Lineage-Specific Alterations in Gynecologic Neoplasms with Choriocarcinomatous Differentiation: Implications for Origin and Therapeutics

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Author Manuscript Published OnlineFirst on April 22, 2019; DOI: 10.1158/1078-0432.CCR-18-4278 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Lineage-specific alterations in gynecologic neoplasms with choriocarcinomatous differentiation: implications for origin and therapeutics

Deyin Xing1,2,#,*, Gang Zheng1,#, §, Aparna Pallavajjala1, J. Kenneth Schoolmeester3, Yuehua Liu1, Lisa Haley1, Yan Hu4, Li Liu5, Lisa Logan5, Yuan Lin6, Kathryn E. Pearce3, Christopher A. Sattler3, Ya Chea Tsai1, Russell S. Vang1,7, Chien-Fu Hung1,2, T.-C. Wu1,2,7, Brigitte M. Ronnett1,7

1Department of Pathology, 2Department of Oncology, 7Department of Gynecology and Obstetrics, The Johns Hopkins Medical Institutions, Baltimore, MD

3Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN

4Department of Pathology, St. Joseph Hospital Health Center, Syracuse, NY

5Department of Pathology, The Mary Catherine Bunting Center, Baltimore, MD

6Clin-Path Associates, PLC, Tempe, AZ

# Contributed equally to this article

* Correspondence: Deyin Xing, MD, PhD, Department of Pathology, The Johns Hopkins Hospital, Weinberg 2242, 410 N. Broadway, Baltimore, MD 21231 (email: [email protected])

§ Current Address: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN

Running title: Gynecologic tumors with choriocarcinomatous differentiation

Keywords: Gynecologic tumors, choriocarcinomatous differentiation, mutations, single nucleotide polymorphisms, PD-L1 

Disclosure of potential conflict of interest: The authors declare no potential conflicts of interest.

Category: Translational Cancer Mechanisms and Therapy

Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 22, 2019; DOI: 10.1158/1078-0432.CCR-18-4278 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ABSTRACT

Purpose: Choriocarcinoma is most commonly gestational (androgenetic or biparental) but can be of germ cell origin or can develop as a component of a somatic neoplasm (genetically related to the patient). The latter type are aggressive neoplasms for which the underlying genetic alterations are not well-characterized.

Experimental Design: To investigate the relationship between the different components of somatic neoplasms with choriocarcinomatous elements, the genetic differences between gestational and non-gestational tumors, and identify potential targetable alterations, we analyzed 23 samples from 11 tumors, including 5 gynecologic-type somatic neoplasms with choriocarcinomatous differentiation (2-3 different components each) and 6 pure choriocarcinomas, for somatic mutations, single nucleotide polymorphisms and PD-L1 expression.

Results: In mixed tumors, gynecologic-type carcinoma components demonstrated lineage- characteristic and lineage-specific alterations, with choriocarcinomatous components sharing some of these as well as demonstrating novel alterations, supporting a clonal relationship with divergent differentiation of the choriocarcinoma from the somatic carcinoma. TP53 mutation only occurred in non-gestational tumors. Diffuse PD-L1 expression was characteristic of choriocarcinoma in both pure and mixed tumors but not seen in the gynecologic-type carcinoma components.

Conclusions: Given that the somatic carcinomatous and choriocarcinomatous components of mixed tumors have distinct genetic alterations and biomarker expression, separate analysis of these components is required to guide targeted therapy. High PD-L1 expression suggests a role for checkpoint inhibitor-based immunotherapy in tumors with a choriocarcinoma component.

The underlying mechanisms by which cancer stem cells reprogram and initiate trophoblastic retrodifferentiation in some somatic tumors warrant further investigation.

TRANSLATIONAL RELEVANCE

Somatic neoplasms comprised of a mixture of a gynecologic-type carcinoma and a component with choriocarcinomatous differentiation are rare but extremely aggressive tumors with a high mortality even with intensive treatment. Due to their rarity, the mechanisms by which these tumors develop and treatment guidelines based on prospective clinical trials are lacking. Our study establishes that gynecologic-type mixed carcinomas with choriocarcinomatous differentiation are somatic tumors in which the choriocarcinomatous component evolves from the carcinoma. Distinct genetic alterations and biomarker expression in different tumor components highlight the importance of separate analysis of all components of mixed tumors to guide molecule/pathway-targeted therapy.

Introduction

Choriocarcinoma, a highly aggressive neoplasm with trophoblastic differentiation, is usually gestational in origin [1, 2]. Unlike gestational choriocarcinoma, non-gestational choriocarcinoma is unrelated to pregnancy; these tumors can be of germ cell origin or somatic as a component of a high-grade malignant tumor [3]. Gestational choriocarcinoma is most commonly androgenetic but can be biparental, whereas germ cell tumors and somatic neoplasms with choriocarcinomatous differentiation are genetically related to the patient. The latter type are aggressive neoplasms for which the specific underlying genetic alterations are not well- characterized. Somatic neoplasms with choriocarcinomatous differentiation are rare but have been described involving certain sites, including breast [4, 5], lung [6, 7], gastrointestinal tract

(esophagus [8], stomach [9, 10], colon [11, 12]), urinary system (bladder [13, 14], renal pelvis

[15, 16]), and the female genital tract (uterine cervix [17, 18], endometrium [19-24], and ovary

[25, 26]). Choriocarcinomatous elements in these somatic tumors display morphologic features essentially identical to those of gestational choriocarcinoma, characterized by cytologically

malignant mononucleate and multinucleate trophoblastic cells which express β-human chorionic gonadotropin (hCG) and other trophoblastic markers. Within the female genital tract, the reported pathologic types of somatic neoplasms that can have choriocarcinomatous differentiation include endometrioid carcinoma [19, 20, 22], clear cell carcinoma [17, 25], serous carcinoma [21] and malignant mixed Müllerian tumor (carcinosarcoma) [23].

While gestational choriocarcinoma typically occurs in women of reproductive age (average age of approximately 30 years) and is often curable by chemotherapy alone [2], the majority of somatic tumors with choriocarcinomatous differentiation affect postmenopausal women and have a poor clinical course [19, 20, 24]. These tumors often manifest with metastatic disease at presentation or during follow-up, often with metastases comprised of only the choriocarcinomatous component [19, 23, 24]. The median survival period is usually less than 1 year [4, 19-23]. Due to their rarity, there is a lack of data on clinicopathologic features, genetic alterations, and therapeutic options for somatic tumors with choriocarcinomatous differentiation.

