Hereditary non-polyposis colorectal cancer (HNPCC; Lynch syndrome (LS)) is an autosomal dominant disorder that is attributable to a germline mutation in one or more of the 4 DNA mismatch repair (MMR) genes: MSH2 and MSH6 (chromosome 2), MLH1 (chromosome 3), and PMS2 (chromosome 7) [8]. The genes may be inactivated, however, by other mechanisms, including somatic MLH1 promoter methylation, miRNA overexpression, or rarely, epigenetic silencing of MSH2 through deletion of the adjacent EPCAM gene.

Dysfunction or loss of the MMR system eventuates in DNA replication errors to the genome, some of which are seen as alterations in the lengths of short repeated sequences known as microsatellites, and some of which eventuate in the development of cancers. Patients with LS are at increased risk for cancers from numerous sites, including a 40–60 % lifetime risk of both endometrial and colorectal cancers, and a 9–12 % lifetime risk of ovarian cancer [9].

Although only 2–5 % of endometrial carcinomas are LS-related, 50–60 % of LS patients will present first with an endometrial carcinoma primary [10–12]. Most endometrial carcinomas associated with the LS have alterations in MSH6, MSH2 or MLH1. The diagnosis of a LS-associated endometrial carcinoma not only allows the possibility of counseling and increased surveillance for the index patient’s relatives who are at risk, but may also allow the earlier detection of second LS-associated malignancy in the index patient [13, 14]. Additionally, an LS-associated carcinoma may have prognostic significance for the index patient, as there is some preliminary evidence that they tend to be more aggressive, although the available data is not entirely congruent on this point [10, 15, 16].

There is currently an ongoing debate about the level of testing of endometrial carcinomas that is required to maximize the identification of the 2–5 % that may be associated with LS. Tumor-associated characteristics have been proposed to identify cases in need of further testing, since MMR-abnormal tumors (with the possible exception of MSH6-altered tumors) tend to show frequent tumor infiltrating lymphocytes, undifferentiated or dedifferentiated areas, and lower uterine segment origin [17, 18]. Other features of LS-associated pathology include type II endometrial carcinomas in younger patients, and an overrepresentation of the endometriosis-associated tumors namely; endometrioid and clear cell carcinoma (CCC) in the ovary [19].

Traditional Amsterdam I and II, Society of Gynecologic Oncologists (SGO), and revised Bethesda criteria are now recognized to be insensitive for identifying older LS patients and morphology-based criteria are also similarly insensitive as a primary strategy [20–26]. The SGO, reflecting a progressive change in sentiment toward a more universal screening approach, has recently endorsed a more expansive strategy, endorsing “A more sensitive strategy” to detect LS that includes “universal molecular tumor testing for either all endometrial cancers or cancers diagnosed at age less than 60 years, regardless of personal or family cancer history” if resources are available [27]. The Stanford group recently noted that “41 % with an LS-associated germline mutation were not associated with any of the traditional indicators that have been recommended for LS screening (i.e., age 50 years or younger, personal/family cancer pedigree that meets Bethesda guideline criteria, presence of MMR-associated tumor morphology, or location in the lower uterine segment)” [28]. The authors recommended universal testing for all newly diagnosed endometrial carcinomas [28]. A long-standing group of European experts recently recommended that all women diagnosed with endometrial cancer up to the age of 70 years be offered tumor testing by MSI or IHC [29]. Others have endorsed a more hybrid strategy that combines universal testing by MSH6 IHC and selective testing of MLH1, PMS2, and MSH2 IHC on the basis of clinicopathologic parameters [30].

A consensus seems to be emerging around universal testing for all histotypes up to the age of 60 years. This approach has been found to display the best performance in two recent studies, and entailed the testing of all endometrial carcinomas by MMR IHC in patients younger than 60 years, tumor MLH1 methylation in patients with MLH1 IHC loss, and germline mutations in patients exhibiting loss of MSH6, MSH2, or PMS2 or loss of MLH1/PMS2 with absence of MLH1 methylation [20, 31].

