—  SPECIALTY CONFERENCE  —

Gynecologic Pathology

Case 4 - Recurrent Postchemotherapy Clear Cell Carcinoma

Ie-Ming Shih
Johns Hopkins University School of Medicine
Baltimore, MD





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Clinical History:
A 42 y/o nulligravid Asian female presented with increasing intra-abdominal lymphadenopathy shortly after carboplatin/paclitaxel treatment for her previous "ovarian carcinoma" in an outside hospital. Unfortunately, the original tumor tissue was not available to review at that moment. She developed postoperative venous thromboembolism. Past history was significant for endometriosis. The sections were obtained from lymph node dissection.


Case 4 - Figure 1
The tumor shows a heterogeneous growth pattern including microcysts, solid and glandular features.
Case 4 - Figure 2
Cytologically, the tumor cells contain moderately atypical nuclei with infrequent mitotic figures. The cytoplasm is not particularly clear. Hyalinization in tumor stroma is also appreciated.
Case 4 - Figure 3
Cytologically, the tumor cells contain moderately atypical nuclei with infrequent mitotic figures. The cytoplasm is not particularly clear. Hyalinization in tumor stroma is also appreciated.


Pathological/Microscopic Findings and any Immunohistochemical or Other Studies:
The tumor shows a heterogeneous growth pattern including microcysts, solid and glandular features. Cytologically, the tumor cells contain moderately atypical nuclei with infrequent mitotic figures. The cytoplasm is not particularly clear. Hyalinization in tumor stroma is also appreciated.

Differential Diagnoses:
High-grade serous carcinoma Low-grade serous carcinoma Clear cell carcinoma

Final Diagnosis:
Recurrent postchemotherapy clear cell carcinoma

Case Discussion:
The diagnosis of this case was based on both clinicopathologic and molecular studies. The outside slides of patient's primary tumor were subsequently available for review and showed a classical ovarian clear cell carcinoma. The tumor is positive for HNF-1beta, but negative for WT1 and PBX1, markers associated with high- grade serous carcinoma. Molecularly, the recurrent tumor, like its primary counterpart, harbored a somatic mutation in PIK3CA gene. The TP53, beta-catenin, KRAS and BRAF were all wild-type. Thus, this case represents an example to demonstrate the many unique aspects of ovarian clear cell carcinoma.

Ovarian Clear Cell Carcinoma

Clinicopathological features
Although ovarian clear cell carcinoma (CCC) represents less than 10% of ovarian cancers in the United States, it appears to occur more frequently in Asian women and its incidence in this population has increased in recent years [1, 2] . Multivariate analysis on a large series of CCC shows that women with CCC present at a younger age and earlier clinical stages than (high-grade) serous carcinoma [1] . The mean age of patients with CCC is 57 years. Approximately 50% of CCCs present as stage I diseases [3, 4] and despite being diagnosed at an early stage they are generally considered highly malignant [5] . Whether CCC has a worse clinical outcome as compared to high-grade serous carcinoma is a subject of controversy. In some studies, the prognosis appears similar to that for other ovarian carcinomas [6] , but in others, the prognosis is reported to be worse. In a multivariate study that analyzed 1,411 CCC patients, CCC had a significantly worse prognosis than serous carcinoma but the difference is only modest at most [1] . In summary, when controlled for stage, the survival of women with CCC may be slightly lower than that of patients with serous carcinoma. Symptoms usually relate to a pelvic or abdominal mass. The most remarkable clinical condition associated with women with CCC is the paraneoplastic syndromes including venous thromboembolism, hypercalcemia and subacute cerebellar degeneration. For example, CCC patients have a 2.5-times greater risk of disease related venous thromboembolism t han women with other histologic types of epithelial ovarian cancer [7] .

Previous morphological and molecular studies have indicated that CCC develops in a stepwise fashion from endometriosis, atypical endometriosis to CCC [8, 9, 10, 11, 12] . In fact, CCC is the most common ovarian carcinoma that is associated with endometriosis. A recent study by analyzing a large series of CCCs proposed dividing CCC into cystic or adenofibromatous types because the investigators found that there was a significant difference in a number of clinicopathologic features including stage at presentation, association with endometriosis, histologic patterns and survival between these two types [8] . Cystic CCCs presented more often at stage I, had a better prognosis, and were more frequently associated with endometriosis compared with adenofibromatous CCC. Histologically, cystic CCC frequently displayed a papillary architecture and was associated with a trend toward a more favorable outcome compared with tumors in which a tubulo-cystic pattern predominated. The latter pattern occurred more frequently in adenofibromatous CCC. Finally, the 5-year survival for stage I cystic CCC (90%) was superior to the adenofibromatous type (50%). By reporting these two types of CCC may have a potential impact in clinical management of CCC patients if future additional studies can confirm these findings.