The genetic basis of choriocarcinomatous differentiation in somatic neoplasms is not well understood. It has been postulated that cancer stem cells can assume aberrant reprogramming that initiates trophoblastic re-differentiation or de-differentiation in terminally differentiated tumor tissues, illustrated by clonal evolution from somatic carcinoma to choriocarcinoma [22]. One analysis demonstrated common genetic changes between the endometrioid and choriocarcinomatous components of 1 tumor, providing evidence of a clonal origin [11]. In addition, the presence of a significant number of additional genetic changes in the latter component provided evidence that the choriocarcinomatous component evolved via marked neoplastic progression from the original endometrioid carcinoma. Other studies have demonstrated that TP53 aberrations, either at the immunohistochemical or molecular level, might be shared in both components, also arguing for a clonal relationship between these

components [21, 23]. We performed next generation sequencing using a large gene panel and other ancillary studies on a set of gynecologic neoplasms with choriocarcinomatous differentiation, with the goals of determining the relationship between the different components of somatic neoplasms with choriocarcinomatous differentiation, understanding the genetic differences between gestational and non-gestational tumors with choriocarcinomatous differentiation, and identifying lineage-specific genetic alterations that might be amenable to targeted therapy.

Materials and Methods

Case selection

Files from the Johns Hopkins Hospital Gynecologic Pathology In-house and Consultation

Service (Baltimore, MD) were searched for cases of choriocarcinoma and choriocarcinomatous/trophoblastic differentiation in women. Five somatic tumors of gynecologic origin with choriocarcinomatous differentiation and 6 pure choriocarcinomas (4 cases described in our previous study [1]) were included in this study. Histologic sections of these cases were re-reviewed by three pathologists (D.X., G. Z. and B.M.R) to confirm the diagnoses. The study was conducted in accordance with the U.S. Common Rule and the tumor specimens and clinicopathologic information were collected with Institutional Review Board (IRB) approval at the Johns Hopkins Hospital (IRB00146977).

Immunohistochemistry (IHC)

IHC staining was performed on formalin-fixed, paraffin-embedded tissue sections, using

Ventana Benchmark automation and the Ultra View detection kit (Ventana Medical Systems,

Tucson, AZ) as previously described [27]. Markers used included: Cyclin D1 (SP4, Cellmarque,

Hot Springs, AZ; prediluted), ER (SP-1, Ventana, Tucson, AZ; prediluted), Gata3 (L50-823,

Biocare, Concord, CA; 1:100 dilution), hCG (mouse polyclonal, Ventana, Tucson, AZ; prediluted), Her2 (4B5, Ventana, Tucson, AZ; prediluted), HSD3B1 (3C11-D4, Novus

Biologicals, Littleton, CO; 1:2,000 dilution), Napsin A (IP64, Leica, Bannock Burn, IL; prediluted), p53 (BP53-11, Ventana, Tucson, AZ; prediluted), Pax8 (Rabbit polyclonal, Proteintech Group;

1:800 dilute), PD-L1 (SP263, Ventana, Tucson, AZ; prediluted), PR (1E2, Leica, Bannock Burn,

IL; prediluted).

DNA extraction

A total of 23 paraffin-embedded tumor tissues and adjacent normal tissue or normal tissues in different blocks, identified by hematoxylin and eosin (H&E) staining of adjacent sections (tumor elements account for more than 80% of section area), were macrodissected, and genomic DNA was extracted using a QIAamp DNA FFPE Tissue Kit with an adapted protocol (Qiagen,

Valencia, CA). For somatic tumors with choriocarcinomatous differentiation, all components with different morphology were well separated. Briefly, slides bearing paraffin embedded tissues were baked at 68°C for 20-30 seconds. The tissues were de-paraffinized 3 times with xylene, and residual xylene was removed by washing through serial dilutions of ethanol. The tumor tissues were separated from adjacent normal tissues and placed in tubes to allow for complete evaporation of residual ethanol. The tissue pellets were resuspended in Buffer ATL with added proteinase K. The rest of the procedure followed the manufacturer’s instruction. Each DNA specimen was assessed by both Qubit and TapeStation analysis to determine both the quality and quantity of template including average genomic fragment length and DNA Integrity Number

(DIN) performance prediction. Normal cut-off thresholds: 100 ng input DNA required in 50 ul volume and DIN  2. In addition to assessment of input DNA template, sequencing quality metrics were also applied (Supplementary Table S1).

Targeted next generation sequencing (NGS)

The targeted next generation sequencing (NGS) was performed as described previously [28].

Libraries were prepared using the Agilent SureSelect-XT Target Enrichment Kit. Briefly, 200-

300 ng of DNA was fragmented to a size of 250-300 bp, using a Covaris M220 sonicator.

The DNA fragments were end-repaired and A-tailed, then adaptors were added by ligation and the fragments were enriched through 10 cycles of polymerase chain reaction (PCR). Each library was then hybridized to a SureSelect custom panel 2.8M bait set (Agilent) according to the manufacturer's protocol. The panel was originally designed as a clinical oncology panel, and covered 637 genes important in oncogenesis. The gene list is available upon request. After stringent washing the captured DNA was amplified with 12 cycles of PCR per manufacturer's protocol. The size and concentration of captured DNA custom was assessed using Tapestation

2200 (Agilent). Captured samples were pooled and sequenced on a single HiSeq flow cell on

HiSeq 2500 (Illumina), using a 2 x 100bp PE Rapid Run v2 protocol. The average unique read depth across the specimens in this study was 577X (Supplementary Table S1).

Mutational analysis

All reads were aligned to the human genome (GRCh37.p13 /hg19), using the Burrows–Wheeler alignment (BWA) algorithm. The final Binary Alignment Map (BAM) file was used for variant calling with our custom variant caller pipeline, which called variants directly from the BAM file with multiple filters, including a filter of >4 mutant reads in both directions, a common SNP filter, and a strand bias filter. In addition, a filter based on a pool of normal FFPE tissue was applied, where variants with variant allele frequency (VAF) falling within 3 standard deviations from mean VAF seen in a pool of normal FFPE tissues, were filtered out. This algorithm was developed for analysis purposes under the assumption that no normal tissue is available. In this study, all 23 samples, including 5 normal tissues from the patients with somatic tumors having choriocarcinomatous components were subjected to NGS. Normal tissues from the patients with somatic tumors were sequenced in order to filter the germline variants. Finally, the Broad

Institute Integrated Genome Viewer (IGV) was used to visually inspect all the variants called by the pipeline, variants with low quality score (median quality score <30) were excluded in this step. Two strand bias scores were calculated: the first one (SB1) considered only strand bias in variant reads: MAX (Var+, Var-)/(Var+ + Var-); the second (SB2) adjusted variant calls for inherent strand bias: (Var+/Ref+)/(Var-/Ref-). A variant call passed the strand bias filter if either

SB1 ≥ 0.7 and/or 2.0 ≥ SB2 ≥ 0.5. The sequencing mean coverage exceeded 300 reads in more than 94% of all regions. Given the estimated tumor cellularity is above 90% of all the cases, we used a variant allele frequency filter of 10%. All variants were separated into 3 categories based on VAF, dbSNP and COSMIC database annotation: 1) pathogenic: loss of function mutations or hotspot mutations based on COSMIC database; 2) likely germline: 40%≤VAF≤60% with or without dbSNP annotation, but present in gnomad database (gnomad.broadinstitute.org) and not fulfilling the criteria as pathogenic; and 3) variants of uncertain significance (VUS): other variants. For SNP analysis, we included both common SNPs (minor allele frequency ≥1%) and rare SNPs with dbSNP annotation.