Testing for MSI is performed by a comparative analysis of the polymerase chain reaction (PCR)-amplified microsatellite fragments between the tumor and normal tissue from the same patient. Paraffin-embedded tissue (tumor) and normal tissue (blood or paraffin-embedded tissue) are used for the test [32]. A panel of five microsatellite markers referred to as the Bethesda panel has been recommended for initial screening. This panel includes two mononucleotide (BAT-25 and BAT-26) and three dinucleotide (D5S346, D2S123, and D17S250) repeats. Samples with instability in at least two of these markers are defined as MSI-High (MSI-H), whereas those with one unstable marker are designated as MSI-Low (MSI-L) and samples with no detectable alterations are microsatellite-stable (MSS) [33]. Testing a secondary panel of mononucleotide markers, such as BAT-40, to exclude MSI-L in cases in which only the dinucleotide repeats are mutated has been recommended [23] (Fig. 15.1). Approximately 20–23 % of endometrial carcinomas show high frequency MSI-H, and approximately 80 % of these are due to an epigenetic inactivation (methylation) of the MLH1 promoter [20]. MSI testing would be ineffective as a primary screening modality without additional testing because (1) approximately 10–30 % of sporadic EECs are MSI-H [34], and less than 20 % of these are true LS-associated cancers [35] and (2) A significant subset of MSH6 mutated endometrial carcinomas are MSS [36, 37].

IHC is a reasonably sensitive and specific approach for identifying the specific MMR that is altered in endometrial carcinoma, and for screening all endometrial carcinoma under a universal strategy [11, 38]. In one analysis, IHC with MLH1 and MSH2 alone detected 69 % of MSI-H tumors with a specificity of 100 %. The inclusion of the other two markers (PMS2 and MSH6) to the panel increased the sensitivity to 91 %, but decreased the specificity to 83 %. MSH6 loss by IHC primarily was responsible for the decreased specificity, as it did not correlate as well as the others with MSI [36]. MSH2 and MSH6 are obligate dimerization partners, as are MLH1 and PMS2. Each member of the pair is generally unstable when unpaired. As such, a defect in one gene that eventuates in loss of expression of its corresponding protein is typically accompanied by loss of expression of its partner [39]. Interpretation of MMR IHC should only be performed in conjunction with internal controls with retained expression [40]. The latter may be stroma, endothelial cells, inflammatory cells, or myometrium. Additionally, care must be taken not to interpret foci of atypical hyperplasia, which are frequently admixed with, or adjacent to endometrial carcinoma. Finally, MMR protein expression may be artifactually altered by the lack of prompt or adequate formalin fixation, and this possibility should be considered especially in cases with only focal or equivocal expression. Parenthetically, MSI has also been seen in approximately 20 % of endometrial hyperplasia, suggesting that MSI may be an early event in the genesis of EEC [41].

In a recent analysis of minimally selected endometrial carcinoma patients, 5.9 % (7/118) showed a germline mutation: 4 had MLH1, 2 had MSH6, and 1 had MSH2 [20]. In another study, germline mutation were identified in 3 % [31]. The current approach includes direct gene sequencing on DNA extracted from fresh tissue or peripheral blood, to detect genomic insertion/deletion rearrangements of the MMR genes and/or deletions in the 3′ end of EPCAM where they most commonly occur [31].

As previously noted, the vast majority of endometrial carcinoma with abnormal MMR protein expression has loss of MLH1 due to methylation of the corresponding MLH1 promoter. As such methylation analysis is generally performed on tumors showing MLH1/PMS2 protein loss. A common approach to methylation analyses is the use of sodium bisulfite conversion and quantitative MethyLight assays [42]. MLH1 methylation is generally quantitatively reported based on the percentage of methylated reference (PMR) calculations, using ≥ 4 % PMR as cutoff for positive methylation status [31]. BRAF (V600E) mutational and/or IHC analyses, which are useful in distinguishing sporadic, MSI-H colorectal cancers from LS-associated MSI-H cancers, are of less utility in endometrial carcinomas, since BRAF mutations are extraordinarily infrequent in endometrial carcinomas [43–45].

