E cadherin, are less frequently mutated, but also their prevalence is encodingCDH1, encoding for E cadherin, for E cadherin, are less frequently are less frequently mutated, mutated, but also their but prevalencealso their pr isevalence higher in is higher in PIK3CA mutated cancers than wild-type ones. LRP1B undergoes, in addition to PIK3CAhigher inmutated PIK3CA cancers mutated than cancers wild-type than ones.wild- LRP1Btype ones. undergoes, LRP1B undergoes, in addition in to addition mutations, to

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deepmutations, deletions deep in deletions cervical cancer in cervical for a cancer total frequency for a total of frequency genetic lesions of genetic of 20%, lesions suggesting of 20%, ansuggesting important an tumor important suppressor tumor role.suppressor role.

PIK3CA mut % mutant % 6 PIK3CA wt

. FigureFigure 5.5. PercentagePercentage of mutations of genes of the the Wnt Wnt//ββ catenincatenin pathway pathway in in PIK3CAPIK3CA mutantmutant and and wild-typewild-type cervicalcervical cancers.cancers. Fisher’sFisher’s exactexact testtest pp == 0.030.03 forfor thethe comparisoncomparison ofof casescases withwith mutationsmutations in anyin any of theseof these genes genes versus versus cases cases with with none none of these of these genes genes mutated mutated in the intwo the groupstwo groups (PIK3CA (PIK3CAmutant andmutantPIK3CA and PIK3CAwild-type). wild Data-type). are Data from are TCGA. from TCGA.

SquamousSquamous cellcell carcinomascarcinomas ofof thethe uterineuterine cervixcervix possesspossess aa lowlow raterate ofof TP53 mutations,mutations, asas thethe functionfunction of of this this seminal seminal tumor tumor suppressor suppressor is debilitated,is debilitated, instead, instead, by by HPV HPV E6 protein.E6 pro- However,tein. However, a small a small number number of cervical of cervical cancers cancers do possess do possess TP53 TP53 mutations, mutations and, and those those are moreare more common common in PIK3CA in PIK3CAmutated mutated cancers cancers (10.4% (10.4% versus versus 5% PIK3CA5% PIK3CAwild-type wild-type cancers, can- Figurecers, Figure6). Other 6). Other proteins proteins involved involved in DNA in DNA damage damage response response and and repair repair have have higher higher mutationmutation ratesrates inin thethe PIK3CAPIK3CA mutatedmutated sub-setsub-set ofof squamoussquamous cervicalcervical carcinomas.carcinomas. BRCA1,BRCA1, BRCA2,BRCA2, BRIP1, ATR ATR,, and and ATM ATM have have all all a amutation mutation frequency frequency of of6% 6% or orhigh higherer (up (up to 9% to 9%for forBRIP1 BRIP1 and and ATM) ATM) in inPIK3CAPIK3CA mutatedmutated cancers, cancers, although although the the overall overall mutation mutation rate rate in inthe the DNA DNA damage damage response response genes genes did did not not reach reach statistical signiﬁcance significance between between the the PIK3CAPIK3CAmutant mutant andandPIK3CA PIK3CAwild-type wild-type groups groups (Fisher’s (Fisher’s exact exact test testp p= = 0.15,0.15, FigureFigure6 ).6). Most Most mutationsmutations observedobserved inin thesethese genesgenes areare currentlycurrently ofof unknownunknown signiﬁcance.significance. MutationsMutations ofof genesgenes involvedinvolved inin mismatchmismatch repairrepair (MMR)(MMR) andand producingproducing microsatellitemicrosatellite instabilityinstability (MSI)(MSI) areare overalloverall rarerare inin cervicalcervical cancerscancers withwith anan individualindividual frequencyfrequency ofof 2%2% oror less.less. However,However, the thePIK3CA PIK3CAmutated mutated group group displays displays higher higher frequencies frequencies ranging ranging from from 3% 3% to 6%to 6% (Figure (Figure7). 7). Similarly, Similarly, the the genes genes for for proofreading proofreading polymerases polymerases POLE POLE and and POLD1 POLD1 are are mutatedmutated inin 7.5% 7.5% and and 4.5% 4.5% of ofPIK3CA PIK3CAmutated mutated cancers cancers versus versus 2.2% 2.2% and and 0.6% 0.6% of PIK3CA of PIK3CAnon- mutatednon-mutated cancers. cancers. Consistent Consistent with thewith higher the higher mutation mutation frequency frequency of MMR of andMMR proofreading and proof- polymerases mutations and the known association of MSI with high total mutation burden reading polymerases mutations and the known association of MSI with high total muta- (TMB), PIK3CA mutated cervical cancers display a higher TMB than PIK3CA wild-type tion burden (TMB), PIK3CA mutated cervical cancers display a higher TMB than PIK3CA counterparts (Figure8). Importantly, almost 40% of PIK3CA mutated cervical cancers have wild-type counterparts (Figure 8). Importantly, almost 40% of PIK3CA mutated cervical a TMB of above 180. This percentage is only 12.3% in unmutated cancers. cancers have a TMB of above 180. This percentage is only 12.3% in unmutated cancers.

