Integrated mutational landscape analysis of poorly differentiated high-grade neuroendocrine carcinoma of the uterine cervix.

High-grade neuroendocrine cervical cancers (NETc) are exceedingly rare, highly aggressive tumors. We analyzed 64 NETc tumor samples by whole-exome sequencing (WES). Human papillomavirus DNA was detected in 65.6% (42/64) of the tumors. Recurrent mutations were identified in PIK3CA, KMT2D/MLL2, K-RAS, ARID1A, NOTCH2, and RPL10. The top mutated genes included RB1, ARID1A, PTEN, KMT2D/MLL2, and WDFY3, a gene not yet implicated in NETc. Somatic CNV analysis identified two copy number gains (3q27.1 and 19q13.12) and five copy number losses (1p36.21/5q31.3/6p22.2/9q21.11/11p15.5). Also, gene fusions affecting the ACLY-CRHR1 and PVT1-MYC genes were identified in one of the eight samples subjected to RNA sequencing. To resolve evolutionary history, multiregion WES in NETc admixed with adenocarcinoma cells was performed (i.e., mixed-NETc). Phylogenetic analysis of mixed-NETc demonstrated that adenocarcinoma and neuroendocrine elements derive from a common precursor with mutations typical of adenocarcinomas. Over one-third (22/64) of NETc demonstrated a mutator phenotype of C > T at CpG consistent with deficiencies in MBD4, a member of the base excision repair (BER) pathway. Mutations in the PI3K/AMPK pathways were identified in 49/64 samples. We used two patient-derived-xenografts (PDX) (i.e., NET19 and NET21) to evaluate the activity of pan-HER (afatinib), PIK3CA (copanlisib), and ATR (elimusertib) inhibitors, alone and in combination. PDXs harboring alterations in the ERBB2/PI3K/AKT/mTOR/ATR pathway were sensitive to afatinib, copanlisib, and elimusertib (P < 0.001 vs. controls). However, combinations of copanlisib/afatinib and copanlisib/elimusertib were significantly more effective in controlling NETc tumor growth. These findings define the genetic landscape of NETc and suggest that a large subset of these highly lethal malignancies might benefit from existing targeted therapies.

Preclinical efficacy of RAF/MEK clamp avutometinib in combination with FAK inhibition in low grade serous ovarian cancer.

OBJECTIVES: Low-grade-serous-ovarian-carcinoma (LGSOC) is characterized by a high recurrence rate and limited therapeutic options. About one-third of LGSOC contains mutations in MAPK pathway genes such as KRAS/NRAS/BRAF. Avutometinib is a dual RAF/MEK inhibitor while defactinib and VS-4718 are focal-adhesion-kinase-inhibitors (FAKi). We determined the preclinical efficacy of avutometinib±VS-4718 in LGSOC patient-derived-tumor-xenografts (PDX). METHODS: Whole-exome-sequencing (WES) was used to evaluate the genetic fingerprint of 3 patient-derived LGSOC (OVA(K)250, PERIT(M)17 and A(PE)148). OVA(K)250 tissue was successfully xenografted as PDX into female CB17/lcrHsd-Prkdc/SCID-mice. Animals were treated with either control, avutometinib, VS-4718, or avutometinib/ VS-4718 once daily five days on and two days off through oral gavage. Mechanistic studies were performed ex vivo using avutometinib±defactinib treated LGSOC tumor samples by western blot. RESULTS: WES results demonstrated wild-type KRAS in all 3 LGSOC. OVA(K)250 PDX showed gain-of-function mutations (GOF) in PTK2 and PTK2B genes, and loss-of-heterozygosity in ADRB2, potentially sensitizing to FAK and RAF/MEK inhibition. The combination of avutometinib/ VS-4718 demonstrated strong tumor-growth inhibition compared to controls starting at day 9 (p < 0.002) in OVA(K)250PDX. By 60 days, mice treated with avutometinib alone and avutometinib/VS-4718 were still alive; compared to median survival of 20 days in control-treated mice and of 35 days in VS-4718-treated mice (p < 0.0001). By western-blot assays exposure of OVA(K)250 to avutometinib, FAKi defactinib and their combination demonstrated decreased phosphorylated FAK (p-FAK) as well as decreased p-ERK. CONCLUSION: Avutometinib, and to a larger extent its combination with FAK inhibitor VS-4718, demonstrated promising in vivo activity against a KRAS wild-type LGSOC-PDX. These data support the ongoing registration-directed study (RAMP201/NCT04625270).