Copy number variation (CNV) analysis

NGS-based copy number detection was performed using an in-house developed algorithm,

TMM-CNV. TMM-CNV first trims the upper 5% and lower 5% based on the coverage in the gene level, and then calculates normalized mean coverage of sequences of 16 normal healthy individuals (PON) in the gene level after trimming. Again, this algorithm was developed for analysis purposes under the assumption that no normal tissue is available. The use of this PON, run under the same conditions as the tumor specimens, allows for a base by base coverage comparison between that expected for 2 copies versus either copy number amplification or loss in the tumor specimen. In this study, both tumor tissues and normal tissues from the patients with somatic tumors having choriocarcinomatous components were analyzed for CNV. For autosomes a log2 value of mean relative coverage depth between sample and pool of controls

was used, and copy number analysis was not performed for sex chromosomes. Conservative thresholds were used to call copy number gains and losses. Final calls were correlated with informative SNP allele frequencies at nearby loci and assessed for number of copies lost or gained with respect to pathology estimate of the tumor cell percentage.

The copy number described here (3-5 copies or > 5 total copies) is a theoretical copy number based on mathematical calculation. For example, given a log2 ratio of 1.4, if tumor cellularity is

100%, there should be 21.4 x 2 = 5.3 copies or 3.3 copy gains (5.3 - 2 = 3.3). With tumor cellularity less than ideal (< 100%) for most of the clinical samples, and a log2 ratio of 1.4 at a certain tumor cellularity may correlate to a theoretical minimum of 5 copies or more. Calculations were validated against known cell lines and clinical samples during clinical validation of the pipeline. We have also performed dilution experiments to verify that the log values reflect the dilution and therefore the copy numbers. Accordingly, we use the following criteria for copy number gains: 0.5849 ≤ log2 ratio < 1.3219, less than 3 copy gain (less than 5 biological copies); 1.3219 ≤ log2 ratio < 1.8073, 3-5 copy gain (5-7 biological copies); log2 ratio ≥

1.8073, ≥ 5 copy gain (≥ 7 biological copies). We use the following criteria for copy number loss:

-1 < log2 ratio < - 0.5849, single copy loss; log2 ratio ≤ -1, 2 copy loss (assumed at 100% tumor cellularity).

Polymerase Chain Reaction (PCR) and Sanger Sequencing

Seven genes, containing 8 mutational sites, were further assessed by Sanger sequencing.

Briefly, 50 ng of DNA was amplified by PCR with Taq DNA Polymerase and Standard Taq

Buffer (New England BioLabs, MA). The reaction for amplification of first-round PCR primers

(F1/R) was carried out in the following conditions: an initial melting step of 2 min at 95°C, followed by 35 cycles of 30 sec at 94°C, 30 sec at 51°C and 45 sec at 72°C and a final elongation of 10 min at 72°C. The PCR products ampliﬁed by F1/R primers were diluted 10 times and used as templates of a second PCR ampliﬁcation (nested PCR) with another pair of

primers F2/R that was carried out in similar reaction conditions except for an annealing temperature of 54 °C. DNA sequencing of the purified DNA products was performed using the

ABI 3730 High-Throughput DNA Sequencer. The mutations and variations were analyzed using

UGENE software. Detailed information regarding primers can be found in Supplementary Table

S2A.

Molecular genotyping

Molecular genotyping was performed using unstained 10μm formalin-fixed paraffin-embedded tissue sections prepared using PCR precautions [1]. A serial H&E-stained 5 μm section was used to identify well-separated areas of tumor and maternal tissue in all cases. An area of each tissue type was circled with a marking pen on the H&E-stained slide which was used to guide microdissection of these tissues for genotyping. PCR amplification of 15 short tandem repeat

(STR) loci from 13 different chromosomes (chromosomes 2, 3, 4, 5, 7, 8, 11, 12, 13, 16, 18, 19, and 21) and the amelogenin locus (for XY determination) was performed, with thermal cycling conditions and capillary electrophoresis carried out according to the manufacturer’s instructions

(Amp FlSTRIdentiﬁler kit; Applied Biosystems, Foster City, CA). Technical details of genotyping and interpretation of genotyping results are provided in previous publications [1, 29].

Fluorescence in situ hybridization (FISH)

FISH analysis was performed as described previously [30]. Briefly, paraffin-embedded tissues were cut at 5 µm thickness and mounted on positively charged glass slides. Using the H&E- stained slide as a reference, target areas were etched with a diamond-tipped etcher on the back of the unstained slide to be assayed. The slides were baked at 90°C for 15 minutes to promote adherence. Following deparaffinization, acid treatment was conducted using 10 mM citric acid for 10 minutes. The slides were pretreated in a 2x saline sodium citrate (2xSSC) solution for 5 minutes at 37°C. Protease digestion was carried out at 37°C for 48 minutes in a 0.9% NaCl/0.2%

pepsin solution. The slides were submerged in graded ethanol solutions (70%, 85%, 100%, each for 3 minutes) for dehydration, then allowed to air dry. Using the ThermoBrite

Denaturation/Hybridization System (Abbott Molecular Inc, Des Plaines, IL), hybridizations were carried out at 37°C for 8 hours. Post-hybridization wash consisted of soaking the slides in 0.1%

NP-40/2xSSC solution at room temperature to remove the cover slips, then placed in 72°C 0.1%

NP-40/2×SSC for 2 minutes, and rinsed in 0.1% NP-40/2Xssc, and immediately counterstained with DAPI (4′,6′-diamidino-2-phenylindole dihydrochloride hydrate). For MYC amplification study, commercially available probe set for MYC and the centromere region of chromosome 8 (D8Z2) were hybridized to the appropriate target areas and 2 technologists each analyze 30 interphase nuclei (60 total) per probe set with the results expressed as a ratio of MYC:D8Z2 signals. MYC locus was reported as amplified when the MYC:D8Z2 ratio of 2.0 or greater and demonstrates 6 or more copies of the MYC locus. A lesion with a MYC:D8Z2 ratio <2.0 or showing a ratio of 2.0 or greater with less than 6 copies of MYC was considered to lack amplification of the MYC locus. Likewise, for HER2 amplification study, commercially available probe set for HER2 and the centromere region of chromosome 17 (D17Z1) were used. HER2 was considered unequivocally amplified when Her2:D17Z1 ≥ 2.00 as well as Her2/cell ≥ 4.0

(2018 ASCO/CAP guidelines).