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12 12

10 10

8 8

6 PIK3CA mut PIK3CA mut

6 PIK3CA wt % mutant % PIK3CA wt % mutant % 4 4

2 2

0 0 TP53 BRCA1 BRCA2 BRIP1 RAD50 RAD51C ATM ATR CDK12 TP53 BRCA1 BRCA2 BRIP1 RAD50 RAD51C ATM ATR CDK12

FigureFigure 6.6. PercentagePercentage ofof mutations of genes involved in DNA damage response and repair pat pathwaysh- inFigurewaysPIK3CA in 6. PIK3CA Percentagemutant mutant and of wild-typemutations and wild- cervicaloftype genes cervical cancers. involved cancers. Fisher’s in DNA Fisher’s exact damage exact test response ptest= 0.15 p = 0.15 for and thefor repair comparisonthe compari- path- of waysson of in cases PIK3CA with mutant mutations and in wild any- typeof these cervical genes cancers. versus Fisher’scases with exact none test of p these= 0.15 genes for the mutated compari- in cases with mutations in any of these genes versus cases with none of these genes mutated in the two sonthe twoof cases groups with (PIK3CA mutations mutant in any and of PIK3CAthese genes wild versus-type). cases Data withare from none TCGA. of these genes mutated in groups (PIK3CA mutant and PIK3CA wild-type). Data are from TCGA. the two groups (PIK3CA mutant and PIK3CA wild-type). Data are from TCGA. 10 10 9 9 8 8 7 7 6 6 5 PIK3CA mut PIK3CA mut 5 PIK3CA wt % mutant % 4 PIK3CA wt % mutant % 4 3 3 2 2 1 1 0 0 MSH6 MSH2 MSH3 MLH1 PMS2 PMS1 POLE POLD1 POLQ MSH6 MSH2 MSH3 MLH1 PMS2 PMS1 POLE POLD1 POLQ Figure 7. Percentage of mutations of mismatch repair (MMR) genes and genes encoding for the FigureFigureproof-reading 7.7. PercentagePercentage polymerases of of mutations mutations epsilon of and of mismatch mismatch delta (POLE repair repair and (MMR) (MMR)POLD1) genes genes in andPIK3CA and genes genes mutant encoding encoding and for wild the for- the proof-readingprooftype cervical-reading cancers. polymerasespolymerases Fisher’s epsilon exact andtest pdelta = 0.0004 (POLE for and the POLD1)comparison POLD1) in in PIK3CAPIK3CA of cases mutant mutantwith mutations and and wild wild-type in- typeany of cervical these genes cancers. versus Fisher’s cases exact with test none p = of 0.0004 these forgenes the mutatedcomparison in the of casestwo groups with mutations (PIK3CA mu-in cervical cancers. Fisher’s exact test p = 0.0004 for the comparison of cases with mutations in any of anytant ofand these PIK3CA genes wild versus-type). cases Data with are none from of TCGA. these genes mutated in the two groups (PIK3CA mu- these genes versus cases with none of these genes mutated in the two groups (PIK3CA mutant and tant and PIK3CA wild-type). Data are from TCGA. PIK3CA wild-type). Data are from TCGA.