Preclinical in vitro and in vivo activity of the RAF/MEK clamp avutometinib in combination with FAK inhibition in uterine carcinosarcomas.

OBJECTIVES: Uterine carcinosarcomas (UCS) are rare, biologically aggressive tumors. Since UCS may harbor mutations in RAS/MAPK pathway genes we evaluated the preclinical in vitro and in vivo efficacy of the RAF/MEK clamp avutometinib in combination with the focal adhesion kinase (FAK) inhibitors defactinib or VS-4718 against multiple primary UCS cell lines and xenografts. METHODS: Whole-exome-sequencing (WES) was used to evaluate the genetic landscape of 5 primary UCS cell lines. The in vitro activity of avutometinib ± FAK inhibitor was evaluated using cell viability and cell cycle assays against primary UCS cell lines. Mechanistic studies were performed using western blot assays while in vivo experiments were completed in UCS tumor bearing mice treated with avutometinib ± FAK inhibitor by oral gavage. RESULTS: WES results demonstrated multiple UCS cell lines harbor genetic alterations including KRAS, PTK2, BRAF, MAP2K, and MAP2K1, potentially sensitizing to FAK and RAF/MEK inhibition. Four out of five of the UCS cell lines demonstrated in vitro sensitivity to FAK and/or RAF/MEK inhibition when used alone or in combination. By western blot assays, exposure of UCS cell lines to the combination of defactinib/avutometinib demonstrated decreased phosphorylated (p)-FAK as well as decreased p-ERK. In vivo, the combination of avutometinib/VS-4718 demonstrated superior tumor growth inhibition and longer survival compared to single agent treatment and controls starting at day 10 (p < 0.002) in UCS xenografts. CONCLUSION: The combination of avutometinib and defactinib demonstrates promising in vitro and in vivo anti-tumor activity against primary UCS cell lines and xenografts.

Integrated mutational landscape analysis of uterine leiomyosarcomas.

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.

Trastuzumab deruxtecan (DS-8201a), a HER2-targeting antibody-drug conjugate with topoisomerase I inhibitor payload, shows antitumor activity in uterine and ovarian carcinosarcoma with HER2/neu expression.

OBJECTIVES: Carcinosarcomas are highly aggressive gynecologic malignancies containing both carcinomatous and sarcomatous elements with heterogeneous HER2/neu expression and limited therapeutic options. We compared the efficacy of trastuzumab deruxtecan (DS-8201a), a novel HER2/neu-targeting antibody-drug conjugate (ADC) to an ADC isotype control (MAAA-9199) against primary uterine and ovarian carcinosarcomas in vitro and in vivo. METHODS: Twelve primary carcinosarcoma (CS) cell lines were evaluated for HER2/neu surface expression by immunohistochemistry (IHC) and by flow cytometry, and gene amplification by fluorescence in situ hybridization (FISH) assays. The in vitro experiments included cytotoxicity and bystander killing effect assays on three cell lines of variable HER2/neu expression. In vivo activity was studied in a mouse CS xenograft model of 3+ HER2/neu uterine CS. RESULTS: In vitro studies showed that DS-8201a was highly effective against uterine and ovarian CS cell lines demonstrating 3+ HER2/neu expression compared to MAAA-9199 control; there was no significant improvement in the 0 HER2/neu CS cell line. However, DS-8201a induced efficient bystander killing of 0 HER2/neu tumor cells when admixed with 3+ HER2/neu cells. In vivo studies confirmed that DS-8201a was more effective than MAAA-9199 in 3+ HER2/neu-expressing CS xenografts. CONCLUSION: DS-8201a may represent a novel and highly effective ADC against HER2/neu-expressing CS.

