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1.
Proc Natl Acad Sci U S A ; 118(15)2021 04 13.
Article in English | MEDLINE | ID: mdl-33876771

ABSTRACT

Uterine leiomyosarcomas (uLMS) are aggressive tumors arising from the smooth muscle layer of the uterus. We analyzed 83 uLMS sample genetics, including 56 from Yale and 27 from The Cancer Genome Atlas (TCGA). Among them, a total of 55 Yale samples including two patient-derived xenografts (PDXs) and 27 TCGA samples have whole-exome sequencing (WES) data; 10 Yale and 27 TCGA samples have RNA-sequencing (RNA-Seq) data; and 11 Yale and 10 TCGA samples have whole-genome sequencing (WGS) data. We found recurrent somatic mutations in TP53, MED12, and PTEN genes. Top somatic mutated genes included TP53, ATRX, PTEN, and MEN1 genes. Somatic copy number variation (CNV) analysis identified 8 copy-number gains, including 5p15.33 (TERT), 8q24.21 (C-MYC), and 17p11.2 (MYOCD, MAP2K4) amplifications and 29 copy-number losses. Fusions involving tumor suppressors or oncogenes were deetected, with most fusions disrupting RB1, TP53, and ATRX/DAXX, and one fusion (ACTG2-ALK) being potentially targetable. WGS results demonstrated that 76% (16 of 21) of the samples harbored chromoplexy and/or chromothripsis. Clinically actionable mutational signatures of homologous-recombination DNA-repair deficiency (HRD) and microsatellite instability (MSI) were identified in 25% (12 of 48) and 2% (1 of 48) of fresh frozen uLMS, respectively. Finally, we found olaparib (PARPi; P = 0.002), GS-626510 (C-MYC/BETi; P < 0.000001 and P = 0.0005), and copanlisib (PIK3CAi; P = 0.0001) monotherapy to significantly inhibit uLMS-PDXs harboring derangements in C-MYC and PTEN/PIK3CA/AKT genes (LEY11) and/or HRD signatures (LEY16) compared to vehicle-treated mice. These findings define the genetic landscape of uLMS and suggest that a subset of uLMS may benefit from existing PARP-, PIK3CA-, and C-MYC/BET-targeted drugs.


Subject(s)
Genotype , Leiomyosarcoma/genetics , Mutation , Oncogene Fusion , Uterine Neoplasms/genetics , Animals , Antineoplastic Agents/therapeutic use , Female , Humans , Leiomyosarcoma/drug therapy , Metabolic Networks and Pathways , Mice , Mice, Inbred C57BL , Molecular Targeted Therapy/methods , Phthalazines/administration & dosage , Phthalazines/therapeutic use , Piperazines/administration & dosage , Piperazines/therapeutic use , Pyrimidines/administration & dosage , Pyrimidines/therapeutic use , Quinazolines/administration & dosage , Quinazolines/therapeutic use , Uterine Neoplasms/drug therapy
2.
Cancer ; 128(6): 1206-1218, 2022 03 15.
Article in English | MEDLINE | ID: mdl-34875107

ABSTRACT

BACKGROUND: Microsatellite instability-high (MSI-H)/mismatch repair deficiency (dMMR) is a biomarker for responses to immune checkpoint inhibitors (ICIs). Whether mechanisms underlying microsatellite instability alter responses to ICIs is unclear. This article reports data from a prospective phase 2 pilot study of pembrolizumab in patients with recurrent MSI-H endometrial cancer (EC) analyzed by whole exome sequencing (WES) and potential mechanisms of primary/secondary ICI resistance (NCT02899793). METHODS: Patients with measurable MSI-H/dMMR EC confirmed by polymerase chain reaction/immunohistochemistry were evaluated by WES and received 200 mg of pembrolizumab every 3 weeks for ≤2 years. The primary end point was the objective response rate (ORR). Secondary end points included progression-free survival (PFS) and overall survival (OS). RESULTS: Twenty-five patients (24 evaluable) were treated. Six patients (25%) harbored Lynch/Lynch-like tumors, whereas 18 (75%) had sporadic EC. The tumor mutation burden was higher in Lynch-like tumors (median, 2939 mutations/megabase [Mut/Mb]; interquartile range [IQR], 867-5108 Mut/Mb) than sporadic tumors (median, 604 Mut/Mb; IQR, 411-798 Mut/Mb; P = .0076). The ORR was 100% in Lynch/Lynch-like patients but only 44% in sporadic patients (P = .024). The 3-year PFS and OS proportions were 100% versus 30% (P = .017) and 100% versus 43% (P = .043), respectively. CONCLUSIONS: This study suggests prognostic significance of Lynch-like cancers versus sporadic MSI-H/dMMR ECs for ORR, PFS, and OS when patients are treated with pembrolizumab. Larger confirmatory studies in ECs and other MSI-H/dMMR tumors are necessary. Defective antigen processing/presentation and deranged induction in interferon responses serve as mechanisms of resistance in sporadic MSI-H ECs. Oligoprogression in MSI-H/dMMR patients appears salvageable with surgical resection and/or local treatment and the continuation of pembrolizumab off study. Clinical studies evaluating separate MSI-H/dMMR EC subtypes treated with ICIs are warranted.