Results

Clinicopathologic features

Clinicopathologic features are summarized in Table 1. For the somatic carcinomas with choriocarcinomatous differentiation (cases 1-5), the patients ranged in age from 44 to 77 years

(mean, 66; median, 69). Tumors were uterine in 4 cases (all postmenopausal) and ovarian in 1

(premenopausal). The somatic components included endometrioid carcinoma (n=2), mixed endometrioid and clear cell carcinoma (n=1), high-grade adenocarcinoma - not otherwise

specified (n=1), and malignant mixed Müllerian tumor/carcinosarcoma (n=1) (see Figures 1A and 2A, Supplementary Figures S1A, S2A, S3A, and S4A). Selected results of immunohistochemical analysis performed as part of the diagnostic evaluation on these tumors to confirm the morphologic interpretations are provided in Supplementary Table S2B. The choriocarcinomatous components of the mixed tumors selectively expressed one or more trophoblastic markers, including hCG in all 5 cases, as well as GATA3 and/or HSD3B1 in 3 of these; the other (non-choriocarcinomatous) components of these tumors lacked expression of these trophoblastic markers (see Figures 1A and 2A, Supplementary Figures S1A, S2A, S3A, and S4A). The non-choriocarcinomatous components generally expressed markers consistent with the morphologic diagnosis, such as Pax8, ER, Napsin A, or HNF1B (Supplementary Table

S2B).

The 6 patients with pure choriocarcinoma ranged in age from 23-46 years (mean, 33; median,

33), and were thus significantly younger than those with somatic carcinomas with choriocarcinomatous differentiation (p < 0.01, unpaired t-test). All of these patients had elevated serum hCG levels. Five tumors were uterine and 1 was pararectal. Three uterine tumors were gestational (androgenetic) and the pararectal tumor was non-gestational based on genotyping results previously reported [1]. One uterine tumor was not genotyped due to lack of normal tissue for comparative analysis; another uterine tumor was not genotyped but consistent with a gestational tumor based to the presence of rare chorionic villi in the specimen. These tumors displayed classical morphologic features of choriocarcinoma.

There was a notable difference in behavior of the different tumor types. Of 2 patients with gestational choriocarcinoma with available follow-up (cases 6 and 10), both were alive without evidence of disease at 14.5 and 30 months. Of note, the other patients with gestational choriocarcinoma in our previous study and with available follow-up (which were not included in

this analysis due to insufficient or unavailable material for further genetic analysis) were all alive and all but one was without evidence of disease [1]. One patient with non-gestational pure choriocarcinoma in the current study died of disease at 18 months. Of note, another patient with non-gestational choriocarcinoma in our previous study and with available follow-up (which was not included in this analysis due to insufficient or unavailable material for further genetic analysis) was alive with progressive disease that was unresponsive to multi-agent and high- dose chemotherapy [1]. Of 4 patients with mixed somatic carcinomas with choriocarcinomatous differentiation with available follow-up, 3 died at intervals of 1, 2 and 13 months after hysterectomy (cases 2, 3, 4) and 1 was alive but had multiple lung metastases at 8 months.

Lineage specific mutational profile in somatic tumors with choriocarcinomatous differentiation

17 samples including each different tumor component as well as a normal tissue control from 5 cases of somatic tumors with choriocaricnomatous differentiation were first sequenced and analyzed (Supplemental Table S2B). For the tumor in case 1, which was comprised of endometrioid carcinoma, clear cell carcinoma, and choriocarcinoma, the mutational profile determined by the targeted next generation sequencing showed that all 3 components harbored somatic mutations of ARID1A, PIK3CA, BRD3, PTPRT, and STAG2, indicating a shared precursor origin (Figures 1B and 1C, Supplemental Tables S2B and S3)., The clear cell carcinoma and choriocarcinoma components contained common somatic mutations of ARID1A,

BRD3, CDKN2A, SRSF2, which were not identified in the endometrioid carcinoma component.

The latter had somatic mutations of HNF1A, KMT2B, TET2, FGFR4, IGF2R, DST, NCOA2, and

ZMYM3, which were not detected in the clear cell carcinoma and choriocarcinoma components.

Both the clear cell carcinoma and choriocarcinoma components each harbored some mutations that were not shared by the other: the former had somatic mutations of ARID1A, SPEN, PALB2,

ATR, and FANCE; the latter had somatic mutations of KMT2B, JARID2, GRM8, AKAP9, and AR.

Somatic mutations of ARID1A and BRD3 were further confirmed by Sanger sequencing (Figure

1D and Supplementary Figure S1B-1E). For the tumor in case 2, the endometrioid carcinoma and choriocarcinoma components shared ERBB3 p.R12W somatic mutation which was confirmed by Sanger sequencing (Figures 2B-2D). AFF3 somatic mutation was detected only in the endometrioid carcinoma component and ERBB4, only in the choriocarcinoma component.

These mutations were not seen in a concurrent breast carcinoma, which harbored somatic mutations of NCOR2, PIK3CA and UBR5 which were not present in either the endometrioid carcinoma or choriocarcinoma components of the uterine tumor, supporting interpretation as independent breast and uterine tumors (Figure 2B). For the tumor in case 3, PPP2R1A, PIK3CA, and NOD1 somatic mutations were identified in both the carcinosarcoma and choriocarcinoma components (supplementary Figures S2B and S2C). For the tumor in case 4, while the high- grade adenocarcinoma and choriocarcinoma components contained somatic mutations of

KAEP1, FOXP1, FBXW7, and EPHA7, a CDH1 mutation was only present in the former component and aTP53 mutation was only in the latter (Supplementary Figures S3B and S3C).

For the tumor in case 5, no shared mutations were found in the endometrioid carcinoma and choriocarcinoma components (Supplementary Figures S4B-S4D). FBXW7 and PIK3R1 mutations were identified in the endometrioid carcinoma component and BCL9, ARID1A, PTEN,

PIK3CA and MAP3K1 mutations were detected in the choriocarcinoma component. Based on the patterns of shared and unique somatic mutations in the different tumor components in each case, and lineage-specific gene expression, diagrams for tumor evolution are proposed (Figures

1E and 2E, Supplementary Figures S2D, S3D, and S4E).