Elimusertib (BAY1895344), a novel ATR inhibitor, demonstrates in vivo activity in ATRX mutated models of uterine leiomyosarcoma.

INTRODUCTION: Uterine leiomyosarcoma (uLMS) is a rare, highly aggressive malignancy. Recent data suggest 50% of uLMS may harbor alterations in the ATRX gene and such mutations may confer sensitivity to ataxia-telangiectasia-and-Rad3-related (ATR) kinase inhibitors. We sought to investigate the in vivo activity of Elimusertib (BAY1895344), a novel ATR-inhibitor, against ATRX-mutated uLMS patient-derived xenografts (PDXs). METHODS: Two fully characterized uLMS (i.e., LEY-11 and LEY-16) were grafted into female CB-17/SCID mice. Treatments with control vehicle or BAY1895344 (20 mg/kg dosed twice daily 3 days on 4 days off) were given via oral gavage and tumor measurements as well as weights obtained twice weekly. Tumor volume differences were calculated with a two-way ANOVA. Mechanistic studies were performed ex vivo using BAY1895344 treated uLMS tumor samples by western blot analysis. RESULTS: Both PDX LEY-11 and PDX LEY-16 harboring ATRX gene mutations demonstrated an aggressive behavior in vivo (i.e., control mice were euthanized on average at day 12.5 for PDX LEY-11 and at day 33 for PDX LEY-16). In both tumor models BAY1895344 20 mg/kg dosed with an intermittent oral schedule was able to induce significant growth inhibition compared to vehicle control treatment (p < 0.001 for both LEY-11 and LEY-16) and prolong median overall survival [PDX LEY-11 (12.5 vs. 42 days, p < 0.001) and PDX LEY-16 (33 vs. 60 days, p < 0.001)]. There were not significant changes in weight between treatment and controls. By western blot assays BAY1895344 exposure decreased phosphorylated-ATR and increased expression of apoptotic molecules in LMS PDXs. CONCLUSIONS: BAY1895344 demonstrates promising in vivo activity against biologically aggressive PDX models of uLMS harboring ATRX mutations, with no significant toxicity. Clinical trials of BAY1895344 in uLMS patients are warranted.

The Poly (ADP-ribose) polymerase inhibitor olaparib and pan-ErbB inhibitor neratinib are highly synergistic in HER2 overexpressing epithelial ovarian carcinoma in vitro and in vivo.

INTRODUCTION: Ovarian cancer (OC) is associated with the highest gynecologic cancer mortality. The development of novel, effective combinations of targeted therapeutics remains an unmet medical need. We evaluated the preclinical efficacy of the Poly (ADP-ribose) polymerase (PARP) inhibitor (olaparib) and the pan-ErbB inhibitor (neratinib) as single agents and in combination in ovarian cancer cell lines and xenografts with variable HER2 expression. METHODS: In vitro cell viability with olaparib, neratinib, and their combination was assessed using flow-cytometry based assays against a panel of OC primary cell lines with variable HER2 expression. Immunoblotting experiments were performed to elucidate the mechanism of activity and synergism. The in vivo antitumor activity of the olaparib/neratinib combination versus single agents was tested in HER2 positive xenograft OC models. RESULTS: HER2 + OC cell lines demonstrated higher sensitivity to olaparib and neratinib when compared to HER2 negative tumors (i.e., IC50: 2.06 ± 0.33 µM vs. 39.28 ± 30.51 µM, p = 0.0035 for olaparib and 19.42 ± 2.63 nM vs. 235.0 ± 165.0 nM, p = 0.0035 for neratinib). The combination of olaparib with neratinib was more potent when compared to single-agent olaparib or neratinib both in vitro and in vivo, and demonstrated synergy in all primary HER2 + OC models. Western blot experiments showed neratinib decreased pHER2/neu while increased Poly(ADP-ribose) (PAR) enzymatic activity; olaparib increased pHER2/Neu expression and blocked PAR activatio. Olaparib/neratinib in combination decreased both pHER2/Neu as well as PAR activation. CONCLUSION: The combination of olaparib and neratinib is synergistic and endowed with remarkable preclinical activity against HER2+ ovarian cancers. This combination may represent a novel therapeutic option for ovarian cancer patients with HER2+, homologous recombination-proficient tumors resistant to chemotherapy.

