To the Editor,
Worldwide, endometrial cancer (EC) is the most common cancer of the female reproductive tract and the fourth most commonly diagnosed cancer in women after breast cancer, lung cancer, and cancer of the colon and rectum; affecting 320,000 women per year . Bokhman has classified EC into two subtypes: type I tumors or estrogen-dependent endometrioid endometrial carcinomas (EECs) and type II tumors or non-endometrioid endometrial carcinomas (NEECs) . EECs that are mediated through estrogen pathways are the most common ECs, accounting for more than 80% of cases . In type I, alterations in the phosphatase and tensin homolog (PTEN), phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA), K-ras, b-catenin (CTNNB1) and/or DNA mismatch repair genes have been reported . However, in type II, the most common genetic alterations were in inactivation of p16 and overexpression of HER-2/neu .
In the current study, we were presented with a case of premenopausal woman suffering from EC and having a cancer family history from both paternal and maternal sides. Our main aim was to accurately classify this case of EC into a subtype, then to report the associated genetic and protein biomarkers, thus individualized treatment strategies can be developed for maximum effectiveness.
We obtained a written informed consent from the affected individual for sequencing analysis, publication of this report and accompanying images.
The 38-year-old premenopausal woman presented to the hospital with EC had undergone hysterectomy. The hysterectomy specimen received in the pathology laboratory, is a 139 g uterus including a 6 cm×6 cm corpus and a 4.5 cm×3.5 cm cervix. The cervix was bulky with a dilated cervical os from which a hemorrhagic granular tissue was protruding. The exocervix was surrounded by a rim of vaginal mucosa. The endocervical canal was patent and lined by a granular, hemorrhagic, 0.8 cm thick mucosa. The endometrial cavity was lined by a 0.5 cm granular, hemorrhagic endometrium with a 1.8 cm×0.7 cm polyp. The right uterine fundus showed a 2.5 cm×1.5 cm ill-defined white myometrial mass.
The histological sections showed atypical hyperplasia and several scattered foci of well to moderately differentiated endometrioid adenocarcinoma (FIGO II) with myometrial invasion. Immunohistochemistry (IHC) was also performed on several sections where positive expression of estrogen receptors (ER), progesterone receptor (PR), topoisomerase II α (TOP2A), transducin-like enhancer of split 3 (TLE3), epidermal growth factor receptor (EGFR), programmed death (PD-1), hepatocyte growth factor receptor (cMET), O6-methylguanine-DNA methyltransferase (MGMT), ribonucleotide reductase M1 (RRM1), thymidylate synthase (TS) was demonstrated as shown in Figure 1a. However, tumor cells were negative for androgen receptor (AR) and PTEN (Figure 1a).
Direct next-generation sequencing (NGS) analysis of 44 genes was performed on genomic DNA isolated from a formalin-fixed paraffin-embedded endometrium sample (Supplementary Table 1). Overall, eight mutations in five genes were detected (Table 1). The c.517C>T p.R173C in PTEN was reported previously as germline and somatic (found in several tumor types) (Table 1) . In cell culture studies by Han et al. , cells harboring this mutation lost their phosphatase activity , implying that this mutation is pathogenic. Two additional novel mutations were identified; c.625G>A, p.G209R and c.696C>A, p.R233X in exons 6 and 7, respectively, and our bioinformatics analysis showed them as possibly pathogenic (Table 1). PTEN is a tumor suppressor gene that prevents cells from proliferating through regulating the PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α) in the receptor tyrosine kinase pathway . Studies have showed that PTEN is an important mediator in signaling downstream of EGFR, and loss of PTEN gene function/expression due to gene mutations or allele loss is associated with reduced benefit to EGFR-targeted monoclonal antibodies . When combining the genetic and IHC results of PTEN, we conclude that the identified mutations lead to loss of function of the encoded phosphatase (PTEN) as regulator of the PIK3CA/AKT pathway .
Going in the same direction, we have identified c.277C>T p.R93W in PIK3CA, this gain of function mutation has been reported in a number of tumors, and it has been shown in cell studies to activate the PIK3CA protein . One additional mutation, p.H1047R in exon 20 of PIK3CA gene was also detected. Based on all the previous findings, we conclude that the identified mutations in PTEN and PIK3CA genes lead to a defective mTOR signaling pathway. This pathway is an active target for drug development especially for everolimus and temsirolimus, both drugs are kinase inhibitors that inhibit cell division and angiogenesis through acting on mTOR pathway . In addition, the above genetic and protein biomarkers classify the case we present as having EEC.
We have also identified a frameshift mutation; p.P291fs*51 leading to a premature termination codon in hepatocyte nuclear factor 1 homeobox A (HNF1A) gene, known to be mutated in various forms of cancers . Finally, we detected two novel missense mutations of unknown significance: V199M in BRCA1 and p.P369S in STK11 genes (Table 1).
CISH analysis using hybridization probes was aslo performed on Her2/Neu and cMET loci, however this technique did not reveal any copy number variation (Supplementary Table 2).
Following the genetic and protein profiling of EEC tumor the clinician prescribed everolimus treatment for 5 months. This treatment is based on previous work describing that affected individuals having mutations in exon 20 of PIK3CA (more specifically H1047R) were benefiting from mTOR inhibitors such as everolimus and temsirolimus . Despite these encouraging observations, our case did not show any signs of improvement. Since the tumor was positive for PD-1, pembrolizumab treatment followed everolimus. Interestingly, our affected individual responded positively after five cycles of treatment (over 24 weeks), and tumor size decreased in size from 7 cm×4.4 cm×10.5 cm to 6.5 cm×3 cm×7.5 cm (Figure 1b). Several emerging studies demonstrate that EC tumors with high mutational burdens exhibit a greater response rate to immune checkpoint blockade [9, 10]. Mehnert et al. , reported that tumors harboring mutations in the gene coding for the catalytic subunit of DNA polymerase (POLE) are good candidates for immune checkpoint inhibitor therapy . Furthermore, Le et al.  proved that microsatellite instability caused by mutations in mismatch repair genes (MSH2, MSH6, MLH1, PMS2, …) makes tumors more susceptible to immune checkpoint blockade . The previously mentioned studies show that mutations in POLE and MMR genes allow the affected individuals to benefit the most from pembrolizumab treatment. Our observations imply that the herein reported case may have mutations in those gene(s), unfortunately, we could not test this hypothesis for two reasons. First, we have used a targeted NGS panel and not whole exome sequencing interrogating the complete set of genes, second to study the microsatellite instability sequencing technology should be performed pre, and post treatment. In conclusion, our case report identified a fraction of biomarkers that might be implicated in the etiology of EEC.
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The online version of this article (https://doi.org/10.1515/cclm-2017-0208) offers supplementary material, available to authorized users.
About the article
Published Online: 2017-06-29
Published in Print: 2017-11-27
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.