Subject(s)
Endometrial Neoplasms , Microsatellite Instability , Antibodies, Monoclonal, Humanized , DNA Mismatch Repair/genetics , Endometrial Neoplasms/drug therapy , Endometrial Neoplasms/genetics , Female , Humans , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Pilot Projects , Prospective Studies
3.
Proc Natl Acad Sci U S A ; 116(2): 619-624, 2019 01 08.
Article in English | MEDLINE | ID: mdl-30584090

ABSTRACT

Ovarian cancer remains the most lethal gynecologic malignancy. We analyzed the mutational landscape of 64 primary, 41 metastatic, and 17 recurrent fresh-frozen tumors from 77 patients along with matched normal DNA, by whole-exome sequencing (WES). We also sequenced 13 pairs of synchronous bilateral ovarian cancer (SBOC) to evaluate the evolutionary history. Lastly, to search for therapeutic targets, we evaluated the activity of the Bromodomain and Extra-Terminal motif (BET) inhibitor GS-626510 on primary tumors and xenografts harboring c-MYC amplifications. In line with previous studies, the large majority of germline and somatic mutations were found in BRCA1/2 (21%) and TP53 (86%) genes, respectively. Among mutations in known cancer driver genes, 77% were transmitted from primary tumors to metastatic tumors, and 80% from primary to recurrent tumors, indicating that driver mutations are commonly retained during ovarian cancer evolution. Importantly, the number, mutation spectra, and signatures in matched primary-metastatic tumors were extremely similar, suggesting transcoelomic metastases as an early dissemination process using preexisting metastatic ability rather than an evolution model. Similarly, comparison of SBOC showed extensive sharing of somatic mutations, unequivocally indicating a common ancestry in all cases. Among the 17 patients with matched tumors, four patients gained PIK3CA amplifications and two patients gained c-MYC amplifications in the recurrent tumors, with no loss of amplification or gain of deletions. Primary cell lines and xenografts derived from chemotherapy-resistant tumors demonstrated sensitivity to JQ1 and GS-626510 (P = 0.01), suggesting that oral BET inhibitors represent a class of personalized therapeutics in patients harboring recurrent/chemotherapy-resistant disease.


Subject(s)
Antineoplastic Agents/pharmacology , Azepines/pharmacology , Mutation , Neoplasm Recurrence, Local , Proteins , Proto-Oncogene Proteins c-myc , Triazoles/pharmacology , Animals , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/metabolism , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases/genetics , Class I Phosphatidylinositol 3-Kinases/metabolism , Female , Humans , Mice , Neoplasm Metastasis , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/metabolism , Ovarian Neoplasms , Proteins/antagonists & inhibitors , Proteins/genetics , Proteins/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays
4.
Proc Natl Acad Sci U S A ; 116(45): 22730-22736, 2019 11 05.
Article in English | MEDLINE | ID: mdl-31624127

ABSTRACT

The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirus-type-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.


Subject(s)
Class I Phosphatidylinositol 3-Kinases/genetics , Exome Sequencing , Mutation , Receptor, ErbB-2/genetics , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/therapy , Animals , Cell Line, Tumor , Combined Modality Therapy , DNA Copy Number Variations , Female , Heterografts , Humans , Polymorphism, Single Nucleotide , Uterine Cervical Neoplasms/pathology
5.
Gynecol Oncol ; 163(2): 334-341, 2021 11.
Article in English | MEDLINE | ID: mdl-34452746

ABSTRACT

OBJECTIVE: Uterine serous carcinoma (USC) is an aggressive histologic variant of endometrial cancer which portends a poor prognosis. DHES0815A is a novel antibody-drug-conjugate (ADC) which binds specifically to HER2 overexpressing tumors at a distinct epitope from that bound by trastuzumab and pertuzumab after which it delivers the toxic payload, PBD-MA, a DNA mono-alkylating agent. The objective of this study was to evaluate the preclinical activity of DHES0815A against primary USC cell lines and xenografts. METHODS: Twelve primary USC cell lines were assessed by immunohistochemistry (IHC) for HER2 protein expression and for C-erbB2 gene amplification using fluorescent in situ hybridization (FISH) analysis. Cell viability and bystander killing in USC cell lines after exposure to DHES0815A, the non-targeted ADC, and the unconjugated antibody (i.e. MHES0488A) were evaluated using flow cytometry-based-assays. In vivo activity of DHES0815A was tested against HER2/neu overexpressing USC xenografts. RESULTS: High HER2/neu protein expression was seen in 25% (3/12) of the primary USC cell lines. USC cell lines overexpressing HER2/neu were significantly more sensitive to DHES0815A when compared to the non-targeted control ADC (p < 0.001). DHES0815A did not induce significant bystander killing of HER2/neu negative tumors when admixed with HER2/neu positive tumors. DHES0815A caused growth-inhibition and increased survival in USC HER2/neu overexpressing xenografts when compared to controls (p < 0.01). CONCLUSIONS: DHES0815A is both highly selective and toxic to USC tumors overexpressing HER2/neu both in vitro and in vivo. HER2-directed ADCs, alone or in combination with other HER2/neu targeted agents may represent a novel treatment option for patients with tumors harboring HER2/neu overexpression refractory to trastuzumab and traditional chemotherapy.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Benzodiazepines/pharmacology , Cystadenocarcinoma, Serous/drug therapy , Immunoconjugates/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Uterine Neoplasms/drug therapy , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal, Humanized/therapeutic use , Benzodiazepines/therapeutic use , Bystander Effect/drug effects , Cell Line, Tumor , Cystadenocarcinoma, Serous/pathology , Drug Resistance, Neoplasm , Female , Humans , Immunoconjugates/therapeutic use , Middle Aged , Primary Cell Culture , Trastuzumab/pharmacology , Trastuzumab/therapeutic use , Uterine Neoplasms/pathology , Xenograft Model Antitumor Assays
6.
Gynecol Oncol ; 158(3): 769-775, 2020 09.
Article in English | MEDLINE | ID: mdl-32600791