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60 60

50 50

40 40

30 PIK3CA mut 30 PIK3CA mut % TMB % PIK3CA wt % TMB % PIK3CA wt 20 20

10 10

0 0 <80 80-180 >180 <80 80-180 >180

Figure 8. Percentage of cases with different levels of total mutation burden (TMB) in PIK3CA mutant andFigureFigure wild-type 8.8.Percentage Percentage cervical cancers of of cases cases (x with with2 p < different 0.001).different Data levels levels are of offrom total total TCGA. mutation mutation burden burden (TMB) (TMB) in inPIK3CA PIK3CAmutant mu- 2 andtant wild-type and wild- cervicaltype cervical cancers cancers (x2 p <(x 0.001). p < 0.001). Data Data are from are from TCGA. TCGA. In contrast to MSI, chromosomal instability as measured by AS is lower in PIK3CA mutated cervicalInIn contrastcontrast cancers toto compared MSI,MSI, chromosomalchromosomal to PIK3CA instabilityinstability wild-type as ascounterparts measuredmeasured by by(Figure ASAS isis 9A). lower lower AS in inbe-PIK3CA PIK3CA low 4 ismutatedmutated observed cervicalcervical in 30.3% cancerscancers mutated compared compared cancers tocompared to PIK3CAPIK3CA towildwild-type 15.9%-type of counterparts PIK3CA counterparts wild (Figure-type (Figure squa- 9A).9A). AS AS be- mous cervicalbelowlow 4 is 4 carcinomas. isobserved observed in In30.3% in addition, 30.3% mutated mutated the cancersPIK3CA cancers compared mutated compared groupto 15.9% to pres 15.9% ofents PIK3CA of aPIK3CA higher wild per--wild-typetype squa- centagesquamousmous of low cervical Frac cervicaltion carcinomas. of carcinomas. Genome In Alteredaddition, In addition, (FGA, the PIK3CA the anotherPIK3CA mutated measuremutated group of groupchromosomal pres presentsents a higher in- a higher per- stability)percentagecentage cases thanof oflow the low Frac PIK3CA Fractiontion of wild ofGenome Genome-type group Altered Altered (Figure (FGA, (FGA, 9B). another another measure measure of ofchromosomal chromosomal in- instability)stability) cases cases than than the the PIK3CAPIK3CA wildwild-type-type group group (Figure (Figure 9B).9B).

90 90 80 80 70 70 60 60 50

50 % %

40 % PIK3CA mut 40 PIK3CA mut PIK3CA wt 30 PIK3CA wt 30 20 20 10 10 0 0 <4 4 to 24 >24 <4 4 to 24 >24 AS AS

(A) (A) Figure 9. Cont.

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30 PIK3CA mut

% FGA % PIK3CA wt

0 <0.05 0.05-0.25 >0.25

(B) Figure Figure9. Percentage 9. Percentage of cases ofwith cases different with different levels of levelschromosomal of chromosomal instability instability as measured as measured by (A) by (A) The AneuploidyThe Aneuploidy Score (AS) Score (x (AS)2 p = (x0.03).2 p = (B 0.03).) The ( Bfraction) The fraction of genome of genome altered altered (FGA) (FGA) in PIK3CA in PIK3CA mu- mutant tant andand wild wild-type-type cervical cervical cancers cancers (x2 (xp 2=p 0.01).= 0.01). Data Data are arefrom from TCGA. TCGA.