Ovarian and uterine carcinosarcomas are sensitive in vitro and in vivo to elimusertib, a novel ataxia-telangiectasia and Rad3-related (ATR) kinase inhibitor.

BACKGROUND: Carcinosarcoma of the ovary (OCS) and uterus (UCS) are rare highly aggressive malignancies. Ataxia-telangiectasia-and-Rad3-related (ATR) kinase and homologous recombination play a pivotal role in DNA damage repair. Homologous recombination deficiency (HRD) has been demonstrated in >30% of OCS/UCS. We investigated the preclinical activity of elimusertib, a selective ATR kinase inhibitor, against carcinosarcoma (CS) cell lines and xenografts. METHODS: Sensitivity to elimusertib was evaluated in vitro against nine whole exome-sequenced (WES) primary CS cell lines and in vivo against HRD CS xenografts. Western blots were performed to determine baseline ATR and p-ATR protein expression in CS, and ATR pathway downstream effectors and apoptosis markers in CS HRD cell lines after Elimusertib treatment. RESULTS: Out of the 9 CS cell lines, 3 harbored HRD and 6 homologous recombination proficient (HRP) features. Most of CS (i.e., 7/9 = 85%) were found to be sensitive to Elimusertib in vitro. Among the 5 primary CS cell lines with a high-grade pure serous epithelial component, HRD cell lines were more sensitive to elimusertib than HRP tumors (mean IC50 ± SEM HRD CS = 61.3 nM ±15.2 vs HRP = 361.6 nM ±24.4 (p = 0.01)). Baseline ATR and p-ATR protein expression was higher in HRD CS cell lines. Elimusertib showed tumor growth inhibition in HRD CS xenografts (p < 0.0001) and increased overall animal survival (p < 0.0001). Western blot demonstrated dose-dependent inhibition of ATR, p-ATR and its downstream effector p-CHK1, and a dose-dependent increase in caspase-3 expression. CONCLUSIONS: Elimusertib is preclinically active in vitro and in vivo against primary CS cell lines and xenografts, respectively. CS models harboring HRD or with pure/mixed endometrioid histology demonstrated higher sensitivity to ATR inhibition. Clinical trials with elimusertib in CS patients are warranted.

Uterine leiomyosarcomas harboring MAP2K4 gene amplification are sensitive in vivo to PLX8725, a novel MAP2K4 inhibitor.

