Natalia Buza

Landscape of somatic single-nucleotide and copy-number mutations in uterine serous carcinoma

Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer. We analyzed the mutational landscape of USC by whole-exome sequencing of 57 cancers, most of which were matched to normal DNA from the same patients. The distribution of the number of protein-altering somatic mutations revealed that 52 USC tumors had fewer than 100 (median 36), whereas 5 had more than 3,000 somatic mutations. The mutations in these latter tumors showed hallmarks of defects in DNA mismatch repair. Among the remainder, we found a significantly increased burden of mutation in 14 genes. In addition to well-known cancer genes (i.e., TP53, PIK3CA, PPP2R1A, KRAS, FBXW7), there were frequent mutations in CHD4/Mi2b, a member of the NuRD–chromatin-remodeling complex, and TAF1, an element of the core TFIID transcriptional machinery. Additionally, somatic copy-number variation was found to play an important role in USC, with 13 copy-number gains and 12 copy-number losses that occurred more often than expected by chance. In addition to loss of TP53, we found frequent deletion of a small segment of chromosome 19 containing MBD3, also a member of the NuRD–chromatin-modification complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (an upstream activator of PIK3CA), and CCNE1 (a target of FBXW7-mediated ubiquitination). These findings identify frequent mutation of DNA damage, chromatin remodeling, cell cycle, and cell proliferation pathways in USC and suggest potential targets for treatment of this lethal variant of endometrial cancer.

In vitro activity of pertuzumab in combination with trastuzumab in uterine serous papillary adenocarcinoma

Background:Uterine serous papillary adenocarcinoma (USPC) is a rare but highly aggressive variant of endometrial cancer. Pertuzumab is a new humanised monoclonal antibody (mAb) targeting the epidermal growth factor type II receptor (HER2/neu). We evaluated pertuzumab activity separately or in combination with trastuzumab against primary USPC cell lines expressing different levels of HER2/neu.Methods:Six USPC cell lines were assessed by immunohistochemistry (IHC), flow cytometry, and real-time PCR for HER2/neu expression. c-erbB2 gene amplification was evaluated using fluorescent in situ hybridisation (FISH). Sensitivity to pertuzumab and trastuzumab-induced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) was evaluated in 5 h chromium release assays. Pertuzumab cytostatic activity was evaluated using proliferation-based assays.Results:Three USPC cell lines stained heavily for HER2/neu by IHC and showed amplification of the c-erbB2 gene by FISH. The remaining FISH-negative USPCs expressed HER2/neu at 0/1+ levels. In cytotoxicity experiments against USPC with a high HER2/neu expression, pertuzumab and trastuzumab were similarly effective in inducing strong ADCC. The addition of complement-containing plasma and interleukin-2 increased the cytotoxic effect induced by both mAbs. In low HER2/neu USPC expressors, trastuzumab was more potent than pertuzumab in inducing ADCC. Importantly, in this setting, the combination of pertuzumab with trastuzumab significantly increased the ADCC effect induced by trastuzumab alone (P=0.02). Finally, pertuzumab induced a significant inhibition in the proliferation of all USPC cell lines tested, regardless of their HER-2/neu expression.Conclusion:Pertuzumab and trastuzumab induce equally strong ADCC and CDC in FISH-positive USPC cell lines. Pertuzumab significantly increases tratuzumab-induced ADCC against USPC with a low HER2/neu expression and may represent a new therapeutic agent in patients harbouring advanced/recurrent and/or refractory USPC.

Serum amyloid A: a novel biomarker for endometrial cancer.

The authors investigated the expression of serum amyloid A (SAA) in endometrial endometrioid carcinoma and evaluated its potential as a serum biomarker.

