The roles of pathology in targeted therapy of women with gynecologic cancers

Abstract

The role of the pathologist in the multidisciplinary management of women with gynecologic cancer has evolved substantially over the past decade. Pathologists’ evaluation of parameters such as pathologic stage, histologic subtype, grade and microsatellite instability, and their identification of patients at risk for Lynch syndrome have become essential components of diagnosis, prognostic assessment and determination of optimal treatment of affected women.

Despite the use of multimodality treatment and combination cytotoxic chemotherapy, the prognosis of women with advanced-stage gynecologic cancer is often poor. Therefore, expanding the arsenal of available systemic therapies with targeted therapeutic agents is appealing. Anti-angiogenic therapies, immunotherapy and poly ADP ribose polymerase (PARP) inhibitors are now routinely used for the treatment of advanced gynecologic cancer, and many more are under investigation. Pathologists remain important in the clinical management of patients with targeted therapy, by identifying potentially targetable tumors on the basis of their pathologic phenotype, by assessing biomarkers that are predictive of response to targeted therapy (e.g. microsatellite instability, PD1/PDL1 expression), and by monitoring treatment response and resistance. Pathologists are also vital to research efforts exploring novel targeted therapies by identifying homogenous subsets of tumors for more reliable and meaningful analyses, and by confirming expression in tumor tissues of novel targets identified in genomic, epigenetic or other screening studies.

In the era of precision gynecologic oncology, the roles of pathologists in the discovery, development and implementation of targeted therapeutic strategies remain as central as they are for traditional (surgery-chemotherapy-radiotherapy) management of women with gynecologic cancers.

INTRODUCTION

In 2017 in the USA, it is estimated that 107,470 women will be diagnosed with gynecologic cancers, and that 31,600 women will die of gynecologic tumors (Table 1) [1]. This corresponds to 12.6% and 11.2%, respectively, of all cancers in women. The traditional management of women with gynecologic cancer largely rests upon surgery, cytotoxic chemotherapy and radiotherapy, singly or in combination as dictated by the clinical circumstances, with the stage of disease largely determining the need for adjuvant or first-line chemotherapy or radiation. In those with recurrent disease, the choice of cytotoxic chemotherapy is generally most dependent upon time since last platinum-based chemotherapy, with the platinum-free interval determining platinum sensitivity versus resistance. More recently, ever-increasing numbers of targeted therapies directed against a variety of molecular targets in gynecologic cancers and their microenvironments are being developed and used in women with these malignancies.

Table 1.

Estimated numbers of gynecologic tract cancers in 2017 (adapted from ¹)

	Estimated new cases	Estimated deaths
Ovary	22,440	14,080
Uterine corpus	61,380	10,920
Cervix	12,820	4,210
Vagina	4,810	1,240
Vulva	6,020	1,150
Total	107,470	31,600

TRADITIONAL ROLES OF PATHOLOGY IN TREATMENT OF GYNECOLOGIC CANCERS

Pathologists have long played a central role in the multidisciplinary management of patients with gynecologic cancer by providing fundamental items of risk-stratification information that guide optimal treatment, such as pathologic stage of disease, histologic subtype and grade [2].

Pathologists are also key to assessment of other parameters that are useful in management. An important example of this is pathologic evaluation of DNA mismatch repair deficiency in endometrial cancer, which has become part of the standard of care for women with these tumors. DNA mismatch repair defects are found in 25–30% of endometrial cancers, and lead to a high-level microsatellite instability (MSI-H) phenotype [3–5]. A few MSI-H endometrial cancers are associated with Lynch syndrome-associated germline alterations in DNA mismatch repair genes (MLH1, PMS2, MSH2, MSH6) or EPCAM, but the majority are due to a sporadic epigenetic change, namely hypermethylation of the promoter region of MLH1, which leads to gene silencing.[3, 6, 7] Both germline and sporadic alterations are associated with loss of expression of protein products of the affected genes [3, 5]. Patients with tumors that exhibit loss of expression of MLH1/PMS2 by immunohistochemistry but which lack MLH1 promoter hypermethylation are likely to harbor germline MLH1 mutations as seen in Lynch syndrome. Should the presence of a MLH1 germline mutation be confirmed, they and their family members would be at increased risk for Lynch-syndrome-associated malignancies, and would require exploration of these risks, including personal and familial genetic counseling and consideration of increased cancer screening. In contrast, tumors that exhibit loss of expression of MLH1/PMS2 by immunohistochemistry and which show MLH1 promoter hypermethylation are likely to be sporadically hypermethylated tumors, and women with these tumors and their families do not have increased cancer risk [8, 9].