PD-L1 expression

Immunohistochemical analysis of PD-L1 expression demonstrated that the choriocarcinomatous components of the mixed somatic tumors highly expressed this marker, with 4 of 5 tumors having more than 90% of tumor cells positive and 1 having approximately 50% of tumor cells

positive (Figures 1A and 2A, Supplementary Figures S2A, S3A, and S4A). In contrast, the non- choriocarcinomatous somatic tumor cells in all mixed tumors were either negative or had limited staining in scattered cells (~1%) which included occasional tumor cells, lymphocytes and macrophages. Similar to the choriocarcinomatous components of the mixed somatic tumors,

PD-L1 expression was present in all pure choriocarcinomas, with extent of expression ranging from 20-90% of tumor cells.

Molecule/pathway-based targeting of somatic tumors with choriocarcinomatous differentiation

Based on the observed lineage-specific mutational changes and PD-L1 expression in somatic tumors with choriocarcinomatous components, we have proposed molecule/pathway-based therapeutic strategies that might be applicable to each of these mixed tumors (Figures 1E and

2E, Supplementary Figures S2D, S3D, and S4E).

Comparative analysis of somatic tumors with choriocarcinomatous differentiation and pure choriocarcinomas

6 pure choriocarcinomas were sequenced for mutational analysis. Surprisingly, only rarely were somatic mutations identified in this group of tumors (Figures 3A and 3B). The 1 pure non- gestational choriocarcinoma (case 9) harbored TP53, LRP1B and NOTCH1 mutations.

Consistent with sequencing results, this tumor displayed a complete loss of nuclear p53 expression (“null” pattern) and contained a nonsense mutation in codon 196 (Figures 3D and

4A). The tumor also highly expressed PD-L1 (~ 50% of tumor cells) and Her2/Neu (diffuse cell membrane staining) (Figure 4A). This tumor was resistant to chemotherapy and was fatal.

CDH2 mutation was detected in one pure gestational type choriocarcinoma and IDH2 mutation was found in another choriocarcinoma of unknown type (Figure 3A). No somatic mutations were detected in the remaining 3 tumors using this gene panel. Of note, TP53 mutation only occurred

in non-gestational tumors, including 1 mixed somatic tumor with choriocarcinomatous differentiation and 1 pure choriocarcinoma (Figures 3C and 3D). The mixed tumor had TP53 mutation in the choriocarcinoma component but none was detected in the adenocarcinoma component even though both components displayed a complete loss of nuclear p53 expression

(“null” pattern).

Copy number variation (CNV) analysis

CNVs in the segments of the genome were analyzed for amplification and deletion from the targeted sequencing data (Supplementary Table S4). A number of candidate driver genes were identified by clinically reportable threshold from the CNVs. Recurrently amplified genes in the choriocarcinomatous components of mixed somatic tumors and pure choriocarcinoma included

CEBPB, CEBPA, FOXL2, EXOSC6, IRS2, SOCS1, SOX11, MAFB, MYCN, MYCL1, RICTOR,

MNX1, ROS1, BCL11B, POT1, DST, LIFR, TOP1 (Supplementary Table S4). In case 2, chromosome region 11q13, which includes Cyclin D1, FGF19, FGF3, was highly amplified in the breast carcinoma but not in the endometrioid carcinoma or choriocarcinoma components of the uterine tumor. In keeping with this sequencing result that showed increased copy number, immunohistochemical staining demonstrated a high level of Cyclin D1 expression in the breast carcinoma (Supplementary Figure S5). The non-gestational pure choriocarcinoma (case 9) displayed frequent copy number changes, illustrated by more than 3 copy gains of Her2, IKZF3,

PGAP3, C-Myc, as well as less than 3 copy gains in 9 genes. A confirmatory FISH study showed marked gene amplification of Her2 and C-Myc in this tumor (Figures 4C and 4D).

Despite overexpression of hCG and PD-L1 in choriocarcinomatous components of mixed somatic tumors and pure choriocarcinomas, a gain-of-copy number was not seen by this analysis (Supplementary Table S4).

Single nucleotide polymorphism (SNP) analysis for origin of choriocarcinomatous components

To further evaluate the clonal relationship of the components of the mixed somatic tumors with choriocarcinomatous differentiation, we analyzed 291 SNP sites in normal tissues and the components of the mixed tumors. We found the pattern of selected SNPs in all components were nearly identical in each case and all alleles were shared in all components in an individual tumor case, suggestive of the same clonal origin from that same individual, rather than a gestational process (Figure 5A). Occasionally, the allele frequency at some loci, for example, rs10413435 C/T, was slightly different in different components, likely due to copy number variation in that locus (Figures 5B and 5C).

Discussion

The current study provides comprehensive molecular data to address the following: (a) histopathologic origin of choriocarcinomatous elements associated with somatic tumors; (b) lineage-specific genetic alterations in individual components of somatic tumors with choriocarcinomatous differentiation; (c) comparative analysis of genetic changes in choriocarcinomatous elements associated with somatic tumors and pure choriocarcinomas; (d) amenable therapeutics based on targeting molecule/pathway-related alterations.

While the mechanism of development of choriocarcinomatous components in somatic tumors remains largely unknown, several case reports have suggested that choriocarcinomatous

elements evolve from the somatic tumor component rather than as independent somatic and gestational neoplastic processes. A whole genome copy number analysis of 1 case revealed a large number of losses commonly present in the endometrioid carcinoma and the component with trophoblastic differentiation, indicating a clonal origin [31]. Likewise, another report used comparative genomic hybridization (CGH) and fluorescence in situ hybridization to elucidate the genetic changes in colorectal adenocarcinoma and associated choriocarcinoma, revealing a loss of chromosomal regions 8p21-pter as well as 18q21-pter, and a gain of 5p and 20q, in both tumor parts [11]. At a molecular level, targeted next-generation sequencing demonstrated an ataxia telangiectasia mutated (p.P604S) missense mutation present in a gastric carcinoma and subsequent colorectal choriocarcinoma [32]. Another report genotyped an endometrioid carcinoma with a choriocarcinomatous component and found that the patterns in both components were nearly identical, with all alleles shared by both tumors [22]. To expand on these limited data from case reports, we performed next generation sequencing using a larger gene panel on a set of gynecologic tumors with choriocarcinomatous differentiation to investigate the origin of these tumors and relationship of the different components from 2 different perspectives.