INTRODUCTION: Uterine leiomyosarcomas (uLMS) are rare, highly aggressive tumors. Up to 30% of uLMS may harbor gain of function (GOF) in the MAP2K4 gene, important for tumor cell proliferation, differentiation and metastasis. We investigated the in vivo activity of a novel MAP2K4 inhibitor, PLX8725, against uLMS harboring MAP2K4 gene-amplification. METHODS: Two fully characterized uLMS (i.e., LEY-11 and LEY-16) were grafted into female CB-17/SCID mice. Treatments with control vehicle or PLX8725 (50 mg/kg) were given via oral gavage daily on weekdays for up to 60 days. Tumor volume differences were calculated with two-way ANOVA. Pharmacokinetic (PK) and mechanistic studies of PLX8725 in uLMS PDX models were also performed. RESULTS: Both uLMS tumors evaluated demonstrated GOF in MAP2K4 (i.e., 3 CNV in both LEY-11 and LEY-16). Tumor growth inhibition was significantly greater in both PDX LEY-11 and PDX LEY-16 treated with PLX8725 when compared to controls (p < 0.001). Median overall survival was also significantly longer in both PDX LEY-11 (p = 0.0047) and PDX LEY-16 (p = 0.0058) treatment cohorts when compared to controls. PLX8725 oral treatment was well tolerated, and PK studies demonstrated that oral PLX8725 gives extended exposure in mice. Ex vivo tumor samples after PLX8725 exposure decreased phosphorylated-ATR, JNK and p38, and increased expression of apoptotic molecules on western blot. CONCLUSION: PLX8725 demonstrates promising in vivo activity against PDX models of uLMS harboring GOF alterations in the MAP2K4 gene with tolerable toxicity. Phase I trials of PLX8725 in advanced, recurrent, chemotherapy-resistant uLMS patients are warranted.

Monitoring Treatment Response, Early Recurrence, and Survival in Uterine Serous Carcinoma and Carcinosarcoma Patients Using Personalized Circulating Tumor DNA Biomarkers.

Uterine serous carcinoma (USC) and carcinosarcomas (CSs) are rare, highly aggressive variants of endometrial cancer. No reliable tumor biomarkers are currently available to guide response to treatment or detection of early recurrence in USC/CS patients. Circulating tumor DNA (ctDNA) identified using ultrasensitive technology such as droplet digital polymerase chain reaction (ddPCR) may represent a novel platform for the identification of occult disease. We explored the use of personalized ctDNA markers for monitoring USC and CS patients. Tumor and plasma samples from USC/CS patients were collected at the time of surgery and/or during the treatment course for assessment of tumor-specific somatic structural variants (SSVs) by a clinical-grade next-generation sequencing (NGS) platform (i.e., Foundation Medicine) and a droplet digital PCR instrument (Raindance, ddPCR). The level of ctDNA was quantified by droplet digital PCR in plasma samples and correlated to clinical findings, including CA-125 serum and/or computed tomography (CT) scanning results. The genomic-profiling-based assay identified mutated "driver" target genes for ctDNA analysis in all USC/CS patients. In multiple patients, longitudinal ctDNA testing was able to detect the presence of cancer cells before the recurrent tumor was clinically detectable by either CA-125 or CT scanning. Persistent undetectable levels of ctDNA following initial treatment were associated with prolonged progression-free and overall survival. In a USC patient, CA-125 and TP53 mutations but not PIK3CA mutations become undetectable in the plasma at the time of recurrence, suggesting that more than one customized probe should be used for monitoring ctDNA. Longitudinal ctDNA testing using tumor-informed assays may identify the presence of residual tumors, predict responses to treatment, and identify early recurrences in USC/CS patients. Recognition of disease persistence and/or recurrence through ctDNA surveillance may allow earlier treatment of recurrent disease and has the potential to change clinical practice in the management of USC and CS patients. CtDNA validation studies in USC/CS patients prospectively enrolled in treatment trials are warranted.

A phase 2 evaluation of pembrolizumab for recurrent Lynch-like versus sporadic endometrial cancers with microsatellite instability.

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.

Randomised phase II trial of weekly ixabepilone ± biweekly bevacizumab for platinum-resistant or refractory ovarian/fallopian tube/primary peritoneal cancer.