TARGETED THERAPY

During the past decade, there have been changes in histologic classification that affect surgical management, adjuvant therapies and prognostic assessment; recognition of areas of diagnostic difficulty (such as histologic subtyping of high-grade endometrial carcinomas); and discovery of molecular genetic alterations and genetically defined prognostic subgroups of gynecologic cancer. Most patients with early stage gynecologic cancer (when many endometrial cancers are diagnosed) have good clinical outcomes. Nevertheless, between 1987 and 2008, it is believed that the number of women who died from endometrial cancer in the US increased substantially, while relative survival has declined over the past decade [10]. The clinical course in patients with advanced-stage gynecologic cancer (which is frequent in ovarian cancer) is often aggressive and the prognosis is poor, despite the use of combination cytotoxic chemotherapy. Furthermore, many ovarian cancer patients who initially respond to chemotherapy suffer tumor recurrence and progressive resistance to therapy [11]. Therefore, there is a need for more effective therapeutic modalities for women with gynecologic cancers, in particular for those with advanced-stage disease.

Targeted therapies are a cornerstone of precision medicine, in which individual patients are treated using agents targeting molecules identified in that patient’s cancer or in the tumor’s microenvironment. Molecular targets include tumor-intrinsic signaling pathways, and aberrations in these pathways may result in expression of mutant/altered proteins, overexpression or loss of expression of normal proteins, and novel fusion proteins resulting from gene rearrangements. Other potential therapeutic targets in gynecologic cancers include homologous recombination deficiency, hormone receptors, angiogenesis and immunologic factors (Table 2).

Table 2.

A selected summary of targeted therapy in gynecologic cancer

Targets	Class	Examples
Signaling pathways	PI3K/AKT/mTOR inhibitors MAPK inhibitors JAK1/JAK2 inhibitors NTRK/ROS1/ALK inhibitors	Temsirolimus Trametinib* Ruxolitinib* Entrectinib*
Homologous recombination deficiency	PARP inhibitors	Olaparib, niraparib, rucaparib, veliparib*
Hormone receptors	Progesterone receptor Estrogen receptor Gonadotropin-releasing hormone agonists Androgen receptor	Progestins Tamoxifen, aromatase inhibitors Leuprolide Enzalutamide*
Angiogenesis	Anti-VEGF/VEGFR	Bevacizumab, cediranib
Immunologic factors	Immune checkpoint inhibitors (e.g. anti- PD1 and anti-CTLA4) Adaptive T cells Vaccines	Pembrolizumab, nivolumab*, ipilimumab*

^*undergoing investigation

TARGETED THERAPY FOR GYNECOLOGIC CANCER

Several targeted therapies are currently available and approved by the US Food and Drugs Administration (FDA) and the European Medicines Agency (EMA) for patients with gynecologic cancer.

Anti-angiogenesis agents, such as bevacizumab (a monoclonal antibody which targets VEGF-A), have been shown to be effective in ovarian cancer when used in conjunction with standard platinum-based chemotherapy [12–15]. Recently, the FDA and EMA approved bevacizumab in combination with paclitaxel plus either cisplatin or topotecan as a treatment for patients with persistent, recurrent or metastatic cervical cancer, based on the extension of overall survival in the GOG-240 study [16]. Bevacizumab is FDA-approved for platinum-resistant ovarian cancer in combination with liposomal doxorubicin, topotecan, or paclitaxel based on results of the AURELIA trial [15]. It is FDA-approved for platinum-sensitive ovarian cancer in combination with carboplatin and gemcitabine based on results of the OCEANS trial [14] and also in combination with carboplatin and paclitaxel based on results of the GOG-0213 trial [17]. Bevacizumab has also shown promising activity in patients with low-grade serous ovarian cancer, a subtype of ovarian cancer that commonly has low response rates to conventional chemotherapy [18].