ABSTRACT

OBJECTIVE: Whole-exome-sequencing (WES) studies reported c-MYC gene-amplification and HUWE1 gene deletion/mutations in a significant number of cervical-cancer-patients (CC) suggesting HUWE1/c-MYC pathway as potential therapeutic target. We investigated HUWE1/c-MYC expression in fresh-frozen-CC and the activity of the novel BET inhibitor GS-626510 (Gilead-Science-Inc) against primary WES CC-cultures and CC-xenografts. METHODS: HUWE1 and c-MYC expression were evaluated by qRT-PCR in 23 CC including 12 fresh-frozen-tumor-tissues and 11 primary-cell-lines. c-Myc expression was also evaluated by Western-Blot (WB) and fluorescence-in-situ-hybridization (FISH) in all 11 fully sequenced primary-CC-cell-lines. Primary tumors were evaluated for sensitivity to GS-626510 in-vitro using proliferation and viability-assays. siRNA experiments were used to evaluate the effect of HUWE1 silencing on primary-CC-cell-line growth and sensitivity to GS-626510. Finally, the in-vivo activity of GS-626510 was studied in CC-CVX8-mouse-xenografts. RESULTS: Fresh-frozen-CC and primary-CC-cell-lines overexpressed c-MYC when compared to normal tissues (p = .01). FISH demonstrated amplification of c-MYC in 9/11 (82%) of the primary-CC-cell-lines. Cell-lines with derangements in HUWE1/c-MYC pathway were highly sensitive to GS-626510, with a dose-response decrease in cell proliferation and viability. siRNA silencing of HUWE1 significantly increased c-MYC expression and CC cell-proliferation and enhanced the in-vitro sensitivity to GS-626510. Twice-daily oral doses of GS-626510 were well tolerated in-vivo and highly effective in decreasing tumor-growth (p = .004) and increasing survival (p = .004) of CC-CVX8 xenografts. CONCLUSIONS: Downregulation/inactivation of HUWE1 may increase c-MYC expression and proliferation in primary-CC-cell-lines. GS-626510 may represent a novel, potentially highly effective therapeutic agent against CC overexpressing c-MYC and/or harboring HUWE1 mutations. Clinical studies with BET inhibitor in CC-patients harboring radiation/chemotherapy-resistant disease are warranted.


Subject(s)
Isoxazoles/pharmacology , Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-myc/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/metabolism , Adult , Aged , Animals , Cell Line, Tumor , Female , Humans , Imidazoles/pharmacology , In Situ Hybridization, Fluorescence , Mice , Middle Aged , Proteins/metabolism , Proto-Oncogene Proteins c-myc/biosynthesis , Proto-Oncogene Proteins c-myc/genetics , Signal Transduction/drug effects , Tumor Suppressor Proteins/biosynthesis , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/biosynthesis , Ubiquitin-Protein Ligases/genetics , Uterine Cervical Neoplasms/genetics , Xenograft Model Antitumor Assays , Young Adult
7.
Gynecol Oncol ; 156(2): 430-438, 2020 02.
Article in English | MEDLINE | ID: mdl-31839338

ABSTRACT

OBJECTIVE: Uterine serous carcinoma (USC) is an aggressive variant of endometrial cancer with poor prognosis. Sacituzumab govitecan (SG) is a novel antibody-drug-conjugate (ADC) targeting trophoblast cell-surface antigen 2 (Trop-2), a transmembrane-calcium-signal-transducer, to deliver SN-38, the active metabolite of irinotecan. The objective of this study was to evaluate the expression of Trop-2 in USC and the preclinical activity of SG against primary USC cell-lines and xenografts. METHODS: We used immunohistochemistry (IHC) and flow-cytometry-based assays to evaluate Trop-2 expression and cell-viability in USC tissue and primary tumor-cell-lines after exposure to SG, non-targeting control ADC, and naked antibody hRS7-IgG. Antibody-dependent-cell-cytotoxicity (ADCC) against Trop-2+ and Trop-2- USC cell-lines was evaluated in vitro using 4-hr-Chromium-release-assays. In vivo activity of SG was tested against Trop-2+ USC xenografts by intravenous administration of SG, control ADC, and hRS7. RESULTS: Trop-2 expression by IHC was detected in 95.1% of USC samples (99/104). Primary tumor cell-lines overexpressing Trop-2 were significantly more sensitive to SG when compared to control ADC (p <0.05). Both SG and hRS7 mediated ADCC in Trop2+ USC cell-lines while no cytotoxicity was detected against Trop-2- cells. SG induced significant bystander killing of Trop-2- tumors when admixed with Trop-2+ tumors. SG caused growth-inhibition and increased survival in SG treated mice harboring Trop-2+ xenografts when compared to controls (p <0.05). CONCLUSIONS: SG is remarkably active against USC overexpressing Trop-2 in vitro and in vivo. Our results combined with SG clinical responses recently reported against multiple chemotherapy resistant human tumors further support clinical development of SG in USC patients with advanced/recurrent disease.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antigens, Neoplasm/immunology , Camptothecin/analogs & derivatives , Cell Adhesion Molecules/immunology , Immunoconjugates/pharmacology , Uterine Neoplasms/drug therapy , Animals , Antibodies, Monoclonal, Humanized/immunology , Antibody-Dependent Cell Cytotoxicity , Antigens, Neoplasm/biosynthesis , Camptothecin/immunology , Camptothecin/pharmacology , Cell Adhesion Molecules/biosynthesis , Cell Line, Tumor , Cystadenocarcinoma, Serous , Female , Flow Cytometry , Humans , Immunoconjugates/immunology , Immunohistochemistry , Mice , Mice, SCID , Molecular Targeted Therapy , Random Allocation , Tissue Array Analysis , Uterine Neoplasms/immunology , Uterine Neoplasms/metabolism , Xenograft Model Antitumor Assays
8.
Gynecol Oncol ; 155(1): 144-150, 2019 10.
Article in English | MEDLINE | ID: mdl-31434613

ABSTRACT

OBJECTIVES: Cervical cancer (CC) remains a major health problem worldwide. Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi) have emerged as a promising class of chemotherapeutics in ovarian cancer. We explored the preclinical in vitro and in vivo activity of olaparib against multiple primary whole exome sequenced (WES) CC cells lines and xenografts. METHODS: Olaparib cell-cycle, apoptosis, homologous-recombination-deficiency (HRD), PARP trapping and cytotoxicity activity was evaluated against 9 primary CC cell lines in vitro. PARP and PAR expression were analyzed by Western blot assays. Finally, olaparib in vivo antitumor activity was tested against CC xenografts. RESULTS: While none of the cell lines demonstrated HRD, three out of 9 (33.3%) primary CC cell lines showed strong PARylation activity and demonstrated high sensitivity to olaparib in vitro treatment (cutoff IC50 values < 2 µM, p = 0.0012). Olaparib suppressed CC cell growth through cell cycle arrest in the G2/M phase and caused apoptosis (p < 0.0001). Olaparib activity in CC involved both PARP enzyme inhibition and trapping. In vivo, olaparib significantly impaired CC xenografts tumor growth (p = 0.0017) and increased overall animal survival (p = 0.008). CONCLUSIONS: A subset of CC primary cell lines is highly responsive to olaparib treatment in vitro and in vivo. High level of PARylation correlated with olaparib preclinical activity and may represent a useful biomarker for the identification of CC patients benefitting the most from PARPi.