Our results demonstrated patterns of shared and divergent somatic mutations that support interpretation of the choriocarcinomatous components of mixed tumors as evolving from the associated gynecologic-type somatic carcinoma components in a clonal fashion. The presence of some shared/identical mutations in the somatic tumor and choriocarcinomatous components in 4 of 5 mixed tumors supports a clonal origin in these cases. In addition, the patterns of differences in mutational profiles between different components suggest pathways of lineage- specific evolution of these components. For example, for the mixed tumor in case 1, the endometrioid carcinoma, clear cell carcinoma, and choriocarcinoma components harbored identical somatic mutations of ARID1A, PIK3CA, PTPRT, BRD3 and STAG2, indicating a clonal

relationship. The clear cell carcinoma and choriocarcinoma components shared common mutations of ARID1A (different codon), SRSF2, BRD3, and CDKN2A, which were not present in the endometrioid carcinoma, supporting subsequent divergent development from the endometrioid carcinoma component. The clear cell carcinoma and choriocarcinoma components also harbored, respectively, unique mutations in ARID1A (different codon), SPEN,

PALB2, ATR, FANCE, KMT2B, JARID2, GRM8, AKAP9, and AR, supporting yet further evolution into their specific tumor types, with the choriocarcinoma component having unique

PD-L1 over-expression (Figure 1A). ARID1A and PIK3CA are found to be frequently disrupted in ovarian clear-cell and endometrioid carcinomas [33, 34], which have a known relationship with endometriosis, and somatic mutations of these 2 genes have been found in endometriosis without cancer, suggesting that mutations of ARID1A and PIK3CA occur at a very early stage.

[35] ARID1A is a tumor suppressor that acts as a component of the SWI/SNF chromatin remodeling complex. PIK3CA is an oncogene located at chromosome 3q26 which encodes the p110 catalytic subunit of phosphatidylinositol 3-kinase (PI3K). These findings support the development first of an endometrioid carcinoma in the setting of endometriosis, which acquired additional mutations to evolve into a high-grade component which developed additional unique lineage-specific mutations to evolve into clear cell carcinoma and choriocarcinoma components.

Three of the other mixed tumors have mutational profiles in the individual components that support a similar model of clonal evolution of the choriocarcinomatous components from the somatic carcinomatous components. The one exception is the lack of any shared mutations in the components of the mixed tumor in case 5. While this finding does not refute a relationship between the components, an independent origin cannot be excluded. Of note, there was no evidence that the choriocarcinomatous component was an independent gestational tumor.

The second evidence we obtained from this study to support the somatic origin of the choriocarcinomatous components in these mixed tumors is a comparative SNP analysis.

Choriocarcinoma is most often gestational and genotyping demonstrates novel/obligate paternal alleles in both androgenetic and biparental gestational choriocarcinoma. [1]. In contrast, non- gestational choriocarcinoma is somatic and genotyping demonstrates that tumor DNA matches the patient’s DNA - that is, all alleles are shared between the tumor and the patient. Similarly,

SNP analysis is expected to demonstrate that a choriocarcinoma of gestational type would have some non-shared SNPs compared with a patient’s normal tissue whereas complete sharing of all SNPs would indicate that a choriocarcinoma is somatic. Indeed, our data showed that all selected SNPs (291 sites) were shared between normal control tissue, somatic tumor components and choriocarcinomatous components in all mixed tumors, without any evidence of non-maternal SNPs identified. These findings provide convincing evidence to support a somatic origin for the choriocarcinomatous components of these mixed tumors.

Our findings suggest that there is a common precursor of somatic origin from which both the non-choriocarcinomatous and choriocarcinomatous components evolve. Thus, choriocarcinoma can be separated into 3 categories based on their origins and oncogenic pathways ([11, 22] and

Figure 6). The most common subtype is gestational choriocarcinoma which is thought to originate from cytotrophoblastic cells that undergo neoplastic transformation from a molar pregnancy (androgenetic subtype) or in a non-molar placenta (biparental subtype). Non- gestational choriocarcinomas include those arising from germ cells and those arising from somatic tumors. It has been proposed that choriocarcinomatous differentiation in a somatic tumor represents “retrodifferentiation” by which rejuvenation to early embryonic development may occur when differentiated cells lose their specific properties acquired during previous steps of maturation [36, 37]. Interestingly, a similar phenomenon has been observed in somatic neoplasms with yolk sac differentiation. While most yolk sac tumors are of germ cell origin, these tumors can originate from somatic tumors, especially endometrioid tumors. This latter type provides additional evidence of “retrodifferentiation” [38-40].

The common genetic changes between the somatic tumor and choriocarcinoma components of mixed tumors, as well as shared SNPs between the normal tissue and tumor components, are evidence of clonal somatic origin. We also found distinct additional genetic changes in different tumor type components of mixed tumors indicative of lineage-specific evolution, as discussed above. The choriocarcinomatous components of mixed tumors contained somatic mutations of

TP53, PIK3CA, ARID1A, ERBB4, PTEN, MAP3K1, KMT2B, JARID2, GRM8, AKAP9, AR, BCL-

9 which were not seen in the corresponding non-choriocarcinomatous components. These findings highlight the importance of using next generation sequencing technology on specific tumor components of mixed tumors to precisely assess for amenable targets. We expected to identify some unique/specific genetic changes that might provide some insight into how choriocarcinomatous differentiation occurs in these mixed somatic tumors. To our surprise, except for TP53 mutation, which was found in 1 pure non-gestational choriocarcinoma and the choriocarcinomatous component of 1 mixed tumor, we failed to identify any other recurrent somatic mutations that occurred in both pure choriocarcinoma and the choriocaricnomatous components of somatic tumors. Moreover, pure choriocarcinomas, which were mostly of gestational type, exhibited only extremely rare somatic mutations based on the gene panel used in this analysis. Of note, the 1 pure choriocarcinoma with several mutations, including TP53,

NOTCH1 and LRP1B mutations, was non-gestational and resistant to chemotherapy. This component also harbored Her2 and c-Myc amplification and highly expressed PD-L1. Some gain-of-function alterations with shared gene amplification, especially those that function as oncogenic drivers, such as IRS2, MYCN, MYCL1, ROS1, were found in both the choriocarcinomatous components of mixed tumors and pure choriocarcinomas, indicating some common histopathologic pathways. Given the rarity of somatic mutations identified in the pure choriocarcinomas based on the gene panel used in this analysis, we speculate that genetic alterations such as copy number variations, as described here, gene

overexpression/inactivation, and DNA and/or histone-related epigenetic regulation, may play a critical role in the oncogenesis of gestational choriocarcinomas.

Despite the findings in our study, the underlying mechanisms by which the choriocarcinomatous phenotype develops are not clear. Gene imprinting is critical for normal human growth and lineage-specific organ development. It has been demonstrated that both types of hydatidiform moles (sporadic and familial) are associated with aberrant genomic imprinting [41, 42]. Since most choriocarcinomas are related to hydatidiform moles [43], epigenetic regulation likely plays a vital role in the development of choriocarcinoma [44]. While some studies have assessed genome-wide methylation profiles of molar tissue, a comprehensive investigation of epigenetic changes in choriocarcinoma, particularly in comparison with choriocarcinomatous components of somatic origin, is lacking. We are currently comparatively ascertaining the genome-wide methylation profile of pure choriocarcinomas and choriocarcinomatous components of mixed tumors, with the goal of identifying epigenetic alterations that might be associated with the development of tumors with a choriocarcinomatous phenotype.