Poly ADP ribose polymerase (PARP) inhibition of cells containing a defect in homologous recombination pathways (e.g. those with BRCA1/2 mutations) results in the death of target tumor cells while sparing normal cells. Recently, both the FDA and EMA approved olaparib, a PARP inhibitor, as effective maintenance therapy in patients with platinum-sensitive ovarian cancer who are in complete or partial response following platinum-based chemotherapy; this follows its original approval in 2014 for the treatment of patients with deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer who have been treated with three or more lines of chemotherapy [19]. Other FDA-approved PARP inhibitors that have shown objective responses include rucaparib (for patients with deleterious germline and/or somatic BRCA mutation-associated advanced ovarian cancer who have been treated with two or more chemotherapies [20]) and niraparib (for maintenance treatment of adult patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in complete or partial response to platinum-based chemotherapy [21]).

In endometrial cancer, POLE-mutated and mismatch repair deficient tumors have higher neoantigen load, increased tumor-infiltrating lymphocytes, and increased expression of several immune checkpoint genes [22, 23]. Immune checkpoint regulators such as programmed death receptor 1 (PD1) promote escape from tumor immune surveillance, and 80% of endometrial cancers express high levels of PD1, or its ligand, PDL1 [24]. Data suggest that POLE-mutated and mismatch repair deficient endometrial tumors might be excellent candidates for PD1-directed immune therapies [22]. Immune checkpoint blockade with the antibody pembrolizumab to PD-1 has shown responses in patients with POLE-mutated[23] and mismatch repair deficient cancers, including endometrial cancer [25]. Pembrolizumab has been approved by the FDA for metastatic cancers exhibiting mismatch repair deficiency, regardless of their histologic subtype. In endometrial cancer patients, pembrolizumab shows durable anti-tumor activity in a subset [26], including rare exceptional responses [23].

Endocrine therapy with progestins and tamoxifen for endometrial cancer is associated with overall response rates reported to range between 9% and 55% (summarized in [27]). Even though patients with lower grade tumors express estrogen and/or progesterone receptors, the Cochrane review in 2010 reported no improvement in survival in women with advanced endometrial cancer who received hormone therapy [27]. Hormonal therapy (LHRH agonists, tamoxifen, aromatase inhibitors) for ovarian cancer has been evaluated in small trials, which have reported inconsistent results [28–30]. A Cochrane review of hormonal therapy in women with ovarian cancer reported overall response rates ranging from 0% to 56%, but there was insufficient data to analyze duration of response or survival [31]. The data in both Cochrane reviews were limited by the lack of any large randomized trials.

In addition to the aforementioned approved agents, several targeted therapies are currently under investigation or undergoing trials in patients with gynecologic cancers. A detailed description of these agents is beyond the scope of this review, but a selected few are summarized in Table 2. One of the challenges for many targeted therapies in advanced gynecologic cancers is the large number of genetic alterations which is often seen in advanced tumors [32] and which worsens due to selective pressures during tumor progression, especially following therapy. This phenomenon may result in derangements in multiple oncogenic or tumor-promoting pathways, and raises doubts that targeted agents directed at one or two genetic alterations would significantly alter outcomes. The acquisition of epigenetic alterations in tumors and adaptations of the tumor microenvironment compound the challenges in achieving durable responses with targeted agents. In attempts to circumvent this problem, combinations of agents targeting different pathways, or combinations of targeted agents with cytotoxic chemotherapy or immunotherapy are also undergoing trials, in addition to novel single therapeutic agents.

ROLES OF PATHOLOGY IN TARGETED THERAPY OF GYNECOLOGIC CANCERS

With the development and implementation of increasing numbers of targeted and other novel therapies, pathologists continue to have important roles in multidisciplinary teams managing patients with gynecologic cancer (Fig. 1).

Figure 1.

An overview of the roles of pathology in the management of women with gynecologic cancer.

Pathology and patient management

Certain pathologic features of gynecologic cancers are associated with genotype and underlying pathogenetic mechanisms, which may serve as targets for therapeutic agents. Identification by pathologists of these phenotypic features, a few examples of which are briefly discussed below, can be very helpful by focusing confirmatory testing of tumors that may be amenable to targeted agents.