As mentionedAs mentioned above,above, about 15% about of 15% cervical of cervical cancers cancers bear amplifications bear amplifications of PIK3CA of PIK3CA as as part ofpart a commonly of a commonly amplified amplified locus at locus 3q26 at-28. 3q26-28. This locus This contains locus contains also other also oncogenes, other oncogenes, includingincluding MECOM, MECOM, encoding encoding for oncogene for oncogene EVI1, SOX2, EVI1, SOX2,the p53 the family p53 familymember member TP63, and TP63, and ETV5. ETV5.The area The of area the of long the longarm armof chromosome of chromosome 3 at 3 3q26 at 3q26-28-28 is iscommonly commonly amplified amplified in in other other squamoussquamous carcinoma carcinomass such such as as lung lung and and head head and and neck neck cancers. cancers. A A comparison comparison of of the the rate rate ofof PIK3CAPIK3CA mutationsmutations in in 3q26 3q26-28-28 amplifiedamplified andand non-amplifiednon-amplified cervical cervical cancers cancers disclosed dis- no closed significantno significant difference, difference, with with the the amplified amplified group group harboring harboringPIK3CA PIK3CAmutations mutations in 26.3%in of 26.3% casesof cases and and the th non-amplifiede non-amplified group group harboring harboring such such mutations mutations in 27.3%in 27.3% of cases.of cases. SurvivalSurvival analysis analysis of TCGA of TCGAcohort cohortdisclosed disclosed that neither that neither mutations mutations nor amplifications nor amplifications of PIK3CAof PIK3CA are prognosticare prognostic for disease for disease free survival free survival (DFS) (DFS) or overall or overall survival survival (OS) (not (OS) (not shown).shown). However, However, when whenlesions lesions of PIK3CA of PIK3CA were werecombined combined a trend a trend towards towards a better a better DFS DFS in in the thegroup group with with PIK3CAPIK3CA alterationsalterations that that did did not not reach reach statistical statistical significance significance (p (p= =0.06) 0.06) was was observedobserved (Figure (Figure 10). 10 OS). OS remained remained not not different different between between groups. groups. These These results results sug- suggest gest thatthat despite despite the the key key role role of of PI3K PI3K kinase in oncogenesis, itit isis notnot prognostic prognostic for for outcomes out- in comes squamousin squamous cervical cervical carcinoma. carcinoma. However, However, the the fact fact that that DFS DFS was was worse, worse, albeit albeit not not reaching reachingstatistical statistical significance, significance, in cancersin cancers with with PI3K PI3K lesions lesions is intriguing is intriguing and and suggests suggest thats PI3K that PI3Kdysfunction dysfunction may may have have prognostic prognostic implications implications in these in these cancers. cancers. This This could could be confirmed be confirmedwith with a more a more extensive extensive study study size. size.

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FigureFigure 10.10.Disease Disease freefree survivalsurvival (DFS)(DFS) ofof cervicalcervical cancercancer patientspatients withwith mutationsmutations oror ampliﬁcationsamplifications ofofPIK3CA PIK3CAcompared compared withwith patients patients with with no no molecular molecular lesions lesions in in the the gene. gene.