One characteristic feature of somatic tumors with choriocarcinomatous differentiation is very poor outcome, with most patients experiencing tumor progression with distant metastasis and fatal outcome. The choriocarcinomatous components of these mixed tumors, as the most common metastatic component, is thought to confer disease-specific death. Because of its rarity, the clinicopathologic features that relate to clinical outcome remain largely unknown and standard treatment protocols that guide clinical management are lacking. Our previous results showed that gestational and non-gestational choriocarcinomas have distinct clinical behavior, sensitivity to chemotherapy, and prognosis [1]. Unlike gestational choriocarcinoma, non- gestational choriocarcinoma originates entirely from the patient and has poorer immunogenicity which results in less sensitivity to chemotherapy. We speculate that the nature of the

choriocarcinomatous component of mixed somatic tumors is likely similar to that of pure non- gestational choriocarcinoma, with similar resistance to chemotherapy likely responsible for the high mortality. One important finding of the current study, similar to that reported for pure gestational trophoblastic tumors [45, 46], is that all choriocarcinomatous components of mixed tumors in this series demonstrated a high level of PD-L1 expression (all tumors with at least 50% of tumor cells positive and most with over 90% expression). This observation provides a rationale for immunotherapy using checkpoint inhibitors [47]. In fact, a case study reported a strong anti-tumor activity of nivolumab, a checkpoint inhibitor disrupting PD-1-mediated signaling to restore anti-tumor immunity, in a patient with pulmonary carcinoma with choriocarcinomatous features which expressed PD-L1 in more than 50% of tumor cells [7].

Given that the mixed somatic tumors with choriocarcinomatous components analyzed in our study have demonstrated distinct genetic alterations and biomarker expression, clinical management in the era of personalized medicine and targeted therapy should specifically address all tumor components. Our results have revealed some common altered molecular pathways that can be precisely targeted in clinical trials, potentially broadening the spectrum of therapeutic options for these rare yet aggressive somatic tumors with choriocarcinomatous differentiation, potentially even regardless of the histopathologic type of the somatic tumor. For example, mutations of ARID1A and PIK3CA were found in all 3 components in the tumor in case 1. In particular, the clear cell carcinoma component harbored 3 different ARID1A mutations; choriocarcinoma 2 different ARID1A mutations; and the endometrioid carcinoma component had 1 ARID1A mutation. The latter was also shared by the clear cell carcinoma and choriocarcinoma components. It has been demonstrated that inhibition of the EZH2 methyltransferase, DNA damage checkpoint kinase ATR, or aurora kinase A (AURKA) acts in a synthetic lethal manner in ARID1A-mutated ovarian and colorectal cancer cells and that

ARID1A mutational status correlated with response to these inhibitors [48-50]. Inhibition of these

enzymes could be effective in all these different components since all 3 have ARID1A mutations.

Similarly, targeted inhibitors for PIK3CA, ERBB, PTEN/AKT, MAP3K1 are available and may be worthy of clinical trials to test the efficacy of these inhibitors to treat these rare tumors.

In summary, we report a series of gynecologic neoplasms with choriocarcinomatous differentiation. We provide comprehensive molecular evidence, including identical somatic mutations and shared SNPs, to demonstrate the somatic origin of the choriocarcinomatous components of these mixed tumors. Importantly, understanding lineage-specific genetic alterations provides a rationale for pathway/molecule-based therapeutics and immunotherapy that target all tumor components. The underlying mechanisms by which cancer stem cells reprogram their evolutionary direction and initiate trophoblastic retrodifferentiation in somatic tumors warrant further investigation.

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Grant Supports: The present study was supported by the Career Development Award by the

Cervical Cancer SPORE program (5P50CA098252) at Johns Hopkins (D.X.)

Authors’ Contributions: Conceptualization & Methodology, Writing, Review & Editing and

Project management: D.X., G.Z. and B.M.R.; Investigation: A.P., J.K.S., Y.L., L.H., K.P., C.S.,

Y.C.T., R.S.V., C.F.H., T.C.W.; Materials and reagents: Y.H., L.L., L.L., Y.L. and Supervision and Funding acquisition: D.X.

Table and Figure legends

Table 1. Clinicopathologic features.

Figure 1. Common and lineage-specific alterations of clear cell carcinoma (CCC), endometrioid carcinoma (EMCA), and choriocarcinomatous component (ChorioCa) in case 1. A.

Representative histologic images of CCC (a, b) that lacks hCG expression (c) and only shows scattered PD-L1 positive cells (d); EMCA (e, f) with no expression of hCG (g) or PD-L1 (h);

ChorioCa (i, j) with strong and diffuse expression of hCG (k) and PD-L1 (l, more than 90% of tumor cells). B. Somatic mutations detected by targeted next-generation sequencing. The mutated activating oncogenic and caretaker tumor suppressor genes are plotted in an order of mutations shared by all 3 tumors (CCC, ChorioCa, EMCA), shared by 2 tumors (CCC, ChorioCa) and by individual tumor. All somatic mutations are not detected in normal tissue. C.

Representative ARID1A pS.2113fs mutation (C insertion). D. By Sanger sequencing, the above mutation was detected in 3 tumors but not in normal tissue. *Mutation site. E. Schematic of tumor evolution is depicted as a tree inferred from the somatic mutations (plotted in B). All 3 tumors in this patient were speculated to originate from endometriosis with shared identical

somatic mutations in 5 genes. Further, CCC and ChorioCa are postulated to be derived from a progenitor cell that underwent an oncogenic pathway different from that of EMCA. ARID1A,

PIK3CA and PD-L1 can be targeted by synthetic lethal inhibitors or checkpoint inhibitors.

*Second ARID1A mutation; **Third ARID1A mutation.

Figure 2. Common and lineage-specific alterations of endometrioid carcinoma (EMCA), breast carcinoma (Breast Ca) and choriocarcinomatous component (ChorioCa) in case 2. A.

Representative histologic images of EMCA (a, b) that lacks expression of hCG (c) and PD-L (d);

Breast Ca (e, f) with no expression of hCG (g) or PD-L1 (h); ChorioCa (i, j) with strong and diffuse expression of hCG (k) and patchy PD-L1 (l, ~50% of tumor cells). B. Somatic mutations detected by targeted next-generation sequencing. The mutated genes are plotted in an order of mutations shared by 2 tumors (EMCA, ChorioCa) and by individual tumor. Mutations in Breast

Ca were not present in EMCA or ChorioCa. All somatic mutations were not detected in normal tissue. C. Representative ERBB3 p.R12W missense mutation. D. By Sanger sequencing, the above mutation was detected in EMCA and ChorioCa but not in normal tissue or Beast Ca.