Mismatch repair-deficient gynecologic tumors

Mismatch repair deficiency, in the setting of Lynch syndrome and occurring sporadically, has been reported in endometrial [4, 5, 33] and ovarian [33–35] carcinomas. In addition to the importance of pathologists screening endometrial cancers for mismatch repair deficiency to identify those women who are at risk for Lynch syndrome (as described earlier), this screen is also likely to become increasingly important for planning treatment. A recent study showed that microsatellite instability analysis is effective as a predictive biomarker for the effect of immune checkpoint inhibitors, including anti-PD1 antibody and anti-PDL1 antibody.¹⁶ MSI-H endometrial cancers show higher neoepitope levels and higher expression of PD-1 and PDL-1, when compared with microsatellite-stable cancer,¹³ and MSI-H ovarian clear cell carcinomas also show elevated expression of PDL-1 [35]. This suggests that microsatellite instability analysis may be a useful predictive biomarker for response to immunotherapy.

Histopathologic features that are significantly more commonly associated with MSI-H endometrial cancers than microsatellite-stable tumors include localization in the lower uterine segment, low-grade endometrioid histology, mucinous differentiation, tumor-infiltrating lymphocytes and peritumoral lymphocytes [9, 36–38]. Mucinous differentiation and tumor-infiltrating lymphocytes [38], and undifferentiated histology in a subset of cases [9] are features that characterize endometrial carcinomas with sporadic MLH1 promoter methylation. One study found that tumor-infiltrating lymphocyte counts of >40 per 10 high-power fields were associated with sensitivity and specificity of 85% and 46%, respectively in predicting microsatellite instability status in endometrioid endometrial carcinomas [36]. These features are readily assessable in routine histologic sections, and are helpful in guiding focused microsatellite instability testing of tumors harboring these morphologic hallmarks. In the same study, morphologic heterogeneity (presence of two or more clearly separate morphologic patterns, each constituting at least 10% of the tumor) was more frequent in microsatellite-unstable tumors, but the difference did not reach statistical significance [36].

Mismatch repair deficiency has been reported in approximately 10% of ovarian carcinomas [34, 39] of endometrioid [40], clear cell [35] and serous [33] histologic types. Although specific histologic features of ovarian endometrioid adenocarcinomas in one study did not correlate with mismatch repair status [40], a recent study of mismatch repair deficient clear cell carcinomas of ovary showed significantly higher number of CD8-positive and PD1-positive tumor-infiltrating lymphocytes with higher CD8⁺/CD4⁺ ratios compared with microsatellite stable tumors [35].

Microsatellite instability can also be assessed in histologic sections using immunohistochemistry. Detection of DNA mismatch repair deficiencies by immunohistochemistry using antibodies directed against MLH1, PMS2, MSH2 and MSH6 can effectively diagnose microsatellite instability in endometrial carcinomas [3, 5]. Indeed, many institutions have already implemented universal testing for DNA mismatch repair deficiency in women with newly diagnosed endometrial cancer using immunohistochemistry. Immunonohistochemistry is readily available and interpretable, both in specialized/tertiary institutions as well as smaller community hospitals and pathology laboratories. In larger institutions, there is a trend toward universal genomic profiling (including PCR-based microsatellite instability analysis) of newly diagnosed endometrial cancer. Concordance between the results of immunohistochemistry and PCR-based microsatellite instability analysis is high [41], and immunohistochemistry, allied with histopathologic assessment of microsatellite instability-associated morphological features, is a useful and cost-effective means of screening tumors for microsatellite instability. These approaches can rapidly identify those tumors that may be susceptible to immune checkpoint inhibitors. Expression of PDL1 can also be assessed by immunohistochemistry. However, this is an area of active study, and varying results have been reported with different antibodies, staining platforms and scoring criteria [42]. Until these elements are standardized after rigorous testing and validation in gynecologic cancers, it would be premature to use PDL1 immunohistochemistry as part of clinical management, except in the setting of investigative studies and clinical trials with clearly established parameters.

Gynecologic tumors with POLE mutations

The seminal Cancer Genome Atlas study described four major genomically-defined groups of endometrial cancers (POLE ultramutated, MSI hypermutated, copy-number-low, and copy-number-high). These groups were also clinically significant, as they correlated with progression-free survival, with POLE-mutated tumors having an excellent prognosis [43]. A subset of primary ovarian carcinomas also harbor POLE mutations [44–47].