Erb-b2 receptor tyrosine kinase 2 (ERBB2 also known as HER2), is a member of the human epidermal growth factor receptor (EGFR) family of tyrosine kinases. A ligand binding of these transmembrane receptors result in their dimerization and intracellular domain phosphorylation, with eventual activation of pathways associated with proliferation and differentiation. In general, ERBB2 (HER2) alterations are significantly more frequently found in non-endometrioid (NEEC) as compared with EECs [46]. ERBB2 alterations have been most extensively studied in ESC, where ERBB2 overexpression as assessed by immunohistochemistry (IHC) has reportedly ranged from 14 % to 80 %, and ERBB2 gene amplification as assessed by fluorescent in situ hybridization (FISH) has reportedly ranged from 21 % to 47 % [47–63]. The significance of ERBB2 overexpression and/or amplification (alterations) is controversial. Regarding prognosis, most studies have reported an association between ERBB2 alterations and poor survival [50, 54, 55, 62] but other studies have found no such association [56, 64]. The role of targeting ERBB2 protein has also been assessed in isolated reports [47, 65, 66]. However, to date, prospective trials have failed to show any significant therapeutic effect to this approach in patients with ESC. A randomized phase II study (ClinicalTrials.gov Identifier: NCT01367002) is currently being conducted in patients with advanced stage or recurrent 3+ ERBB2 overexpressing or FISH-amplified ESC, assessing carboplatin/paclitaxel with or without the anti-ERBB2 (HER2) agent trastuzumab. Nonetheless, in vitro studies continue to demonstrate the effectiveness of therapeutic approaches targeting the ERBB2 protein in serous carcinoma cell lines, including T-DM1, an antibody–drug conjugate, and neratinib, an ERBB1 and ERBB2 inhibitor [67, 68]. Taselisib, a selective inhibitor of PIK3CA, has been shown to be effective on PIK3CA-mutated and ERBB2 amplified ESC in vitro and in vivo, whereas GDC-0980, an inhibitor of class I PI3 kinase and mTOR kinase (TORC1/2), and AZD8055, a novel dual mTORC1/2 inhibitor have similarly been shown to be effective in ESC cell lines showing oncogenic PIK3CA mutations and ERBB2 gene amplification in vitro [69–71].

There are currently no well-established guidelines for ERBB2 testing in endometrial carcinoma. The staining pattern by IHC is often basolateral or lateral, without the circumferential staining that is characteristic of ERBB2-amplified breast cancers, and the staining is often heterogeneous within a given tumor [72, 73]. The latter suggests that large tumor sections are preferable for this testing, and that results from small samples, such as biopsies and curettages be viewed with some skepticism. Direct extrapolation of breast scoring criteria to the endometrium probably results in a significant overestimation of the rate of ERBB2 amplification when IHC is used as the primary means of its estimation [73]. One recent study found that the overall concordance rate between chromogenic in situ hybridization (CISH) and IHC was 64 % with the 2007 ASCO/CAP criteria [74] and 32 % with the 2013 ASCO/CAP criteria for breast cancers [75]. Another study, using FISH and IHC and the 2007 ASCO/CAP criteria [74] found the concordance rate to be approximately 75 % [72]. Therefore, the breast 2007 ASCO/CAP scoring criteria [74] appears to be more applicable to ESC. Ultimately, responsiveness to therapy is the gold standard for assessing the effectiveness of these modalities for assessing ERBB2 alterations, and future studies will be required to determine the criteria that show the strongest correlation with therapeutic efficacy.

PTEN is a tumor suppressor gene located on 10q23 that encodes a dual-specificity phosphatase. The primary substrate of the PTEN protein is the phospholipid phosphatidylinositol-[3–5]-trisphosphate (PIP3) [76]. Production of PIP3 leads to translocation and activation of the protein kinase AKT which mediates cell survival and proliferation. PTEN acts as the negative check-point on the level of PIP3 to down regulate cell proliferation [77]. Germline mutations in the PTEN gene have been identified in Cowden Syndrome and Lhermitte-Duclos disease [78, 79]. On the other hand, somatic mutations have been demonstrated in a wide variety of tumors, including prostate cancer [80] and melanoma [81]. In addition to gene mutation, PTEN can be inactivated by deletion, epigenetic silencing, disruption of competitive endogenous RNA networks, micro-RNA regulation, posttranslational modifications, and aberrant localization [82]. PTEN mutations appear to be early events in type I endometrial carcinogenesis, being present in 50 % of complex atypical hyperplasia [83]. In endometrial carcinomas, PTEN gene mutations are more frequently identified in EECs (80 %) than non-EECs, and accordingly may have prognostic significance [84–87].

PTEN mutations have been identified in ~80 % of EECs with MSI-H compared to ~30 % of tumors that are MSI-stable [87]. It appears that replication error may predispose tumors to develop mutations in the PTEN gene even before clinicopathologic detection of cancer or precancerous lesion [88]. It has been reported that cells with PTEN inactivation in EEC are sensitive to mTOR inhibitors such as everolimus and temsirolimus [89, 90]. In a phase II study in patients with advanced or recurrent endometrial cancer, 26 % patients had a partial response and 63 % had stable disease on temsirolimus [89, 91].