4.4. DiscussionDiscussion SquamousSquamous cervicalcervical carcinomas carcinomas present present a particulara particular molecular molecular landscape landscape characterized character- byized low by prevalence low prevalence of mutations of mutations in the in tumor the tumor suppressor suppressor p53. Instead, p53. Instead, in these in carcinomas, these carci- thenomas, multifaceted the multifaceted functions functions of p53 in of suppressing p53 in suppressing virtually virtually all enabling all enabling characteristics character- of canceristics of are cancer neutralized are neutralized by the HPV by proteinthe HPV E6 protein [21]. E6 E6 promotes [21]. E6 proteasomepromotes proteasome degradation deg- of wild-typeradation p53of wild by facilitation-type p53 of by ubiquitination facilitation of of ubiquitinationp53 by ubiquitin of ligase p53 UBE3A, by ubiquitin also known ligase asUBE3A, E6-AP also (E6-Associated known as E6 Protein)-AP (E6 [22-Associated]. In addition, Protein) the viral [22]. protein In addition, E7 has the been viral implicated protein inE7 HPV has been related implicated tumorigenesis in HPV by related interference tumorigenesis with the function by interference of the tumor with suppressorthe function RB of andthe tumor neutralization suppressor of cell RBcycle and neutralization checkpoints. As of acell result, cycle cancer checkpoints. cell proliferation As a result, becomes cancer independentcell proliferation from becomes physiologic independent signals that from normally physiologic regulate signals untimely that normally cell cycle regulate entry. PIK3CAuntimelyencodes cell cycle for entry. the catalytic PIK3CA sub-unit encodes of for lipid the kinase catalytic PI3K, sub which-unit isof anlipid internal kinase part PI3K of, signalingwhich is cascadesan internal that part connect of signaling receptor cascades tyrosine kinasesthat connect to the mTORreceptor pathway tyrosine and kinases protein to synthesis,the mTOR inhibition pathway and of cell protein cycle synthesis, arrest, and inhibition inhibition of of cell apoptosis cycle arrest [3]., Itand also inhibition transmits of signalsapoptosis through [3]. It thealso TGF transmitsβ/Smad signals signaling through pathway the TGFβ and the/Smad Wnt/ signalingβ-catenin pathway pathway, and both the importantWnt/β-catenin transducers pathway of, oncogenesis-relatedboth important transducers signals and of alteredoncogenesis in various-related cancers. signals Thus, and inaltered cervical in various carcinomas, cancers.PIK3CA Thus,activation in cervical through carcinomas, mutations PIK3CA could activation assist viral-induced through mu- oncogenesistations could by assist further viral incurring-induced oncogenesis on p53 inhibition by further and enhancingincurring on proliferation p53 inhibition by celland cycleenhancing promotion. proliferation by cell cycle promotion. PIK3CAPIK3CA isis thethe secondsecond mostmost frequentlyfrequently mutatedmutated oncogeneoncogene acrossacross cancerscancers [[23].23]. InIn thethe TCGATCGA cohortcohort ofof squamoussquamous cervicalcervical carcinomas,carcinomas,it it isis mutatedmutated inin 24%,24%, showingshowing thethe highesthighest prevalence of mutations of all oncogenes in these cancers. Clinically, PIK3CA mutations prevalence of mutations of all oncogenes in these cancers. Clinically, PIK3CA mutations are associated with a more advanced age at presentation. This association with older are associated with a more advanced age at presentation. This association with older age age at presentation had also been observed in another series of cervical carcinomas that at presentation had also been observed in another series of cervical carcinomas that in- included both squamous and adenocarcinoma histologies [24]. The subset of cervical cluded both squamous and adenocarcinoma histologies [24]. The subset of cervical can- cancers with PIK3CA mutations tend to have additional mutations in cancer-associated cers with PIK3CA mutations tend to have additional mutations in cancer-associated genes genes of oncogenic pathways more frequently than patients without PIK3CA mutations. of oncogenic pathways more frequently than patients without PIK3CA mutations. Con- Consistent with this observation, the percentage of patients with PIK3CA mutations and a sistent with this observation, the percentage of patients with PIK3CA mutations and a high high TMB is higher than in patients without PIK3CA mutations. MMR and proofreading