Potential diagnostic pitfalls and differential diagnosis
Because CCCs respond poorly to conventional chemotherapy and there is enthusiasm to initiate clinical trials for CCC, it is important for pathologists to make an accurate diagnosis. Diagnosis of CCC is usually not a problem [13] because most cases display the typical features of CCC, namely those with papillary, tubulo-cystic and solid patterns containing polygonal tumor cells with clear (glycogen rich) or, less commonly, eosinophilic (granular) cytoplasm. When the latter feature becomes diffuse, the term "oxyphilic" CCC could be used. In some cases, cytoplasmic vacuoles harbor homogeneous eosinophilic globules, creating a "targetoid" appearance, but their presence is not diagnostic for CCC. In CCCs with predominant tubulopapillary architecture, the cells are often columnar with a hobnail appearance. Another classical feature of CCC is the hyalinized and homogeneous eosinophilic stromal fibrosis which can be detected in the majority of cases. Given the characteristic morphologic features of CCC, the diagnosis can be at times difficult for various reasons, in particular when the typical clear cell features are not clear. It is not infrequently to encounter mixed ovarian epithelial carcinomas with clear cell and serous components. These mixed tumors have been studied based on clinicopathological and immunostaining features. The conclusion is that the mixed tumors may likely represent variants of high-grade serous carcinoma [13] . Future molecular studies are required to support this view. It has also been reported that neoadjuvant chemotherapy may be associated with extensive clear cell changes in a serous carcinoma [14] . Moreover, the case as discussed in this specialty section represents another diagnostic pitfall that CCC may show serous differentiation after chemotherapy. Besides, the "classical" differential diagnosis of CCC includes dysgerminoma, york sac tumor, endometrioid carcinoma with secretory change and metastatic clear cell carcinoma from kidney and gastrointestinal tract [15] . Of note, metastatic clear cell neoplasms from outside the female genital tract do occur but they are exceptionally rare. When oxyphilic tumor cells predominate, steroid cell tumor, hepatoid york sas tumor and hepatoid carcinoma may also be considered. Clinicopathological features are important to distinguish CCC from other morphologic mimickers. The presence of typical CCC morphology and association with endometriosis or an adenofibromas or an admixture with endometrioid carcinoma may be helpful to diagnose CCC. Patients with york sac tumor with or without hepatoid features and dysgerminoma are usually much younger than CCC and have elevated serum AFP levels. As compared to york sac tumors, the papillary structures in CCCs appear more complex and have hyalinized stromal cores. Hepatoid carcinoma lacks the foci of typical CCC. Endometrioid carcinoma with secretory changes can be confused with CCC, especially when the changes are extensive. The tall columnar epithelium with sub- or supranuclear vacuoles resembling early secretory endometrium suggests an endometrioid carcinoma with secretory changes. Finally, immunohistochemistry could be helpful in the differential diagnosis and this will be discussed in the following section.

Recent advances in molecular genetic findings
The most common molecular genetic changes known so far in CCC is the somatic activating mutation of PIK3CA [16] , which were detected in nearly 48% of affinity-purified fresh tumors and cell lines. In contrast, the frequency of PIK3CA mutations is relatively low in the conventional high-grade serous carcinoma. Besides, PTEN, a tumor suppressor gene involved in the PIK3CA signaling pathway, decreases its expression in approximately one third of CCC [17] , supporting the role of aberrant PIK3CA pathway in the development of CCC. LOH at the PTEN locus in both the carcinoma and associated endometriosis has been reported in some CCCs, suggesting that inactivation of the PTEN tumor suppressor, when it occurs, is a relatively early event in the development of CCC [11] . Although mutations in KRAS, BRAF, and TP53 are present in some CCCs their frequency is generally very low (review in [18] ) . For example, TP53 mutation and BRAF mutation were found in 8.3% and 6.3% of CCCs, respectively. Although studies to date are rather limited, the CCCs do not appear to share many other changes with endometrioid carcinomas, as canonical Wnt signaling pathway defects and microsatellite instability have rarely been in CCC [18] .