Subject(s)
Phthalazines/pharmacology , Piperazines/pharmacology , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/enzymology , Adult , Animals , Apoptosis/drug effects , Cell Growth Processes/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Female , G2 Phase Cell Cycle Checkpoints/drug effects , Humans , M Phase Cell Cycle Checkpoints/drug effects , Mice, SCID , Middle Aged , Uterine Cervical Neoplasms/pathology , Xenograft Model Antitumor Assays , Young Adult
9.
Gynecol Oncol ; 153(1): 158-164, 2019 04.
Article in English | MEDLINE | ID: mdl-30630630

ABSTRACT

OBJECTIVE: Aberrant expression of HER2/neu and PIK3CA gene products secondary to amplification/mutations are common in high-grade-serous-endometrial (USC) and ovarian-cancers (HGSOC). Because scant information is currently available in the literature on the potential negative effect of PIK3CA mutations on the activity of afatinib, in this study we evaluate for the first time the role of oncogenic PIK3CA mutations as a potential mechanism of resistance to afatinib in HGSOC and USC overexpressing HER2/neu. METHODS: We used six whole-exome-sequenced primary HGSOC/USC cell-lines and three xenografts overexpressing HER2/neu and harboring mutated or wild-type PIK3CA/PIK3R1 genes to evaluate the role of PI3K-mutations as potential mechanism of resistance to afatinib, an FDA-approved pan-c-erb-inhibitor in clinical trials in USC. Primary-USC harboring wild-type-PIK3CA gene was transfected with plasmids encoding oncogenic PIK3CA-mutations (H1047R/E545K). The effect of afatinib on HER2/PI3K/AKT/mTOR pathway was evaluated by immunoblotting. RESULTS: We found PI3K wild-type cell-lines to be significantly more sensitive (lower IC50) than PI3K-mutated cell-lines p = 0.004). In vivo, xenografts of primary cell-line USC-ARK2, transfected with the PIK3CA-H1047R or E545K hotspot-mutations, exhibited significantly more rapid tumor growth when treated with afatinib, compared to mice harboring ARK2-tumors transfected with wild-type-PIK3CA (p = 0.041 and 0.001, respectively). By western-blot, afatinib effectively reduced total and phospho-HER2 proteins in all cell-lines. However, H1047R/E545K-PIK3CA-transfected-ARK2-cells demonstrated a greater compensatory increase in phosphorylated-AKT proteins after afatinib exposure when compared to controls ARK2. CONCLUSIONS: Oncogenic PI3K mutations may represent a major mechanism of resistance to afatinib. Combinations of c-erb with PIK3CA, AKT or mTOR inhibitors may be necessary to more efficiently block the PIK3CA/AKT/mTOR pathway.


Subject(s)
Afatinib/pharmacology , Class I Phosphatidylinositol 3-Kinases/genetics , Genital Neoplasms, Female/drug therapy , Phosphatidylinositol 3-Kinases/genetics , Adult , Aged , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases/biosynthesis , Class Ia Phosphatidylinositol 3-Kinase , Drug Resistance, Neoplasm/genetics , Female , Genital Neoplasms, Female/enzymology , Genital Neoplasms, Female/genetics , Humans , Mice , Mice, SCID , Middle Aged , Mutation , Phosphatidylinositol 3-Kinases/biosynthesis , Protein Kinase Inhibitors/pharmacology , Receptor, ErbB-2/biosynthesis , Receptor, ErbB-2/genetics , Transfection , Xenograft Model Antitumor Assays
10.
Proc Natl Acad Sci U S A ; 113(43): 12238-12243, 2016 10 25.
Article in English | MEDLINE | ID: mdl-27791010

ABSTRACT

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.


Subject(s)
Histones/genetics , Ovarian Neoplasms/genetics , Tumor Suppressor Protein p53/genetics , Uterine Neoplasms/genetics , Aged , Aged, 80 and over , Carcinosarcoma/genetics , Carcinosarcoma/pathology , Class I Phosphatidylinositol 3-Kinases/genetics , DNA-Binding Proteins/genetics , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Middle Aged , Mutation , Ovarian Neoplasms/pathology , PTEN Phosphohydrolase/genetics , Telomerase/genetics , Uterine Neoplasms/pathology
11.
Gynecol Oncol ; 149(3): 585-591, 2018 06.
Article in English | MEDLINE | ID: mdl-29572027