BACKGROUND: This multi-center RP2 study assessed activity/safety of ixabepilone + bevacizumab compared to ixabepilone in platinum-resistant/refractory ovarian/fallopian tube/primary peritoneal cancer. Additional objectives were to examine the role of prior bevacizumab and taxanes, and explore class III-ß-tubulin (TUBB3) as a predictive biomarker. METHODS: Participants were randomised to receive ixabepilone 20 mg/m2 days 1, 8, 15 with (IXA + BEV) or without (IXA) bevacizumab 10 mg/kg days 1, 15 every 28 days. Patients were stratified by prior BEV. The primary endpoint was PFS. OS, safety, and ORR served as secondary endpoints. RESULTS: Among 76 evaluable patients who received IXA + BEV (n = 39) compared to IXA (n = 37), the ORR was 33% (n = 13) versus 8% (n = 3)(P = 0.004), durable at 6 months in 37% (n = 14) and 3% (n = 1) (P < 0.001). BEV significantly improved PFS (median:5.5 vs 2.2 months, HR = 0.33, 95%CI 0.19-0.55, P < 0.001) and OS (median:10.0 vs 6.0 months, HR = 0.52, 95%CI 0.31-0.87, P = 0.006). Both regimens were well-tolerated. TUBB3 expression did not predict response. Subgroup analyses revealed minimal effect of prior BEV or taxane resistant/refractory status on response to IXA + BEV. CONCLUSIONS: IXA + BEV is a well-tolerated, effective combination for platinum/taxane-resistant ovarian cancer that extends PFS and likely OS relative to IXA monotherapy. Prior receipt of BEV should not preclude the use of IXA + BEV. TUBB3 is not a predictive biomarker. CLINICAL TRIAL REGISTRATION: NCT3093155.

Synergistic activity of neratinib in combination with olaparib in uterine serous carcinoma overexpressing HER2/neu.

INTRODUCTION: Uterine serous carcinoma (USC) is an aggressive variant of endometrial cancer with a poor prognosis. Approximately 30% of USC overexpress HER2/neu, a recognized target for trastuzumab in advanced/recurrent HER2/neu-positive USC. We evaluated the efficacy of the pan-c-erb inhibitor neratinib and the poly (ADP-ribose)-polymerase (PARP) inhibitor olaparib as single agents and in combination against USC cell lines and xenografts. METHODS: In-vitro cell-viability assays with olaparib, neratinib, and olaparib/neratinib were assessed using flow-cytometry based assays against a panel of USC cell lines with high and low HER2/neu expression. Homologous recombination deficiency (HRD) signatures were evaluated as described by Alexandrov et al. (Nature;2020;578:94-101) while downstream signaling affected by neratinib/olaparib exposure was assessed with immunoblotting. Efficacy of single- versus dual-agent inhibition was evaluated in-vivo using two USC-xenografts with 3+ HER2/neu expression. RESULTS: Neratinib was more potent than olaparib in suppression of in-vitro growth of HER2/neu 3+ cell lines (ARK1: p = 0.0047; ARK2: p = 0.0428) while no difference was noted against HER2/neu 1+ tumors (ARK4). Importantly, the combination of olaparib with neratinib synergistically improved tumor suppression compared to either single-agent in vitro. USC cells exposed to olaparib upregulated HER2/neu expression, while neratinib treatment increased PARP activity (ARK1: p < 0.0001; ARK2: p < 0.0001). Single-agent neratinib transiently inhibited in vivo growth of USC xenografts harboring HER2/neu gene amplification (ARK1: p < 0.05; ARK2: p < 0.05). In contrast, the combination of the two inhibitors caused a stronger and durable growth inhibition in both USC xenografts (ARK1: p < 0.05; ARK2: p < 0.05). CONCLUSION: The combination of olaparib and neratinib is active and synergistic against primary HER2/neu + USC. This combination may represent a novel therapeutic option for USC patients with HER2/neu+, homologous recombination-proficient tumors resistant to chemotherapy.

Homologous recombination deficiency (HRD) signature-3 in ovarian and uterine carcinosarcomas correlates with preclinical sensitivity to Olaparib, a poly (adenosine diphosphate [ADP]- ribose) polymerase (PARP) inhibitor.