DNA copy number alteration is another common molecular genetic hallmark in human cancer and such change can be detected at a genome-wide scale by several techniques including high-density SNP array. Based on SNP array analysis on purified tumor cells isolated from fresh cancerous tissue, Kuo et al. have recently reported frequent amplification of the ZNF217 (zinc finger protein 217) locus and deletion of the CDKN2A/2B locus in CCCs, suggesting that pathways involving these two genes are important in CCC tumorigenesis [19] . ZNF217 encodes a nuclear protein with Krüppel-like zinc finger domains and serves as a common protein in forming transcriptional repressor complexes with HDAC2 and LSD1. Previous reports have shown frequent amplification and overexpression of ZNF217 in other carcinomas including breast, colorectal, and prostate. In breast carcinomas, increased ZNF217 expression was found to be associated with a poor prognosis and ectopic expression of ZNF217 immortalized epithelial cells and resulted in the loss of TGF-beta responsiveness [20] . Like in other cancer types [21, 22] , ZNF217 expression has been shown to be essential role in maintaining cellular proliferation and survival in CCC, further supporting a role for ZNF217 in CCC tumorigenesis.

The degree of overall DNA copy number changes has been used to reflect the chromosomal instability (CIN) levels in cancer. Based on the CIN index (the total numbers of DNA copy number gains or loss in an individual tumor), CCC is much more similar to low-grade serous carcinoma than to high-grade serous carcinoma. The molecular genetic analysis of CCCs described herein reflects an initial step toward revealing the genomic landscape of this disease and provides a useful foundation for future studies aimed at elucidating the molecular pathogenesis of CCC and at development of target-based therapeutics for this highly aggressive ovarian malignancy.

Gene expression profile and immunohistochemistry markers
Analyses of gene expression patterns have demonstrated that CCCs are distinct from other histological types of ovarian carcinomas (review in [18] ) . When compared to normal tissues including colon, endometrium, and fallopian tube, the overall gene expression profile of CCC is most similar to that in normal endometrium, supporting the view that the cell of origin of CCC may likely come from endometriosis [12] . An increasing number of genes have been reported to be preferentially expressed in CCC. A very recent study based on a comprehensive gene expression analysis proposes the "CCC signature" which consists of HNF-1beta, versican (VCAN), and several genes involved in oxidative stress [23] . Interestingly, expression of those CCC signature genes is induced by treatment of immortalized ovarian surface epithelial cells with the contents of endometriotic cysts, suggesting that the CCC signature may be dependent on the tumor microenvironment and its pathogenesis is related to endometriosis. Other upregulated genes in CCC include glutathione peroxidase 3, plasma membrane-associated sialidase (NEU3), osteopontin, annexin A4, tissue factor pathway inhibitor 2 and MAP3k5/ask1, CD26, hypoxia-inducible protein 2, and genes involved in nucleotide excision repair, just to name a few [18] . Among these upregulated genes, HNF-1beta is most consistently reported and appears to be a relatively specific marker associated with CCC [24] . HNF-1beta is a nuclear homeodomain protein that is responsible for regulating transcriptional activity including genes relatively specific to CCC genes and those involved in glucose/glycogen metabolism. It is likely that HNF-1beta up-regulation may participate in the generation of the glycogen-rich, clear-appearing cytoplasm, a cytological feature characteristic of CCC. Knockdown of HNF-1beta expression in CCC cells induces apoptosis, suggesting that HNF-1beta is essential for survival of cancer cells [24] . DNA hypomethylation may be one of the mechanisms involved in upregulating HNF-1beta and other CCC-associated genes [23] .

Although the CCC associated genes can be the clues to study the biology of this tumor type, their roles in differential diagnosis remain largely unclear except HNF-1beta. From a practical perspective, several "classical" markers have been proven useful in distinguishing CCC from other tumor types that may be confused with CCC. For example, as compared to germ cell tumors, CCCs almost always are CK7+, EMA+, AFP-, CD10-, OCT-. A diagnostic panel consisting of WT1, ER, and HNF-1beta has been reported to be useful in distinguishing CCCs (HNF-1beta+, WT1-, ER-) from high-grade serous carcinoma (HNF-1beta-, WT1+, ER+) [25] . Of note, the rare expression of WT1 in CCC may be attributed to the frequent WT1 promoter methylation in CCCs but not in high-grade serous carcinoma [26, 27] .

