Small cell carcinoma of the ovary of the hypercalcemic type (SCCOHT) is a rare, highly aggressive tumor that affects mainly young women (median age: 24 years). Prognosis is poor, as most patients die within two years of diagnosis [1
]. The histogenesis of SCCOHT remains unclear, although there is increasing evidence in favor of a germ cell origin [2
]. In addition, it has been proposed that SCCOHT may represent the ovarian counterpart of malignant rhabdoid tumors [4
While there is no international consensus regarding the optimal treatment of SCCOHT, it usually involves multimodal chemotherapy, radical surgery and possibly, radiotherapy [5
]. However, no randomized studies have been conducted to date and the available data consist of case reports or small retrospective series with heterogeneous management strategies. The only prospective clinical study in SCCOHT, a multicenter phase II trial conducted at Institut Gustave Roussy, tested combination chemotherapy (PAVEP: cisplatin, adriamycin, vepeside and cyclophosphamide) followed by radical surgery and high dose chemotherapy with autologous stem cell transplant, and demonstrated a three year survival rate of 49% among 27 SCCOHT patients [6
]. This shows that even with intensive regimens, prognosis remains dismal, and that despite frequent initial response to chemotherapy, relapses are almost inevitable and tend to be refractory to second line chemotherapy.
The literature describing the genomic features of SCCOHT was scarce until 2014, when four groups identified loss-of-function mutations in SMARCA4
(Brahma-related gene 1, BRG1) as a highly recurrent event in SCCOHT [3
encodes one of the two possible catalytic subunits of the Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling complex. Others have since confirmed this finding, with SMARCA4
mutations being found in over 90% of cases [9
]. Nevertheless, effective treatment options to target this rare and lethal disease are still lacking.
We aimed to conduct an integrated genomic analysis of an independent cohort of SCCOHT by WES, RNA-Seq and aCGH to check for the presence of additional recurrent genomic alterations, which could allow the proposal of alternative treatment strategies.
SCCOHT are rare tumors that occur in young women and their prognosis remains poor, despite aggressive multimodal therapy. We present an integrated molecular characterization of additional cases of SCCOHT from an independent cohort.
Intriguingly, our findings and the previously published data show that these aggressive tumors carry a diploid DNA content, which is a rare phenomenon in a highly lethal malignancy [34
]. In addition, we show that SCCOHT have a very low mutation load (mean, 5.43 mut/Mb) and lack mutations in genes most altered across various cancer types. Collectively, these observations support the hypothesis that SCCOHT are largely driven by epigenetic deregulation and not by genomic instability.
Importantly, our results underscore marked inter- and intra-tumor homogeneity of SCCOHTs. Combined WES and aCGH analysis revealed a recurrent copy-neutral LOH (CN-LOH) at the 19p13.2-3 locus. CN-LOH can account for inactivation of tumor suppressor genes and likely implicates the loss of the normal allele and duplication of the mutated copy. Notably, 19p LOH has previously been detected in SCCOHT by WES [4
], but our results provide additional evidence for a copy-neutral nature of this event. Of note, telomeric CN-LOH has been linked to meiotic errors occurring during cross-over [37
], which could be in line with the postulated germ cell origin of SCCOHT tumors [2
], although further studies are needed to support this hypothesis. The 19p CN-LOH associated with inactivating SMARCA4
mutations has also been reported in non-small cell lung cancer [38
In line with previous studies, we found that SMARCA4
mutations were present in all but one SCCOHT (5/6) in our series. SMARCA4
encodes one of the enzymatic (ATP-ase) subunits of mammalian SWI/SNF, a chromatin remodeling complex which directs nucleosomes and modulates gene expression. The importance of SWI/SNF alterations in oncogenesis or tumor progression is being increasingly acknowledged, as alterations in SWI/SNF subunits are found in over 20% of human cancers [36
]. Early preclinical studies suggested that SMARCA4
-mutated tumors (such as non-small cell lung cancers) were critically reliant on the SMARCA2
]. Conversely, SCCOHT do not seem amenable to this synthetic lethality strategy, given the complete loss of SMARCA2
expression demonstrated in our series and in previous studies [22
]. This loss of expression is not explained by mutations in the coding sequence of SMARCA2
. In an effort to explore the underpinnings of SMARCA2
silencing, we report for the first time that homozygous polymorphisms at the two SMARCA2
promotor polymorphism sites (−741 bp and −1321 bp), previously linked to SMARCA2
silencing in cancer [15
], do not seem to be a recurrent event responsible for SMARCA2
silencing in SCCOHT. Nevertheless, since most tumors in our study showed a heterozygous promotor polymorphism site status, further explorations are warranted to elucidate whether heterozygous polymorphisms can contribute to SMARCA2
silencing. In particular, in malignant rhabdoid tumor cell lines, increased binding of epigenetic silencers HDAC9 and MEF2D at SMARCA2
promoter sites has been associated with such heterozygous polymorphisms [41
One case (IGR-03) from our series exhibited concomitant inactivating mutations in ARID1A
, two paralog DNA-binding subunits of SWI/SNF, but did not show SMARCA4
mutations. This tumor was also the only case in which 19p CN-LOH was not present. Concomitant ARID1A/B
alterations occur in ~25% of dedifferentiated endometrial and ovarian carcinomas [42
]. While case IGR-03 could illustrate the challenges of differential diagnosis between SCCOHT and dedifferentiated ovarian carcinoma, another possibility is the existence of a molecular and morphologic overlap between those two entities, both of which are characterized by a poorly differentiated, aggressive tumor and a critically deregulated SWI/SNF complex.