Ovarian carcinomas with POLE mutations are characterized by endometrioid histology and rare cases show morphologic heterogeneity [44–46]. Features of endometrial carcinomas harboring POLE mutations that help in their identification are: occurrence in younger women; high grade; frequent lymphovascular space invasion; frequent endometrioid histology; conspicuous tumor-infiltrating lymphocytes and/or peri-tumoral lymphocytes, morphologic heterogeneity/ambiguity and bizarre/giant tumor cell nuclei [48, 49]. POLE mutations have also been reported in endometrial clear cell carcinomas [50], undifferentiated carcinomas [51] and carcinosarcomas [52]. Algorithms for identifying patients for adjuvant chemotherapy and radiation therapy rely on tumor grade, stage and lymphovascular space invasion, and based on these criteria, a significant proportion of patients with POLE-mutated tumors would receive adjuvant therapy [49]. However, their apparently high-risk characteristics belie their excellent outcomes [43]. This raises the possibility that aggressive adjuvant chemoradiation for these tumors may not be required.

In addition to their selection for immunotherapy (as discussed earlier), identification of patients with POLE-mutated tumors may be important to avoid over-treatment. The Leiden [53] and Vancouver [54] groups have proposed diagnostic algorithms for molecular classification of endometrial cancers. The Vancouver group algorithm is particularly applicable to diagnostic pathology specimens; it involves, in sequence, immunohistochemistry for DNA mismatch repair proteins, sequencing of mismatch-repair-intact tumors for POLE mutations, and immunohistochemistry for p53 in the POLE-wild-type tumors. This algorithm accurately classifies tumors as mismatch repair-deficient (MSI-H), POLE-mutated, p53-wild type (copy-number-low) and p53-aberrant (copy-number-high) [41, 54], and allied with morphologic assessment, may represent a useful means of classifying endometrial carcinomas into genomically distinct and clinically relevant subgroups.

Ovarian carcinomas associated with homologous recombination deficiency

Aberrations of homologous recombination repair are identified in approximately half of all high-grade serous carcinomas. Homologous recombination deficiency may be due to germline or somatic mutations in BRCA1/BRCA2, as well as mutations in genes such as ATM, BRIP1, CHEK2, RAD51 and PALB2 [55, 56]. Compared with homologous recombination-competent tumors, homologous recombination-deficient ovarian carcinomas are associated with significantly more frequent variant (solid, endometrioid, and transitional cell carcinoma, SET-like) morphology, greater mitotic activity, more tumor-infiltrating lymphocytes, and more frequent necrosis, and associated with a lower frequency of serous tubal intraepithelial carcinoma, younger patient age and improved survival. SET features are also more common in BRCA2-mutant tumors [57–59]. Pathologic recognition of homologous recombination-deficient tumors can be useful in identifying patients who might benefit from PARP inhibitors.

Tumors associated with mitogen-activated protein kinase (MAPK) pathway activation

Mutations in the mitogen-activated protein kinase (MAPK) pathway are commonly observed in gynecologic cancers. This pathway is potentially targetable using agents such as BRAF inhibitors and MEK inhibitors [60, 61].

KRAS mutations are associated with mucinous differentiation. KRAS mutations are common in endometrial cancer [43], and endometrial cancers with mucinous differentiation appear to be more frequently associated with KRAS mutations [62]. Primary ovarian mucinous carcinomas also frequently harbor KRAS mutations and ERBB2 amplifications [63]; the latter are also identified in endometrial serous carcinomas [64]. Although KRAS is not a direct molecular therapeutic target, identification of tumors that are likely to harbor KRAS mutations might make them amenable to therapy directed against other components of the MAPK/ERK pathway, such as members of the EGFR family.