Mutational landscape of uterine and ovarian carcinosarcomas implicates histone genes in epithelial–mesenchymal transition

Significance Some cancers, termed carcinosarcomas (CSs), have mixed cell types, with either epithelial or mesenchymal features. Sequencing the genomes of uterine and ovarian CSs demonstrated that these different cell types derive from a common precursor cell that has many mutations typical of epithelial cancers. In addition, we find that these tumors have a significant burden of point mutations and amplification of histone genes, suggesting a potential role of these mutations in sarcomatous transformation. Consistent with this finding, expression of specific histone gene mutations in uterine carcinoma cells changed gene expression toward a mesenchymal state. These findings have potential implications for the treatment of these cancers. Carcinosarcomas (CSs) of the uterus and ovary are highly aggressive neoplasms containing both carcinomatous and sarcomatous elements. We analyzed the mutational landscape of 68 uterine and ovarian CSs by whole-exome sequencing. We also performed multiregion whole-exome sequencing comprising two carcinoma and sarcoma samples from six tumors to resolve their evolutionary histories. The results demonstrated that carcinomatous and sarcomatous elements derive from a common precursor having mutations typical of carcinomas. In addition to mutations in cancer genes previously identified in uterine and ovarian carcinomas such as TP53, PIK3CA, PPP2R1A, KRAS, PTEN, CHD4, and BCOR, we found an excess of mutations in genes encoding histone H2A and H2B, as well as significant amplification of the segment of chromosome 6p harboring the histone gene cluster containing these genes. We also found frequent deletions of the genes TP53 and MBD3 (a member with CHD4 of the nucleosome remodeling deacetylase complex) and frequent amplification of chromosome segments containing the genes PIK3CA, TERT, and MYC. Stable transgenic expression of H2A and H2B in a uterine serous carcinoma cell line demonstrated that mutant, but not wild-type, histones increased expression of markers of epithelial–mesenchymal transition (EMT) as well as tumor migratory and invasive properties, suggesting a role in sarcomatous transformation. Comparison of the phylogenetic relationships of carcinomatous and sarcomatous elements of the same tumors demonstrated separate lineages leading to these two components. These findings define the genetic landscape of CSs and suggest therapeutic targets for these highly aggressive neoplasms.

HER2/neu in Endometrial Cancer: A Promising Therapeutic Target With Diagnostic Challenges.

CONTEXT In the era of targeted cancer therapy, there is growing interest in developing novel therapeutic strategies against endometrial carcinoma, especially its most biologically aggressive variant, serous adenocarcinoma. Several publications have demonstrated that a significant proportion of uterine serous carcinomas show HER2 overexpression and/or amplification, suggesting that HER2 may be a promising therapeutic target. Case reports have already shown clinical response to trastuzumab, a humanized monoclonal immunoglobulin (Ig) G1 antibody against HER2, and patients are currently being enrolled in a multi-institutional prospective randomized trial to evaluate the therapeutic efficacy of trastuzumab. OBJECTIVE To review current data on HER2 testing and targeted therapy against HER2/neu in endometrial carcinoma. DATA SOURCES Review of the literature and personal experience of the authors. CONCLUSIONS Parallel to the clinical studies, there is a need to develop standardized criteria for HER2 testing in endometrial carcinoma that reflect the unique biological and pathogenetic features of these tumors and correlate with clinical response to therapy. This article presents a comprehensive review of the current state of HER2-based therapy and HER2 testing in endometrial carcinoma.

Toward standard HER2 testing of endometrial serous carcinoma: 4-year experience at a large academic center and recommendations for clinical practice