ABSTRACT

INTRODUCTION: Since the majority of patients are diagnosed at an advanced stage, ovarian cancer remains the most lethal gynecologic malignancy. There is no single biomarker with the sensitivity and specificity required for effective cancer screening; therefore, we investigated a panel of novel biomarkers for the early detection of high-grade serous ovarian carcinoma. METHODS: Twelve serum biomarkers with high differential gene expression and validated antibodies were selected: IL-1Ra, IL-6, Dkk-1, uPA, E-CAD, ErbB2, SLPI, HE4, CA125, LCN2, MSLN, and OPN. They were tested using Simple Plex™, a multi-analyte immunoassay platform, in samples collected from 172 patients who were either healthy, had benign gynecologic pathologies, or had high-grade serous ovarian adenocarcinomas. The receiver operating characteristic (ROC) curve, ROC area under the curve (AUC), and standard error (SE) of the AUC were obtained. Univariate ROC analyses and multivariate ROC analyses with the combination of multiple biomarkers were performed. RESULTS: The 4-marker panel consisting of CA125, HE4, E-CAD, and IL-6 had the highest ROC AUC. When evaluated for the ability to distinguish early stage ovarian cancer from a non-cancer control, not only did this 4-marker panel (AUC=0.961) performed better than CA 125 alone (AUC=0.851; P=0.0150) and HE4 alone (AUC=0.870; P=0.0220), but also performed significantly better than the 2- marker combination of CA125+HE4 (AUC=0.922; P=0.0278). The 4-marker panel had the highest average sensitivity under the region of its ROC curve corresponding to specificity ranging from 100% down to ~95%. CONCLUSION: The four-marker panel, CA125, HE4, E-CAD, and IL-6, shows potential in detecting serous ovarian cancer at earlier stages. Additional validation studies using the biomarker combination in ovarian cancer patients are warranted.


Subject(s)
Biomarkers, Tumor/metabolism , Cystadenocarcinoma, Serous/diagnosis , Ovarian Neoplasms/diagnosis , Biomarkers, Tumor/biosynthesis , Biomarkers, Tumor/genetics , Case-Control Studies , Cystadenocarcinoma, Serous/metabolism , Cystadenocarcinoma, Serous/pathology , Female , Gene Expression , Humans , Mesothelin , Middle Aged , Neoplasm Grading , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , ROC Curve
12.
Gynecol Oncol ; 146(1): 179-186, 2017 07.
Article in English | MEDLINE | ID: mdl-28473206

ABSTRACT

BACKGROUND: Epithelial ovarian cancer (EOC) is an aggressive and heterogeneous disease. <10% of EOC demonstrate HER2/neu 3+ receptor over-expression. However, moderate to low (i.e., 2+ and 1+) HER2/neu expression is reported in up to 50% of EOC. The objective of this study was to compare the anti-tumor activity of SYD985, a novel HER2-targeting antibody-drug conjugate (ADC), to trastuzumab emtansine (T-DM1) in EOC models with differential HER2/neu expression. METHODS: The cytotoxicity of SYD985 and T-DM1 was evaluated using ten primary EOC cell lines with 0/1+, 2+, and 3+ HER2/neu expression in antibody-dependent cellular cytotoxicity (ADCC), proliferation, viability and bystander killing experiments. Finally, the in vivo activity of SYD985 and T-DM1 was also studied in ovarian cancer xenografts. RESULTS: SYD985 and T-DM1 induced similar ADCC in the presence of peripheral blood lymphocytes (PBL) against EOC cell lines with differential HER2/neu expression. In contrast, SYD985 was 3 to 42 fold more cytotoxic in the absence of PBL when compared to T-DM1 (p<0.0001). Unlike T-DM1, SYD985 induced efficient bystander killing of HER2/neu 0/1+ tumor cells when admixed with HER2/neu 3+ EOC cells. In vivo studies confirmed that SYD985 is significantly more active than T-DM1 against HER2/neu 3+ EOC xenografts. CONCLUSIONS: SYD985 is a novel ADC with remarkable activity against EOC with strong (3+) as well as moderate to low (i.e., 2+ and 1+) HER2/neu expression. SYD985 is more potent than T-DM1 in comparative experiments and unlike T-DM1, it is active against EOC demonstrating moderate/low or heterogeneous HER2/neu expression.


Subject(s)
Immunotoxins/pharmacology , Indoles/administration & dosage , Neoplasms, Glandular and Epithelial/drug therapy , Ovarian Neoplasms/drug therapy , Receptor, ErbB-2/immunology , Ado-Trastuzumab Emtansine , Adult , Aged , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Antibody-Dependent Cell Cytotoxicity , Antineoplastic Agents, Alkylating/administration & dosage , Bystander Effect/immunology , Carcinoma, Ovarian Epithelial , Cell Line, Tumor , Duocarmycins , Female , Humans , Immunotoxins/immunology , Maytansine/analogs & derivatives , Maytansine/pharmacology , Mice , Mice, SCID , Middle Aged , Neoplasms, Glandular and Epithelial/enzymology , Neoplasms, Glandular and Epithelial/immunology , Ovarian Neoplasms/enzymology , Ovarian Neoplasms/immunology , Pyrrolidinones/administration & dosage , Random Allocation , Receptor, ErbB-2/biosynthesis , Receptor, ErbB-2/genetics , Trastuzumab , Xenograft Model Antitumor Assays
13.
Gynecol Oncol ; 147(1): 145-152, 2017 10.
Article in English | MEDLINE | ID: mdl-28705408

ABSTRACT

BACKGROUND: Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. The objective of this study was to compare the anti-tumor activity of HER2/neu-targeting monoclonal antibodies, trastuzumab (T), pertuzumab (P), combination of trastuzumab and pertuzumab (T+P) and trastuzumab-emtansine (T-DM1) in EOC with high HER2/neu expression. METHODS: Primary EOC cell lines were established and cell blocks were analyzed for HER2/neu expression. Cytostatic, apoptotic and antibody-dependent cell-mediated cytotoxicity (ADCC) activities of T, P, T+P and T-DM1 were evaluated in vitro. The in vivo antitumor activity was tested in xenograft models with 3+ HER2/neu expression. RESULTS: High (3+) HER2/neu expression was detected in 40% of the primary EOC cell lines. T, P, T+P, and T-DM1 were similarly effective in inducing strong ADCC against primary EOC cell lines expressing 3+ HER2/neu. The combination of T and P was more cytostatic when compared with that of T or P used alone (p<0.0001 and p<0.0001, respectively). T-DM1 induced significantly more apoptosis when compared with T+P (p<0.0001). Finally, T-DM1 was significantly more effective in tumor growth inhibition in vivo in EOC xenografts overexpressing HER2/neu when compared to T alone, P alone and T+P (p=0.04). CONCLUSION: In vitro and in vivo experiments with 3+ HER2/neu expressing EOC revealed limited anti-tumor activity of T or P. T-DM1 showed superior anti-tumor activity to T and P as single agents and as a combination. Our preclinical data support the design of clinical studies with T-DM1 for the treatment of chemotherapy-resistant EOC overexpressing HER2/neu.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma/drug therapy , Ovarian Neoplasms/drug therapy , Adult , Aged , Animals , Antibodies, Monoclonal, Humanized/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Carcinoma/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Drug Combinations , Female , Humans , Maytansine/administration & dosage , Maytansine/analogs & derivatives , Mice , Mice, SCID , Middle Aged , Ovarian Neoplasms/metabolism , Receptor, ErbB-2/metabolism , Trastuzumab/administration & dosage
14.
Gynecol Oncol ; 144(1): 146-152, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27894751