KRAS and BRAF mutations have been reported in 17–36% [65–67] and 30–45% [66–68], respectively, of serous borderline tumors. Low-grade serous carcinomas of the ovary also harbor mutations in KRAS (16–41%) as well as BRAF (<10%). Tumors with V600E BRAF mutations have been found to have a better prognosis than those with wild-type BRAF [67–70]. These tumors can also bear other alterations that result in MAPK pathway activation [61]. While a phase II study of the MEK inhibitor selumetinib in patients with advanced low-grade serous ovarian cancer showed promising results, a phase III study comparing the MEK inhibitor binimetinib with physicians’ choice of chemotherapy (the MILO study [71]) recently closed after a planned interim analysis showed that the hazard ratio for progression-free survival crossed the predefined futility boundary. Additional analyses are ongoing in order to determine if any molecular biomarkers were associated with response in the patients treated in that study.

BRAF-mutated serous borderline tumors are characterized by a distinctive subpopulation of cells with abundant eosinophilic cytoplasm, well-defined cell borders and bland nuclei. In addition, the tumors exhibit cuboidal and columnar cells that line papillae and bud from their surfaces, leading to the appearance of individual cells and clusters of detached cells above the papillae [72]. The eosinophilic cells are highly correlated with BRAF mutation status in serous borderline tumors but are seen only in rare BRAF-mutated low-grade serous carcinomas [72]. Immunohistochemistry using a monoclonal antibody (VE1) specific for the BRAF V600E protein has been shown to be sensitive and specific for the detection of BRAF^V600E mutation in ovarian serous tumors [73]. The eosinophilic cells in serous borderline tumors also exhibit markers of senescence [72], and the comparatively low frequency of BRAF mutations in low-grade serous carcinomas may indicate that BRAF mutation in serous borderline tumors identifies tumors at low risk of progression.

Value of pathologists in integrating genotype and phenotype for optimal patient management

Pathologists are ideally positioned to synthesize the clinico-pathologic phenotype with genomic data from molecular pathology. For instance, in the setting of high-grade adnexal serous carcinomas, accurate pathologic classification and assessment of morphologic features can help identify the presence of homologous recombination deficiency, suggest the need for germline testing for BRCA1/BRCA2 mutations, provide an indication of chemosensitivity to conventional agents, and offer the option of PARP inhibitors or clinical trials for other suitable targeted therapies. Other settings in which pathology-driven genotype-phenotype correlations are important in patient management are listed in Table 3.

Table 3.

Pathologic-molecular correlations in gynecologic cancers and their implications for patient management

Histopathologic finding(s)	Ancillary pathologic test(s) (IHC/ISH)	Associated molecular abnormality	Intervention/Management implications
Endometrial: Lower uterine segment location; endometrioid or undifferentiated histotype; mucinous differentiation; tumor-infiltrating lymphocytes; peri-tumoral lymphocytes Ovarian: endometrioid or clear cell histotype; tumor- infiltrating lymphocytes	Immunohistochemistry for MLH1, MSH2, MSH6, PMS2; MLH1 promoter methylation analysis	DNA mismatch repair deficiency due to: 1) germline mutations in MLH1, MSH2, MSH6, PMS2 or EPCAM 2) somatic mutations or MLH1 promoter methylation	1) Genetic counseling and family screening for Lynch syndrome 2) Consider immune therapy e.g. checkpoint inhibitors for recurrent disease
Endometrial: High grade; frequent lymphovascular space invasion; endometrioid, clear cell, undifferentiated or carcinosarcoma histotype; conspicuous tumor- infiltrating lymphocytes and/or peri-tumoral lymphocytes; morphologic heterogeneity/ambiguity; bizarre/giant tumor cell nuclei Ovarian: endometrioid histotype; morphologic heterogeneity		POLE mutations	Consider immune therapy e.g. checkpoint inhibitors for recurrent disease Consideration of avoidance of overtreatment in adjuvant setting
Ovarian: Variant (solid, endometrioid, and transitional cell carcinoma, SET-like) morphology; tumor-infiltrating lymphocytes; necrosis		Homologous recombination deficiency e.g. BRCA1/2 mutations	Consider germline BRCA1/BRCA2 testing and PARP inhibitor therapy in recurrent setting
Ovarian: Serous borderline tumors with eosinophilic cells; cuboidal and columnar cells that line papillae and bud from their surfaces	BRAF VE1 immunohistochemistry	MAPK pathway activation	Presence of V600E BRAF mutation may portend improved prognosis