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polymerases mutations are more often seen in PIK3CA mutated cancers. However, the great majority of PIK3CA mutations are concentrated in a few hotspot codons and almost the totality of the remaining mutations is classified as probably oncogenic, strongly suggesting that they are not passenger mutations occurring by chance in hypermutated cancers. Surprisingly, increased TMB in PIK3CA mutated cancers does not translate to clearly better survival outcomes, implying that any positive influence of the hypermutated phenotype may be neutralized by other factors in the PIK3CA mutated cellular micro-environment. In colorectal cancers, MSI are associated with a better prognosis than microsatellite stable cancers [25]. Thus, other factors present in the cervical cancer microenvironment, beyond the increased number of antigens presented to immune effectors, are at play in MSI related cancers and hinders an improved prognosis. In that respect, the oncogenic HPV-encoded E5 protein interferes with antigen presentation and attenuates immune responses [26]. In another series of mostly squamous cervical cancers, early stage IB and II PIK3CA mutated cancers had worse survival than patients with same stages PIK3CA wild-type cancers [27]. In contrast, no survival difference was observed in stage III and IVA cancers. Yet in a series with advanced and metastatic pretreated patients with squamous cervical carcinomas who had participated in phase I trials, patients with PIK3CA mutations had a better survival than non-mutated patients [28]. Amplifications of the locus of PIK3CA at chromosome arm 3q26 are the most common copy number alterations in squamous cervical cancers in TCGA. Mutations of the gene occur in equal frequencies in 3q26 amplified and non-amplified cases, suggesting that PIK3CA mutations are non-redundant with gene amplifications. This is corroborated by the fact that several cases in TCGA with PIK3CA mutations but without concomitant amplification display increased expression of PIK3CA at the mRNA level. PI3K inhibitors are currently available and have obtained regulatory approval for ER-positive, HER2-negative breast cancers bearing PIK3CA mutations [11]. The PI3Kα specific inhibitor alpelisib was studied in a phase I study in various solid tumors with PIK3CA mutations [29]. Interestingly, among the five cervical cancer patients included, three patients obtained a partial response. Combinations of PI3K inhibitors with other targeted therapies could be of interest for further development. There is, for example, a suggestion from preclinical studies that PIK3CA mutations may sensitize cervical cancer cells to PARP inhibitors [30]. High risk HPV strains associated with cervical cancer promote defects in homologous recombination in cervical cancer cells [31]. The HPV oncoproteins E6 and E7 sequester protein RAD51 away from double strand lesions and significantly impair the ability of the cell to repair double strand breaks. Activity of alpelisib with olaparib has been observed in platinum refractory pretreated ovarian cancer patients with a response rate of 36% [32]. The combination would be a rational therapy to be investigated in cervical cancers with DDR defects derived from the presence of viral oncogenes and mutations of genes encoding for DDR proteins, which as shown here are present in a significant minority of squamous cervical carcinomas, mostly with PIK3CA mutations. Increased TMB has been arising as a marker for response to immunotherapy with PD-1/PD-L1 and CTLA-4 inhibitors [33,34]. The PD-1 inhibitor pembrolizumab has obtained accelerated approval in patients with metastatic PD-L1 positive (combined positive score ≥ 1%) cervical cancer who had progressed on chemotherapy [35]. The great majority (83.7%) of patients had PD-L1 positive cancers. The response rate was 14.6%, and thus predictive biomarkers beyond PD-L1 are needed. It would be interesting to clarify whether PIK3CA mutations, associated with higher TMB, could be such a biomarker. However, given the low response rates with PD-1 inhibitor immunotherapy therapy in cervical cancer that have also been confirmed in an early nivolumab trial, combination therapies are the most promising and preferable development strategy [36]. In this regard, the combination of nivolumab with ipilimumab has produced response rates between 32% and 46%, de- pending on the schedule, in a phase I/II multi-cohort trial [37]. Trials of combinations of PD-1 inhibitors with other targeted therapies such as PI3K inhibitors, guided by molecular markers such as PIK3CA mutations, should be considered as one of the next steps of cervical J. Clin. Med. 2021, 10, 220 12 of 13

cancer therapeutics. An advantage of the combination of PI3K inhibitors with PD-1/PD-L1 is the non-overlapping adverse effect profile of kinase inhibitors with immunotherapeutics. In addition, with a better biomarker targeting, such combinations may increase the sub-sets of patients who derive benefit, without a decrease in quality of life. The adverse effect profile of combinations of targeted therapies will be a key factor in their development especially in the metastatic setting, given the importance of quality of life maintenance in these patients.

Funding: No funding was received to perform this study. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: No additional data are available. Conﬂicts of Interest: The author declares no conﬂict of interest regarding this study.

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