HER2 overexpression and/or amplification have been reported in endometrial serous carcinoma, suggesting that HER2 may be a promising therapeutic target. However, there is considerable variation in the reported rates of HER2 overexpression and amplification, likely—at least in part—resulting from variability in the testing methods, interpretation, and scoring criteria used. Unlike in breast and gastric cancer, currently there are no established guidelines for HER2 testing in endometrial carcinoma. A total of 108 endometrial carcinoma cases—85 pure serous carcinomas and 23 mixed endometrial carcinomas with serous component—were identified over a 4-year period. All H&E and HER2 immunohistochemical slides were reviewed and HER2 FISH results (available on 52 cases) were retrieved from pathology reports. HER2 immunohistochemical scores were assigned according to the FDA criteria and the current breast ASCO/CAP scoring criteria. Clinical information was retrieved from the patients’ medical records. Thirty-eight cases (35%) showed HER2 overexpression and/or gene amplification, 20 of which (53%) had significant heterogeneity of protein expression by immunohistochemistry. Lack of apical membrane staining resulting in a lateral/basolateral staining pattern was observed in the majority of HER2-positive tumors. Five of the HER2-positive cases (13%) demonstrated discrepant immunohistochemical scores when using the FDA versus ASCO/CAP scoring system. The overall concordance rate between HER2 immunohistochemistry and FISH was 75% (39/52) when using the FDA criteria, compared with 81% (42/52) by the ASCO/CAP scoring system. In conclusion, in this largest comprehensive study, 35% of endometrial serous carcinoma harbors HER2 protein overexpression and/or gene amplification, over half of which demonstrate significant heterogeneity of protein expression. The current breast ASCO/CAP scoring criteria provide the highest concordance between immunohistochemistry and FISH. Assessment of HER2 immunohistochemistry on multiple tumor sections or sections with large tumor areas is recommended, due to the significant heterogeneity of HER2 protein expression.

Tubulin-β-III overexpression by uterine serous carcinomas is a marker for poor overall survival after platinum/taxane chemotherapy and sensitivity to epothilones

Uterine serous carcinoma (USC) is a subtype of endometrial cancer associated with chemoresistance and poor outcome. Overexpression of tubulin‐β‐III and p‐glycoprotein has been linked to paclitaxel resistance in many cancers but has been undercharacterized among USCs. Epothilones have demonstrated activity in certain paclitaxel‐resistant malignancies. In this study, relationships are clarified, in USCs relative to ovarian serous carcinomas (OSCs), between tubulin‐β‐III and p‐glycoprotein expression, clinical outcome, and in vitro chemoresponsiveness to epothilone B, ixabepilone, and paclitaxel.

Downregulation of membrane complement inhibitors CD55 and CD59 by siRNA sensitises uterine serous carcinoma overexpressing Her2/neu to complement and antibody-dependent cell cytotoxicity in vitro : implications for trastuzumab-based immunotherapy

Background:We evaluated the expression of CD46, CD55 and CD59 membrane-bound complement-regulatory proteins (mCRPs) in primary uterine serous carcinoma (USC) and the ability of small interfering RNA (siRNA) against these mCRPs to sensitise USC to complement-dependent cytotoxicity (CDC) and antibody (trastuzumab)-dependent cellular cytotoxicity (ADCC) in vitro.Methods:Membrane-bound complement-regulatory proteins expression was evaluated using real-time PCR (RT–PCR) and flow cytometry, whereas Her2/neu expression and c-erbB2 gene amplification were assessed using immunohistochemistry, flow cytometry and fluorescent in-situ hybridisation. The biological effect of siRNA-mediated knockdown of mCRPs on HER2/neu-overexpressing USC cell lines was evaluated in CDC and ADCC 4-h chromium-release assays.Results:High expression of mCRPs was found in USC cell lines when compared with normal endometrial cells (P<0.05). RT–PCR and FACS analyses demonstrated that anti-mCRP siRNAs were effective in reducing CD46, CD55 and CD59 expression on USC (P<0.05). Baseline complement-dependent cytotoxicity (CDC) against USC cell lines was low (mean±s.e.m.=6.8±0.9%) but significantly increased upon CD55 and CD59 knockdown (11.6±0.8% and 10.7±0.9%, respectively, P<0.05). Importantly, in the absence of complement, both CD55 and CD59, but not CD46, knockdowns significantly augmented ADCC against USC overexpressing Her2/neu.Conclusion:Uterine serous carcinoma express high levels of the mCRPs CD46, CD55 and CD59. Small interfering RNA inhibition of CD55 and CD59, but not CD46, sensitises USC to both CDC and ADCC in vitro, and if specifically targeted to tumour cells, may significantly increase trastuzumab-mediated therapeutic effect in vivo.