Pathology and clinical trials of targeted agents

Based on morphologic assessment and judicious ancillary testing for biomarkers, as detailed above, allied with limited sequencing, pathologists can help identify phenotypically and genotypically homogenous sets of tumors, and optimize the selection of patients with these tumors for entry into clinical trials of suitable targeted agents. The example of high-grade endometrial carcinomas highlights the benefits of such an integrated pathologic-genomic-clinical approach. In the past, high-grade endometrial cancers were treated as a homogenous group. Pathologic refinements in morphologic grading and classification of these tumors, initially into type I and type II tumors, and subsequently into specific histotypes (endometrioid, serous, clear cell) improved risk stratification and allowed the formulation of informed treatment algorithms for these patients. However, morphologic limitations became apparent, particularly in high-grade endometrial carcinomas, which are difficult to subclassify reproducibly due to ambiguities in their morphologic features [74]. For example, 82 tumors diagnosed as FIGO grade 3 endometrioid carcinoma in TCGA study of endometrial carcinoma [43] were subsequently reviewed independently by two specialist gynecologic pathologists, who re-classified 20–25% of the tumors as serous carcinoma. The subsequent discovery of genomic classes of endometrial carcinoma [43] and their associations with phenotypic features [36, 37, 48, 49] have revealed that morphologically high-grade tumors include copy-number-high, MSI-H and POLE-mutated subsets, each of which is associated with specific pathologic features and clinical implications, as described earlier. Given the challenges in reproducible classification of high-grade tumors in particular, pathologic review by specialist gynecologic pathologists would be valuable for accurate assignation of histotype. Integration of the pathologic phenotype with judicious use of selected biomarkers [54] allows subcategorization of high-grade endometrial carcinomas into biologically homogenous subgroups in which specific therapeutic targets can be explored in clinical trials.

Pathology in monitoring response and resistance in tumors treated with targeted agents

Primary and acquired resistance, driven by intratumor heterogeneity as well as other tumor-specific and tumor microenvironmental factors, has been documented in a variety of tumors and represents a key challenge to delivering enduring responses to targeted therapy [75, 76]. Pathologic evaluation of tumor responses to targeted agents in the course of clinical trials can help quantify tumor responses and changes in the tumor microenvironment post-treatment. Allied with ancillary techniques such as immunohistochemistry and in-situ hybridization, pathologists can potentially help identify additional biomarkers of tumor sensitivity and resistance to therapy. Furthermore, increasing experience with ancillary testing (e.g. flow cytometry and single-cell sequencing) of cytologic specimens offers the promise of using cytologic material, such as effusions or cervicovaginal material, to monitor treatment response and resistance [77, 78].

Pathology in research and discovery of novel therapeutic targets

In the research setting, there are numerous studies and clinical trials attempting to identify or to implement novel targets for therapy in patients with gynecologic cancers.

Pathologists can help explore expression of novel targets identified in genomic, epigenetic or other screening studies in tumor tissues, using techniques such as immunohistochemistry and in-situ hybridization. Confirmation of tissue expression of protein products of mutated genes, or localization of their expression in specific tissues or tissue compartments can provide insights into the biology and mechanisms of action of these molecules, which in turn can aid in tailoring agents directed against them for optimal efficacy.

Correlative analyses of genotype or epigenetic or immune profiles with pathologic phenotypes can be helpful in fine-tuning the application of novel targeted therapies, by selecting for those patients who are most likely to benefit from agents targeting specific alterations whose histopathologic correlates are identified in their tumors (as described above).

Based on histopathologic evaluation and judicious ancillary testing, pathologists can help identify phenotypically homogenous subsets of tumors for analysis, which facilitates reliable and reproducible interpretation of molecular and genomic findings. A recent example is the case of ovarian small cell carcinoma of hypercalcemic type; the uniform histopathologic phenotype of these tumors led to the hypothesis that they harbor a common driver mutation. Subsequent sequencing analyses identified recurrent driver mutations in SMARCA4 [79]. BRG1, the protein product of SMARCA4, is a component of the SWI/SNF chromatin remodeling complex, and EZH2 inhibitors may prove to be useful therapeutic agents for these tumors.