*Mutation site. E. Schematic of tumor evolution is depicted as a tree inferred from the somatic mutations (plotted in B). EMCA and ChorioCa are postulated to be derived from a progenitor cell; Breast Ca is likely an independent event that is not related to the other tumor. ERBB3,

ERBB4, PIK3CA and PD-L1 can be targeted by specific inhibitors or checkpoint inhibitors.

Figure 3. Comparative analysis of somatic mutations in choriocarcinomatous component of somatic tumor and pure choriocarcinoma. A.B. In comparison with the former, extremely rare somatic mutations were identified in pure choriocarcinomas. One pure choriocarcinoma (case 9) harbored TP53, LRP1B and NOTCH1 mutations. C and D. TP53 is the only gene that contained mutations in both a choriocarcinomatous component of a mixed tumor (C, case 4) and a pure choriocarcinoma (D, case 9).

Figure 4. A non-gestational pure choriocarcinoma. A. Representative histologic images of choriocarcinoma (a) that display a “null” TP53 mutational staining pattern (see Figure 3) and diffuse expression of PD-L1 (c, more than 90% of tumor cells) and Her2 (d, 3+). B. Genotyping demonstrates that the tumor (lower portion) matches the maternal DNA (upper portion), at all loci with no novel alleles identified. C. Representative FISH image with probes for Her2 (Orange) and D17Z1. HER2 is considered unequivocally amplified when Her2:D17Z1 > or = 2.00 as well as Her2/cell > or = 4.0. D. Representative FISH image with probes for MYC (Orange) and D8Z2.

MYC locus is reported as amplified when the MYC:D8Z2 ratio of 2.0 or greater and demonstrates 6 or more copies of the MYC locus.

Figure 5. Single nucleotide polymorphisms (SNPs) detected by next-generation sequencing. A.

Total 291 SNPs in normal tissue and each component of tumors are plotted for cases 1 to 5. All

SNPs in choriocarcinomatous components (ChorioCa) can be found in corresponding normal tissue and somatic tumor components. B. Representative SNP rs10413435 C/T in PPP2R1A gene (Chromosome 19). C. Relative allele frequency of rs10413435 C/T in normal tissue and tumor components. Allele frequency was slightly different in different components, likely due to copy number variation in that locus.

Figure 6. Model for choriocarcinoma development. (a). Gestational choriocarcinoma is related to pregnancy and originates from cytotrophoblastic stem cells in either a hydatidiform mole or a placenta. (b). Non-gestational choriocarcinoma can be of germ cell origin, with or without other germ cell tumors. (c). Somatic tumors can reprogram their evolutionary direction and initiate choriocarcinomatous retrodifferentiation (for which the underlying mechanisms remain unknown) to develop a clonally-related choriocarcinomatous component.

Table 1. Clinicopathologic features Treatment and Age Clinical presentation and β-hCG at Tumor location Case Procedure Diagnosis Additional pathologic findings follow-up if (years) presentation if available (mIU/mL) and size available

Bilateral ovarian endometriotic Alive with Primary infertility, complex cystic pelvic Mixed clear cell carcinoma and Right ovary, cysts with endometrial-type multiple lung 1 44 mass, abnormal uterine bleeding; TAH/BSO endometrioid carcinoma with 11.5 cm complex atypical hyperplasia in metastases, 8 β-hCG 3000 choriocarcinomatous differentiation left side months

Infiltrating breast carcinoma with Chemotherapy; New onset heart failure likely secondary TAH/BSO; Endometrioid carcinoma with focal micropapillary features; 2 68 Uterus, 9 cm Dead of disease, to metastatic cancer Mastectomy choriocarcinomatous differentiation Metastatic choriocarcinoma in 2 months axillary lymph node

Cervix, 10.5 Malignant mixed mullerian tumor Chemotherapy; cm; Metastatic adenocarcinoma in 3 77 Cervical mass TAH/BSO (carcinosarcoma) with Dead of disease, endometrium, left ovary choriocarcinomatous differentiation 13 months 5.2 cm

TAH/BSO, Metastatic adenocarcinoma in Postmenopausal vaginal bleeding and Uterus, 6.0 cm High-grade adenocarcinoma with Dead of disease, 4 70 Tumor left ovary and abdominal soft endometrial mass choriocarcinomatous differentiation 1 month debulking tissue

Uterus, 10.5 Endometrioid carcinoma with Metastatic choriocarcinoma in 5 69 Postmenopausal vaginal bleeding Hysterectomy Lost to follow-up cm choriocarcinomatous differentiation pelvic lymph nodes and lung

Positive pregnancy test, right cornual Chemotherapy; Right uterine # ectopic pregnancy with complete Cornual mass No evidence of 6 25 cornu, 10.6 cm Choriocarcinoma -- hydatidiform mole with lung metastasis resection disease, 15 aggregate 5 years prior; β-hCG 3398 months Endometrial Uterus, 8 cm 7 46 Incomplete abortion and endometriosis Choriocarcinoma -- Lost to follow-up curettage aggregate

Heavy vaginal bleeding, normal Endometrial Uterus, 9.5 cm Rare viable and necrotic 8 23 Choriocarcinoma Lost to follow-up delivery 6 months prior; β-hCG 2315 curettage aggregate chorionic villi

Vaginal bleeding, rising β-hCG despite Pararectal # Para-rectal soft Dead of disease, 9 27 chemotherapy for presumptive ectopic mass Choriocarcinoma -- tissue, 4.5 cm 18 months pregnancy resection Intrauterine fetal demise, bleeding, Chemotherapy; # retained products of conception, right Right uterine Separate 21-week placenta; lung No evidence of 10 40 Hysterectomy Choriocarcinoma cornual mass; β-hCG rising to 224,900 cornu, 4 cm metastases disease, 30 at 2 days after delivery months Concurrent complete # Vaginal bleeding, heterogeneous Endometrial Uterus, 30 cm 11 39 Choriocarcinoma hydatidiform mole; metastases in Lost to follow-up endometrial mass; β-hCG > 273,000 curettage aggregate vagina and lung #Cases from previous study [Reference 1] TAH/BSO: total abdominal hysterectomy with bilateral salpingo-oophorectomy 1

Lineage-specific alterations in gynecologic neoplasms with choriocarcinomatous differentiation: implications for origin and therapeutics

Deyin Xing, Gang Zheng, Aparna Pallavajjala, et al.

Clin Cancer Res Published OnlineFirst April 22, 2019.

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