ABSTRACT

OBJECTIVE: Up to 12% of all endometrial-carcinomas (EC) harbor DNA-polymerase-ε-(POLE) mutations. It is currently unknown whether the favorable prognosis of POLE-mutated EC is derived from their low metastatic capability, extraordinary number of somatic mutations thus imparting immunogenicity, or a high sensitivity to chemotherapy. METHODS: Polymerase-chain-reaction-amplification and Sanger-sequencing were used to test for POLE exonuclease-domain-mutations (exons 9-14) 131 EC. Infiltration of CD4+ and CD8+ T-lymphocytes (TIL) and PD-1-expression in POLE-mutated vs POLE wild-type EC was studied by immunohistochemistry (IHC) and the correlations between survival and molecular features were investigated. Finally, primary POLE-mutated and POLE-wild-type EC cell lines were established and compared in-vitro for their sensitivity to chemotherapy. RESULTS: Eleven POLE-mutated EC (8.5%) were identified. POLE-mutated tumors were associated with improved progression-free-survival (P<0.05) and displayed increased numbers of CD4+ (44.5 vs 21.8; P=0.001) and CD8+ (32.8 vs 13.5; P<0.001) TILs when compared to wild-type POLE EC. PD-1 receptor was overexpressed in TILs from POLE-mutated vs wild-type-tumors (81% vs 28%; P<0.001). Primary POLE tumor cell lines were significantly more resistant to platinum-chemotherapy in-vitro when compared to POLE-wild-type tumors (P<0.004). CONCLUSIONS: POLE ultra-mutated EC are heavily infiltrated with CD4+/CD8+ TIL, overexpress PD-1 immune-check-point (i.e., features consistent with chronic antigen-exposure), and have a better prognosis when compared to other molecular subtypes of EC patients. POLE-mutated tumor-cell lines are resistant to platinum-chemotherapy in-vitro suggesting that the better prognosis of POLE-patients is not secondary to a higher sensitivity to chemotherapy but likely linked to enhanced immunogenicity.


Subject(s)
Carcinoma/genetics , Carcinoma/immunology , DNA Polymerase II/genetics , Drug Resistance, Neoplasm/genetics , Endometrial Neoplasms/genetics , Endometrial Neoplasms/immunology , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/pharmacology , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Carboplatin/pharmacology , Carcinoma/chemistry , Cell Survival/drug effects , DNA Polymerase II/analysis , Disease-Free Survival , Endometrial Neoplasms/chemistry , Female , Humans , Microsatellite Instability , Middle Aged , Mutation , Poly-ADP-Ribose Binding Proteins , Tumor Cells, Cultured
15.
Br J Cancer ; 115(3): 303-11, 2016 07 26.
Article in English | MEDLINE | ID: mdl-27351214

ABSTRACT

BACKGROUND: Clinical options for patients harbouring advanced/recurrent uterine serous carcinoma (USC), an aggressive variant of endometrial tumour, are very limited. Next-generation sequencing (NGS) data recently demonstrated that cyclin E1 (CCNE1) gene amplification and pik3ca driver mutations are common in USC and may therefore represent ideal therapeutic targets. METHODS: Cyclin E1 expression was evaluated by immunohistochemistry (IHC) on 95 USCs. The efficacy of the cyclin-dependent kinase 2/9 inhibitor CYC065 was assessed on multiple primary USC cell lines with or without CCNE1 amplification. Cell-cycle analyses and knockdown experiments were performed to assess CYC065 targeting specificity. Finally, the in vitro and in vivo activity of CYC065, Taselisib (a PIK3CA inhibitor) and their combinations was tested on USC xenografts derived from CCNE1-amplified/pik3ca-mutated USCs. RESULTS: We found that 89.5% of the USCs expressed CCNE1. CYC065 blocked cells in the G1 phase of the cell cycle and inhibited cell growth specifically in CCNE1-overexpressing USCs. Cyclin E1 knockdown conferred increased resistance to CYC065, whereas CYC065 treatment of xenografts derived from CCNE1-amplified USCs significantly reduced tumour growth. The combination of CYC065 and Taselisib demonstrated synergistic effect in vitro and was significantly more effective than single-agent treatment in decreasing tumour growth in xenografts of CCNE1-amplified/pik3ca-mutated USCs. CONCLUSIONS: Dual CCNE1/PIK3CA blockade may represent a novel therapeutic option for USC patients harbouring recurrent CCNE1-amplified/pi3kca-mutated tumours.