The computer science term ‘Garbage In, Garbage Out’ [80], which refers to the reliance of the quality of post-analytical data upon the quality of the input, applies to research studies of biomarkers and therapeutic targets. The importance of rigor in the pre-analytical selection of tumors for molecular genetic analyses and clinical trials is highlighted by the following examples. In the Cancer Genome Atlas analysis of ovarian carcinomas [55], 96% of high-grade serous carcinomas were reported to harbor TP53 mutations. In this study, cases were included based on the original pathology diagnosis. Although specimens were subsequently reviewed in a centralized laboratory, specific histologic criteria that were used were not reported. A subsequent review of 14 of 15 TP53-wild-type cases from this cohort found that 5 specialized gynecologic pathologists rendered a unanimous diagnosis of high-grade serous carcinoma in only one (7%) case, which was associated with BRCA1 germline mutation and a homozygous TP53 deletion [81]. The authors concluded that all de novo high-grade serous carcinomas contain TP53 somatic mutations or deletions, with the exception of rare tumors that develop from an antecedent low-grade serous tumor. They proposed that molecular alterations of TP53 are the sine qua non for a diagnosis of ovarian high-grade serous carcinoma [81]. Similarly, in the TCGA studies of cervical carcinomas, adenocarcinomas were analyzed together as a single group [82], or subdivided into adenocarcinoma, clear cell carcinoma and serous carcinoma [83]. Since it is recognized that HPV-negative adenocarcinomas (such as gastric-type adenocarcinomas) appear to be biologically, clinically and pathologically distinct from HPV-associated adenocarcinomas [84, 85], analysis of adenocarcinomas without subclassification based on HPV status may not yield results that are universally applicable to all patients with cervical adenocarcinomas. Furthermore, a subset of HPV-negative carcinomas in this study were characterized by mutations in KRAS, ARID1A and PTEN [82], which are commonly seen in endometrioid tumors; this raises the possibility that at least some of these tumors might represent endometrioid adenocarcinomas arising in the corpus or lower uterine segment rather than being primary cervical adenocarcinomas. These examples illustrates the critical importance of high quality pathology review to ensure pathobiologically-informed selection and phenotypic homogeneity of tumors for study, particularly when attempting to define genotypic or immunophenotypic biomarkers that may be subsequent therapeutic targets.

CONCLUSION

In the era of precision medicine, increasing numbers of targeted therapies are in clinical use and undergoing trials in patients with gynecologic cancer. As described above, the roles of pathologists in the discovery, development and implementation of these novel therapeutic strategies are as central as they are for traditional (surgery-chemotherapy-radiotherapy) management of women with gynecologic cancers. This underscores the importance of pathology as a key component of multidisciplinary approaches to research and deployment of targeted therapy and precision gynecologic oncology.

Figure 2.

Pathologic correlates of molecular/genetic alterations in gynecologic cancers. a) Mismatch repair deficient endometrial carcinoma exhibiting low-grade endometrioid histology, tumor-infiltrating lymphocytes (lymphocytes infiltrating neoplastic epithelium) and peritumoral lymphocytes (lymphocytes present adjacent to tumor). b) POLE-mutated endometrial carcinoma showing conspicuous tumor-infiltrating lymphocytes and peri-tumoral lymphocytes and bizarre/giant tumor cell nuclei.

Figure 3.

Pathologic correlates of molecular/genetic alterations. a) Homologous recombination-deficient high-grade serous carcinoma exhibiting solid, endometrioid, and transitional cell carcinoma (SET)-like morphology. b) Endometrioid adenocarcinoma with mucinous differentiation, which is more frequently associated with KRAS mutations. c–f) BRAF-mutated serous borderline tumor showing complex papillary architecture (c), a subpopulation of cells with abundant eosinophilic (pink) cytoplasm (d, e). Cells bud from the epithelial surface, leading to the appearance of individual cells and clusters of detached cells (e). Immunohistochemistry for BRAF VE1 shows overexpression, which correlates with the presence of V600E BRAF mutation (f).

Highlights.

• Pathologists play a central role in the management (including targeted therapy) of women with gynecologic cancer

• Pathology is important in identification of targetable tumors based on morphologic features and biomarkers

• Pathology is key to monitoring therapeutic response, and to discovery of novel biomarkers and therapeutic targets