OBJECTIVES: Carcinosarcoma (CS) of the ovary and uterus are highly aggressive malignancies associated with poor survival. Poly(ADP-ribose)-polymerase inhibitors (PARPi) are targeted agents impairing DNA repair via homologous-recombination-deficiency (HRD) mechanisms. We used whole-exome-sequencing (WES) data from a cohort of fresh tumor samples of ovarian (OCS) and uterine carcinosarcoma (UCS), primary cell lines and xenografts to investigate the role for olaparib in CSs. METHODS: WES data from 73 CS samples (48 UCS and 25 OCS) were analyzed for HRD signatures. Olaparib activity was evaluated using cell-viability, cell-cycle, apoptosis and cytotoxicity assays against primary CS cell lines. Olaparib antitumor activity was tested in vivo against HRD CS xenografts. RESULTS: Signature-3 (i.e. HRD-related signature) was identified in 60% of OCS (15 of 25) vs 25% of UCS (12 of 48) (p = 0.005). CS cell lines harboring Signature-3/HRD (3 OCS/1 UCS) were significantly more sensitive to olaparib when compared to HRP cell lines (5 UCS/1 OCS) [mean IC50 ± SEM = 2.94 µM ± 0.07 vs mean ± SEM = 23.3 µM ± 0.09, (p = 0.02), respectively]. PARPi suppressed CS cell growth through cell cycle arrest in the G2/M phase and caused more apoptosis in HRD vs HRP primary tumors (p < 0.0001). In vivo, olaparib significantly impaired HRD CS xenografts tumor growth (p = 0.0008) and increased overall animal survival (p < 0.0001). CONCLUSIONS: OCS and UCS cell lines harboring HRD signature-3 were significantly more sensitive to olaparib in vitro and in vivo when compared to HRP CS. Clinical studies with PARPi in CS patients with a dominant signature 3 (HRD-related) are warranted.

Mutational landscape of primary, metastatic, and recurrent ovarian cancer reveals c-MYC gains as potential target for BET inhibitors.

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.

Whole-exome sequencing of cervical carcinomas identifies activating ERBB2 and PIK3CA mutations as targets for combination therapy.

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.

Vessel-Targeting Nanoclovers Enable Noninvasive Delivery of Magnetic Hyperthermia-Chemotherapy Combination for Brain Cancer Treatment.

Despite being promising, the clinical application of magnetic hyperthermia for brain cancer treatment is limited by the requirement of highly invasive intracranial injections. To overcome this limitation, here we report the development of gallic acid-coated magnetic nanoclovers (GA-MNCs), which allow not only for noninvasive delivery of magnetic hyperthermia but also for targeted delivery of systemic chemotherapy to brain tumors. GA-MNCs are composed of clover-shaped MNCs in the core, which can induce magnetic heat in high efficiency, and polymerized GA on the shell, which enables tumor vessel-targeting. We demonstrate that intravenous administration of GA-MNCs following alternating magnetic field exposure effectively inhibited brain cancer development and preferentially disrupted tumor vasculature, making it possible to efficiently deliver systemic chemotherapy for further improved efficacy. Due to the noninvasive nature and high efficiency in killing tumor cells and enhancing systemic drug delivery, GA-MNCs have the potential to be translated for improved treatment of brain cancer.

Evaluation of treatment effects in patients with endometrial cancer and POLE mutations: An individual patient data meta-analysis.