Markers that can predict the treatment outcome in CCC patients have been a subject of intense study. The rationale is that CCC typically presents with stage I or II disease and in this setting prognostic markers could have an impact on clinical management, in particular the decision to treat with adjuvant chemotherapy [28] . IGF2BP3 (IMP3) expression has been reported as an independent marker of reduced disease-specific survival in CCC, but not in high-grade serous or endometrioid carcinomas of the ovary. The prognostic significance of IGF2BP3 expression for reduced disease-specific survival was confirmed in an independent series of cases from three different centers in North America [28] . Similarly, enhanced expression of Annexin A4 in CCC and its association with chemoresistance to carboplatin have been recently reported [29] .

Where CCC stands in the dualistic model of ovarian cancer development?
In an attempt to elucidate the molecular pathogenesis of ovarian carcinoma, a dualistic model of ovarian carcinogenesis has been proposed for ovarian carcinomas [30, 31, 32] . This model is based on extensive clinicopathologic and molecular genetic studies, and classifies ovarian carcinomas into type I and type II tumors. It should be emphasized that the model describes pathways of tumorigenesis and the terms, type I and type II, are not specific histopathologic terms. In this model, low-grade serous, mucinous, and endometrioid carcinomas are designated as "type I" tumors. These neoplasms share many features including development in a stepwise fashion from well-recognized precursors termed "borderline" tumors that originate from cystadenomas/adenofibromas and endometriosis. They typically are low-grade, present as large cystic tumors confined to the ovary, and have an indolent behavior. In contrast, high-grade serous carcinoma, undifferentiated carcinoma and malignant mixed mesodermal tumors (carcinosarcomas) are designated as "type II" tumors. In contrast to type I tumors, these high-grade malignancies are not associated with benign precursor lesions. Recent studies have suggested that type II tumors develop from serous intraepithelial carcinomas in the fimbriated portion of the fallopian tube [33] . Regardless of their origins, type II tumors are invariably highly aggressive and the disease often is in advanced stages at the time of diagnosis. Compared to type I tumors at the molecular level, type II tumors have high chromosome instability and are characterized by frequent TP53 mutations. In contrast, type I tumors have moderate chromosome instability and contain frequent somatic mutations of genes participating in signal transduction pathways, including KRAS/BRAF, PTEN/PIK3CA and CTNNB1. In contrast, the mutations in those genes are rarely detected in type II tumors.

CCC appears to exhibit features of both type I and type II neoplasms. Similar to type I tumors, CCCs develop from well characterized benign precursors (e.g., endometriosis) and present as large cystic tumors confined to the ovary. However, when the disease spreads beyond the ovary CCC is typically high-grade, high-staged, and very aggressive. So, does CCC belong to type I or type II tumor? Recently, Kuo et al. found that CCCs have a chromosomal instability index very similar to that of low-grade serous carcinomas and much lower than that of high-grade serous carcinomas [19] . Rare TP53 mutations but frequent PIK3CA activating mutations have also been detected in CCCs [16] . The above molecular genetic findings are in stark contrast to results obtained in high-grade serous carcinomas and strongly support classifying CCC as a type I tumor from both clinicopathologic and molecular perspectives.

Challenges and new promise in managing advanced stage disease
As discussed above, although most CCCs are indolent, typically presenting in one ovary (stage I) when surgical intervention is effective in eradicating the disease, CCC in advanced stage can be highly aggressive because of its resistance to conventional chemotherapy [3, 4, 34] . This represents a major challenge in treating patients with advanced stage CCC and accounts for their dismal prognosis. The relatively high frequency of PIK3CA mutations holds promise for new therapeutic approaches using small molecule inhibitors targeting PI3K, the protein encoded by PIK3CA. New PI3K targeting drugs, including GDC-0941 [35] , NVP-BEZ235 [36, 37] , PI-103 [38] , and SF1126, a LY294002 prodrug [39] , have recently been developed and are being evaluated in clinical trials [35, 37, 40] . In the future, it will be important to design clinical studies to evaluate the safety and efficacy of such inhibitors by correlating clinical response with PIK3CA mutational status or pathway activation. If such studies show promising results, this would be an important step in the development of customized treatment for ovarian CCC at advanced stages.

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