Of note, one case (IGR-01) showed a p.Arg635* stop gain in the SMARCA1 gene in addition to a deleterious SMARCA4 mutation. SMARCA1 encodes the ATP-ase of another chromatin remodeling complex, ISWI, and is located on the X chromosome, suggesting that this alteration, which was seen at an allele frequency of 0.37, could potentially carry a deleterious impact.
As a complement to the genomic findings, we also show for the first time that SCCOHT are not characterized by SOX2 overexpression, contrary to another aggressive SMARCA4/SMARCA2
-deficient thoracic sarcoma (SMARCA4
-DTS). This emphasizes the existence of biological differences between SMARCA4
-DTS and SCCOHT, in addition to previously described discrepancies, such as higher genomic instability in SMARCA4
], and could have potential implications in diagnostic pathology.
In addition to SMARCA4
loss-of-function alterations, a few variants were seen in genes other than SMARCA4
, all localized in the 19p13.2-3 locus and subject to the CN-LOH event, for which a functional impact could not be ruled out. In particular, the PLK5
p.G223V variant, predicted as potentially damaging by the Polyphen-2 classifier, was found in 3/33 SCCOHT samples of the extended cohort. The protein kinase domain of PLK5
is truncated in humans compared to mice, but the residual protein containing the polo-box binding domain may act as a stress inducible tumor suppressor regulating G1 arrest [29
]. Nevertheless, the relevance of these variants remains to be validated functionally.
Differential expression analysis comparing SCCOHT and benign ovarian tissue allowed to nominate several genes potentially overexpressed in SCCOHT. Nevertheless, it must be kept in mind that the bulk benign ovarian tissue used as the control in this analysis does not represent the exact cell origin of SCCOHT, which remains unknown. Among other findings, we observed significant overexpression of some putative therapeutic targets. Cancer-testis antigens have been proposed as targets for immunotherapy approaches and Melanoma-associated antigen 4 (MAGEA4
), which was the most highly overexpressed gene in SCCOHT, is currently being investigated as a TCR-engineered T-cell target (NCT03247309). The AURKB
gene encodes Aurora B kinase, implicated in mitotic progression, and may be targeted by pharmaceutical inhibitors (e.g., GSK1070916). The overexpression of receptor tyrosine kinase genes, such as ERBB4
(HER4), could potentially be in line with a recent study showing marked vulnerability of SCCOHT cells to multi-kinase inhibition [43
]. Intriguingly, SCCOHT also showed expression of neural differentiation markers and embryonic stem cell markers, in keeping with what has previously been reported in malignant rhabdoid tumors [44
]. Some of these markers could represent treatment opportunities, such as the embryonic cell junction protein Claudin-6 (CLDN6
), against which monoclonal antibodies were recently part of a clinical trial in ovarian cancers (NCT02054351). Nevertheless, further studies are needed to confirm overexpression of these putative treatment targets at the protein level and to validate their functional relevance in SCCOHT.
To further explore putative therapeutic approaches, we found that the SCCOHT cell line BIN67 was exquisitely sensitive to TSA and 5′-dAZAC, while cells with a SMARCA4
mutation and retained SMARCA2
expression were resistant to these epigenetic therapies. In addition, we describe a clinically meaningful response to single agent EZH2 inhibitor in a patient with SCCOHT, in keeping with what has previously been demonstrated in vitro and in vivo [32
]. Collectively, data from our series and from previous studies suggest that SCCOHT tumors characterized by the loss of both SWI/SNF catalytic subunits may be sensitive to treatment with HDAC, DNA methyltransferase and/or EZH2 inhibitors, and that such strategies merit further investigation in this lethal disease.
Since SCCOHT often display initial chemosensitivity, but subsequently show rapid progression, we compared the genomics of treatment-naïve (n = 3) versus chemotherapy-treated tumors (n = 3), to uncover candidate resistance genes. Critically, neither the tumor mutation burden nor the somatic copy number alterations were significantly increased in post-chemotherapy samples. The only alterations seen in more than one post-chemotherapy sample and not in chemotherapy-naïve samples were ADGRV1 (one stop gain and one missense alteration) and FANCD2 (splice region variants). Variants in ADGRV1, which encodes adhesion G protein-coupled receptor 1, were potentially heterozygous, and the implication of this protein in cancer progression is unclear. Variants in FANCD2 were predicted to be of low functional impact by the SnpEff tool. Although differential expression analysis did not reveal any specific genes significantly deregulated between the two groups, GSEA performed on the totality of ranked genes nominated several deregulated gene expression programs, including an enrichment of genes related to eIF4E upregulation and a putative downregulation of the KRAS pathway. Further studies comparing paired samples from the same patient before and after treatment are needed to elucidate molecular underpinnings of treatment resistance in SCCOHT. Nevertheless, our data suggest that it may rely on other mechanisms than acquiring drug resistance mutations, contrary to what has been described in other tumor types.