BACKGROUND: Endometrial cancers (ECs) with somatic mutations in DNA polymerase epsilon (POLE) are characterized by unfavorable pathological features, which prompt adjuvant treatment. Paradoxically, women with POLE-mutated EC have outstanding clinical outcomes, and this raises concerns of overtreatment. The authors investigated whether favorable outcomes were independent of treatment. METHODS: A PubMed search for POLE and endometrial was restricted to articles published between March 1, 2012, and March 1, 2018, that provided individual patient data (IPD), adjuvant treatment, and survival. Following the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) reporting guidelines for IPD, the authors used univariate and multivariate one-stage meta-analyses with mixed effects Cox models (random effects for study cohorts) to infer the associations of treatment, traditional prognostic factors, and outcome, which was defined as the time from first diagnosis to any adverse event (progression/recurrence or death from EC). RESULTS: Three hundred fifty-nine women with POLE-mutated EC were identified; 294 (82%) had pathogenic mutations. Worse outcomes were demonstrated in patients with nonpathogenic POLE mutations (hazard ratio, 3.42; 95% confidence interval, 1.47-7.58; log-rank P < .01). Except for stage (P < .01), traditional prognosticators were not associated with progression/recurrence or death from disease. Adverse events were rare (11 progressions/recurrences and 3 disease-specific deaths). Salvage rates in patients who experienced recurrence were high and sustained, with 8 of 11 alive without evidence of disease (range, 5.5-14.2 years). Adjuvant treatment was not associated with outcome. CONCLUSIONS: Clinical outcomes for ECs with pathogenic POLE mutations are not associated with most traditional risk parameters, and patients do not appear to benefit from adjuvant therapy. The observed low rates of recurrence/progression and the high and sustained salvage rates raise the possibility of safely de-escalating treatment for these patients. LAY SUMMARY: Ten percent of all endometrial cancers have mutations in the DNA repair gene DNA polymerase epsilon (POLE). Women who have endometrial cancers with true POLE mutations experience almost no recurrences or deaths from their cancer even when their tumors appear to have very unfavorable characteristics. Additional therapy (radiation and chemotherapy) does not appear to improve outcomes for women with POLE-mutated endometrial cancer, and this supports the move to less therapy and less associated toxicity. Diligent classification of endometrial cancers by molecular features provides valuable information to inform prognosis and to direct treatment/no treatment.

Trastuzumab tolerability in the treatment of advanced (stage III-IV) or recurrent uterine serous carcinomas that overexpress HER2/neu.

OBJECTIVE: Due to previously reported trastuzumab safety concerns and the scant data available in endometrial cancer patients, we sought to assess the safety, tolerability and toxicity profile of trastuzumab in patients with advanced/recurrent uterine serous carcinoma (USC) that overexpress HER2/neu in our multicenter randomized phase II trial. METHODS: Patients were randomized 1:1 to receive carboplatin/paclitaxel (C/P) for 6 cycles ± trastuzumab (T) with the experimental arm continuing to receive single agent trastuzumab maintenance treatment until disease progression/toxicity. Progression-free-survival was the primary endpoint; overall-survival and toxicity were secondary endpoints. Adverse events (AEs) were compared between treatment arms. RESULTS: There were 28 patients in the C/P arm and 32 patients in the experimental (C/P + T) arm. Fifty-eight patients (97%) experienced 977 treatment-related AEs of which 875 (89.6%) were low-grade (grade 1-2) and 102 (10.4%) were high-grade (grade 3-5). The mean ± standard deviation of AEs per patient was 15.5 ± 16.3 in the C/P arm and 17.0 ± 16.0 in the C/P + T arm. Gastrointestinal AEs were the most common in both arms (n = 155, 15.7%) of which 94.2% were low-grade (n = 146). Importantly, no significant difference between treatment arms was detected in any system-organ class of AE including cardiac AE. Five (17%) of 29 patients who received prolonged trastuzumab maintenance therapy had no sign of cumulative toxicity after an average (range) of 5.1 (4.2-6.3) years. CONCLUSIONS: Trastuzumab appears to be safe and has a manageable toxicity profile both when used in combination with chemotherapy and when used for single agent maintenance in patients with HER2/neu positive USC. This safety profile is reassuring given the proven efficacy of trastuzumab in advanced/recurrent HER2/neu positive USC.

DHES0815A, a novel antibody-drug conjugate targeting HER2/neu, is highly active against uterine serous carcinomas in vitro and in vivo.

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.