Subject(s)
Cyclin E/genetics , Mutation , Oncogene Proteins/genetics , Phosphatidylinositol 3-Kinases/genetics , Uterine Neoplasms/drug therapy , Animals , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases , Cyclin E/metabolism , DNA Copy Number Variations , Female , Gene Knockdown Techniques , Heterografts , Humans , In Situ Hybridization, Fluorescence , In Vitro Techniques , Mice , RNA, Messenger/genetics , Tissue Array Analysis , Uterine Neoplasms/genetics , Uterine Neoplasms/metabolism
16.
Am J Obstet Gynecol ; 214(1): 99.e1-8, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26272866

ABSTRACT

BACKGROUND: Uterine serous carcinoma is an aggressive form of endometrial cancer that carries an extremely poor prognosis. Solitomab is a novel bispecific single-chain antibody construct that targets epithelial cell adhesion molecule on tumor cells and also contains a CD3 binding region. We evaluated the expression levels of epithelial cell adhesion molecule and the in vitro activity of solitomab against primary uterine serous carcinoma cell lines in vitro and ex-vivo in the ascites of patients with uterine serous carcinoma. OBJECTIVE: The purpose of this study was to determine the frequency of expression of epithelial cell adhesion molecule on uterine serous carcinoma cell lines and the ability of solitomab to modulate immune responses (T-cell proliferation, activation, cytokine production, and tumor killing) to tumor cells when it is combined with lymphocytes and epithelial cell adhesion molecule-positive cell lines or epithelial cell adhesion molecule-positive ascitic fluid in vitro. STUDY DESIGN: Epithelial cell adhesion molecule expression was evaluated by flow cytometry in a total of 14 primary uterine serous carcinoma cell lines. Sensitivity to solitomab-dependent cellular-cytotoxicity was tested against a panel of primary uterine serous carcinoma cell lines that express different levels of epithelial cell adhesion molecule in standard 4-hour chromium release assays. The proliferative activity, activation, cytokine secretion (ie, type I vs type II), and cytotoxicity of solitomab in autologous tumor-associated T cells in the ascitic fluid of patients with uterine serous carcinoma was also evaluated by carboxyfluorescein succinimidyl ester and flow-cytometry assays. Differences in epithelial cell adhesion molecule expression, solitomab-dependent cellular-cytotoxicity levels were analyzed with the use of an unpaired t test. T-cell activation marker increase and cytokine release were analyzed by a paired t test. RESULTS: Surface expression of epithelial cell adhesion molecule was found in 85.7% (12 of 14) of the uterine serous carcinoma cell lines that were tested by flow cytometry. Epithelial cell adhesion molecule-positive cell lines were found resistant to natural killer cells or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean killing ± standard of the mean, 2.7% ± 3.1% after incubation of epithelial cell adhesion molecule-positive cell lines with control bispecific antibody construct). In contrast, after incubation with solitomab, epithelial cell adhesion molecule-positive uterine serous carcinoma cells became highly sensitive to T-cell cytotoxicity (mean killing, 25.7% ± 4.5%; P < .0001) by peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with epithelial cell adhesion molecule that expressed malignant cells in ascites with solitomab resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (ie, CD25 and HLA-DR), and a reduction in number of viable uterine serous carcinoma cells in ascites (P < .001). CONCLUSION: Solitomab induces robust immunologic responses in vitro that result in increased T-cell activation, proliferation, production of cytokines, and direct killing of tumor cells. These findings suggest that solitomab may represent a novel, potentially effective agent for the treatment of recurrent/metastatic and/or chemo-resistant uterine serous carcinoma-overexpressing epithelial cell adhesion molecule.


Subject(s)
Antibodies, Bispecific/pharmacology , Antigens, Neoplasm/drug effects , Antigens, Neoplasm/immunology , Antineoplastic Agents/pharmacology , CD3 Complex/immunology , Carcinoma, Papillary/drug therapy , Cell Adhesion Molecules/drug effects , Cell Adhesion Molecules/immunology , Neoplasms, Cystic, Mucinous, and Serous/drug therapy , Uterine Neoplasms/drug therapy , Antigens, Neoplasm/analysis , Ascitic Fluid/pathology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , Carcinoma, Papillary/chemistry , Carcinoma, Papillary/immunology , Cell Adhesion Molecules/analysis , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Coculture Techniques , Cytokines/drug effects , Cytokines/metabolism , Cytotoxicity, Immunologic/drug effects , Epithelial Cell Adhesion Molecule , Female , Flow Cytometry , Humans , Lymphocyte Activation/drug effects , Neoplasms, Cystic, Mucinous, and Serous/chemistry , Neoplasms, Cystic, Mucinous, and Serous/immunology , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , Uterine Neoplasms/chemistry , Uterine Neoplasms/immunology
17.
Int J Cancer ; 137(11): 2618-29, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26060989

ABSTRACT

Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin-3- and -4-overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy-naïve and chemotherapy-resistant human ovarian cancer xenografts or patient-derived xenografts (PDXs) were treated with the carboxyl-terminal binding domain of the Clostridium perfringens enterotoxin (c-CPE) conjugated to FITC (FITC-c-CPE) or the near-infrared (NIR) fluorescent tag IRDye CW800 (CW800-c-CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC-c-CPE and the CW800-c-CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800-c-CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor-to-background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c-CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c-CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real-time detection of micrometastatic ovarian disease overexpressing the Claudin-3 and -4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.


Subject(s)
Enterotoxins/administration & dosage , Fluorescent Dyes/administration & dosage , Neoplasm Micrometastasis/pathology , Ovarian Neoplasms/pathology , Animals , Claudin-3/metabolism , Claudin-4/metabolism , Female , Humans , Mice , Mice, SCID , Xenograft Model Antitumor Assays/methods
18.
Cancer ; 121(3): 403-12, 2015 Feb 01.
Article in English | MEDLINE | ID: mdl-25251053