Overexpression of EpCAM in Uterine Serous Papillary Carcinoma: Implications for EpCAM-Specific Immunotherapy With Human Monoclonal Antibody Adecatumumab (MT201)

We evaluated the expression of epithelial cell adhesion molecule (EpCAM) and the potential of MT201 (adecatumumab), a human monoclonal antibody against EpCAM, in uterine serous papillary carcinoma (USPC). EpCAM expression was evaluated by real-time PCR and immunohistochemistry in a total of 56 USPC fresh-frozen biopsies and paraffin-embedded tissues. EpCAM surface expression was also evaluated by flow cytometry and immunohistochemistry in six USPC cell lines. Sensitivity to MT201 antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity was tested against a panel of primary USPC cell lines expressing different levels of EpCAM in standard 5-h 51Cr release assays. EpCAM transcript was significantly overexpressed in fresh-frozen USPC when compared with normal endometrial cells (NEC). Median (minimum–maximum) copy number was 943.8 (31.5–1568.3) in tumor samples versus 12.9 (1.0–37.0) in NEC (P < 0.001). By immunohistochemistry, EpCAM expression was found in 96% (26 out of 27) of USPC samples with significantly higher expression compared with NECs (P < 0.001). High surface expression of EpCAM was found in 83% (five out of six) of the USPC cell lines tested by flow cytometry. EpCAM-positive cell lines were found highly sensitive to MT201-mediated antibody-dependent cellular cytotoxicity in vitro, whereas primary USPC cell lines were resistant to natural killer cell–dependent cytotoxicity. Human plasma IgG did not significantly inhibit MT201-mediated cytotoxicity against USPC. EpCAM is highly expressed in uterine serous carcinoma at mRNA and protein levels, and primary USPC are highly sensitivity to MT201-mediated cytotoxicity. MT201 might represent a novel therapeutic strategy in patients harboring advanced/recurrent or metastatic USPC refractory to standard treatment modalities. Mol Cancer Ther; 9(1); 57–66

Serum amyloid A (SAA): a novel biomarker for uterine serous papillary cancer

Background:Uterine serous papillary carcinoma (USPC) is a biologically aggressive variant of endometrial cancer. We investigated the expression of Serum Amyloid A (SAA) and evaluated its potential as a serum biomarker in USPC patients.Methods:SAA gene and protein expression levels were evaluated in USPC and normal endometrial tissues (NEC) by real-time PCR, immunohistochemistry (IHC), flow cytometry and by a sensitive bead-based immunoassay. SAA concentration in 123 serum samples from 51 healthy women, 42 women with benign diseases, and 30 USPC patients were also studied.Results:SAA gene expression levels were significantly higher in USPC when compared with NEC (mean copy number by RT–PCR=162 vs 2.21; P=0.0002). IHC revealed diffuse cytoplasmic SAA protein staining in USPC tissues. High intracellular levels of SAA were identified in primary USPC cell lines evaluated by flow cytometry and SAA was found to be actively secreted in vitro. SAA concentrations (μg ml−1) had a median (95% CIs) of 6.0 (4.0–8.9) in normal healthy females and 6.0 (4.2–8.1) in patients with benign disease (P=0.92). In contrast, SAA values in the serum of USPC patients had a median (95% CI) of 15.6 (9.2–56.2), significantly higher than those in the healthy group (P=0.0005) and benign group (P=0.0006). Receiver operating characteristics (ROC) analysis of serum SAA to classify advanced- and early-stage USPC yielded an area under the ROC curve of 0.837 (P=0.0024).Conclusion:SAA is not only a liver-secreted protein but is also a USPC cell product. SAA may represent a novel biomarker for USPC to assist in staging patients preoperatively, and to monitor early-disease recurrence and response to therapy.