PI3Kα (PI3K-alpha) is a protein complex composed of a catalytic, p110α, subunit and regulatory, p85α, subunit encoded by the PIK3CA (3q26.32) and PIK3R1 (5q13.1) genes, respectively [96]. Mutation of PIK3CA results in increase in AKT-dependent or independent signaling and leads to increased cell proliferation, growth, survival, and migration [97]. This pathway is antagonized by PTEN phosphatase [98]. One of the common mechanisms of PI3K-alpha activation is the somatic gain-of-function mutations within PIK3CA, PIK3R1 or loss of the PTEN activity [98, 99]. The product of the PIK3R1 gene, binds to p110α leading to stabilization and inhibition of p110α. Somatic mutations in PIK3R1 have been reported in few tumor types compared to that of PIK3CA [99, 100]. Somatic mutation in PIK3R1 has been reported in 43 % of EECs and 12 % of NEECs especially PIK3CA-wild type EECs than in PIK3CA mutant ones [99].

Somatic PIK3CA mutations have been reported in 52 % of EECs and 33 % of NEECs [92, 101, 102]. Fifty percent of all nonsynonymous PIK3CA mutations were localized in exons 1–7 and the other half were in exons 9 and 20. Approximately 62 % of exons 1–7 mutants and 64 % of exons 9–20 mutants were activating [92, 98, 101, 103].

Hotspots at amino acids 88, 93, and 111 within the ABD, C2 domain and the linker constitute the most common mutations affecting exon 1–7. Mutational analysis by nucleotide sequencing of all coding exons (exons 1–20) of the PIK3CA gene has been reported [92].

Epithelial ovarian cancer is the most common cause of cancer related deaths among gynecological cancers in the western countries with an estimated 21,980 new cases and 14,270 deaths in 2014 [120]. This high mortality is probably because most cases of ovarian cancers present at an advanced stage. The current practice consists of surgical excision of tumors, followed by platinum–taxane based chemotherapy [121]. Unfortunately, despite aggressive treatment, most cancers recur with dismal 5-year survival rate at ~ 45 % [122]. Molecular alterations in ovarian cancer have been the subject of research with the goal of identifying diagnostic, predictive, prognostic, and/or therapeutic targets. Up to 20 % of high grade ovarian cancers are associated with germline mutations in BRCA1 or BRCA2 [123]. On the other hand, somatic alterations in BRCA1/2 are seen in approximately 50 % in ovarian cancers [124].

Morphologically, ovarian epithelial tumors are classified into two groups with distinct molecular profiles (Table 15.4). Type I tumors include low grade serous carcinomas (LGSC), clear cell carcinoma (CCC), low and intermediate grade ovarian endometrioid carcinoma (OEC), and mucinous carcinoma. These tumors usually present at earlier stages, grow slowly and confined to the one or both ovaries. BRAF and KRAS somatic mutations are common molecular alterations in these tumors. Type II ovarian tumors include high grade ovarian serous carcinoma (HGOSC), high grade OEC, carcinosarcoma and poorly differentiated carcinomas. These tumors are aggressive with high metastatic potential at presentation. Approximately 80 % of HGSC exhibit TP53 mutation [124–126]. PIK3CA and RAS are other signaling pathways which have been found to be altered in 45 % of HGOSC [124]. Table 15.5 summarizes the most common molecular testing in ovarian tumors.

TP53 (17p13.1), encoding the p53 (TP53) protein is the most common altered tumor suppressor gene in human neoplasms. P53 is a tetrameric transcription factor that regulates cell growth, death and DNA integrity by controlling several target genes [143]. TP53 mutations, tested by conventional mutational analysis or inferred from overexpression, or complete absence, of protein by IHC, are recognized in HGOSC. Sixty to seventy percent of HGOSC cases that have been tested by full TP53 (p53) gene sequencing harbor TP53 mutations [144]. In contrast, EOC, CCC, and mucinous carcinomas have a much lower incidence of TP53 mutations [145]. TP53 mutations have been reported in serous tubal intraepithelial carcinomas. This suggests that TP53 mutation is an early event in the development of HGOSC of the ovary, fallopian tube, and peritoneum [146]. Inactivation of the TP53 gene may be an important event for the initiation of serous cancers even in those cancers that appear to retain the wild type sequence or IHC staining. Other mechanisms which may lead to TP53 (p53) inactivation include promoter or splicing alterations. Inactivation of TP53 (p53) has been reported to be associated with resistance to cytotoxic therapy [147].