ABSTRACT

BACKGROUND: Solitomab is a novel, bispecific, single-chain antibody that targets epithelial cell adhesion molecule (EpCAM) on tumor cells and also contains a cluster of differentiation 3 (CD3) (T-cell coreceptor) binding region. The authors evaluated the in vitro activity of solitomab against primary chemotherapy-resistant epithelial ovarian carcinoma cell lines as well as malignant cells in ascites. METHODS: EpCAM expression was evaluated by flow cytometry in 5 primary ovarian cancer cell lines and in 42 fresh ovarian tumor cell cultures in ascites from patients with mainly advanced or recurrent, chemotherapy-resistant disease. The potential activity of solitomab against EpCAM-positive tumor cells was evaluated by flow cytometry, proliferation, and 4-hour chromium-release, cell-mediated cytotoxicity assays. RESULTS: EpCAM expression was detected by flow cytometry in approximately 80% of the fresh ovarian tumors and primary ovarian tumor cell lines tested. EpCAM-positive, chemotherapy-resistant cell lines were identified as resistant to natural killer cell-mediated or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean±standard error of the mean, 3.6%±0.7% of cells killed after incubation of EpCAM-positive cell lines with control bispecific antibody). In contrast, after incubation with solitomab, EpCAM-positive, chemotherapy-resistant cells became highly sensitive to T-cell cytotoxicity (mean±standard error of the mean, 28.2%±2.05% of cells killed; P<.0001) after exposure to peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with EpCAM-expressing malignant cells in ascites with solitomab resulted in a significant increase in T-cell activation markers and a reduction in the number of viable ovarian tumor cells in ascites (P<.001). CONCLUSIONS: Solitomab may represent a novel, potentially effective agent for the treatment of chemotherapy-resistant ovarian cancers that overexpress EpCAM.


Subject(s)
Antibodies, Bispecific/pharmacology , Neoplasms, Glandular and Epithelial/drug therapy , Ovarian Neoplasms/drug therapy , Adult , Aged , Aged, 80 and over , Antibodies, Bispecific/immunology , Antigens, Neoplasm/biosynthesis , Antigens, Neoplasm/immunology , CD3 Complex/immunology , Carcinoma, Ovarian Epithelial , Cell Adhesion Molecules/biosynthesis , Cell Adhesion Molecules/immunology , Cell Line, Tumor , Cytotoxicity, Immunologic/drug effects , Drug Resistance, Neoplasm , Epithelial Cell Adhesion Molecule , Female , Flow Cytometry , Humans , Middle Aged , Neoplasms, Glandular and Epithelial/immunology , Ovarian Neoplasms/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology
19.
Br J Cancer ; 113(7): 1020-6, 2015 Sep 29.
Article in English | MEDLINE | ID: mdl-26325104

ABSTRACT

OBJECTIVES: We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. METHODS: Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. RESULTS: Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/neu not amplified cell lines (P=0.01). HER2/neu-amplified/PIK3CA-mutated USC were highly resistant to trastuzumab when compared with HER2/neu-amplified/wild-type-PIK3CA cell lines (P=0.02). HER2/neu-amplified/PIK3CA wild-type cell lines transfected with oncogenic PIK3CA-mutations increased their resistance to trastuzumab (P<0.0001). Trastuzumab was effective in reducing tumour growth (P=0.001) and improved survival (P=0.0001) in mouse xenografts harbouring HER2-amplified/PIK3CA wild-type USC but not in HER2-amplified/PIK3CA-mutated tumours. CONCLUSIONS: Oncogenic PIK3CA mutations are common in HER2/neu-amplified USC and may constitute a major mechanism of resistance to trastuzumab treatment.


Subject(s)
Antineoplastic Agents/administration & dosage , Cystadenocarcinoma, Serous/drug therapy , Phosphatidylinositol 3-Kinases/genetics , Receptor, ErbB-2/genetics , Trastuzumab/administration & dosage , Uterine Neoplasms/drug therapy , Animals , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/genetics , Cell Survival/drug effects , Class I Phosphatidylinositol 3-Kinases , Cystadenocarcinoma, Serous/genetics , Female , Gene Amplification , Humans , Mice , Mutation , Trastuzumab/therapeutic use , Uterine Neoplasms/genetics , Xenograft Model Antitumor Assays
20.
Tumour Biol ; 36(7): 5505-13, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25669172

ABSTRACT

Uterine serous carcinoma (USC) is an aggressive subtype of endometrial cancer that carries an extremely poor prognosis. Up to 35 % of USC may overexpress the epidermal growth factor receptor-2 (HER2/neu) at strong (i.e., 3+) level by immunohistochemistry (IHC) or harbor HER2/neu gene amplification by fluorescence in situ hybridization (FISH). In this study, we assessed the sensitivity of a panel of USC cell lines with and without HER2/neu gene amplification to dacomitinib (PF-00299804), an irreversible pan-human epidermal growth factor receptor tyrosine kinase inhibitor. Eight primary cell lines (i.e., four harboring HER2/neu gene amplification by FISH and four FISH- cell lines), all demonstrating similar in vitro growth rates, were evaluated in viability/proliferation assays. The effect of dacomitinib on cell growth, cell cycle distribution, and signaling was determined using flow cytometry-based assays. Dacomitinib caused a significantly stronger growth inhibition in HER2/neu FISH+ USC cell lines when compared to FISH- USC (dacomitinib half maximal inhibitory concentration (IC50) mean ± SEM = 0.02803 ± 0.003355 µM in FISH+ versus 1.498 ± 0.2209 µM in FISH- tumors, P < 0.0001). Dacomitinib growth inhibition was associated with a significant and dose-dependent decline in phosphorylated HER2/neu and S6 transcription factor and a dose-dependent and time-dependent cell cycle arrest in G0/G1 in FISH+ USC. Dacomitinib is remarkably effective against chemotherapy-resistant HER2/neu gene-amplified USC. Clinical studies with dacomitinib in HER2/neu FISH+ USC patients resistant to standard salvage chemotherapy are warranted.


Subject(s)
Cystadenocarcinoma, Serous/drug therapy , Protein Kinase Inhibitors/administration & dosage , Quinazolinones/administration & dosage , Receptor, ErbB-2/genetics , Uterine Neoplasms/drug therapy , Antibodies, Monoclonal, Humanized/administration & dosage , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cystadenocarcinoma, Serous/genetics , Cystadenocarcinoma, Serous/pathology , Female , Gene Amplification , Humans , In Situ Hybridization, Fluorescence , Receptor, ErbB-2/antagonists & inhibitors , Uterine Neoplasms/genetics , Uterine Neoplasms/pathology
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