Macrophage Checkpoint Nanoimmunotherapy Has the Potential to Reduce Malignant Progression in Bioengineered In Vitro Models of Ovarian Cancer.

Most ovarian carcinoma (OvCa) patients present with advanced disease at the time of diagnosis. Malignant, metastatic OvCa is invasive and has poor prognosis, exposing the need for improved therapeutic targeting. High CD47 (OvCa) and SIRPα (macrophage) expression has been linked to decreased survival, making this interaction a significant target for therapeutic discovery. Even so, previous attempts have fallen short, limited by CD47 antibody specificity and efficacy. Macrophages are an important component of the OvCa tumor microenvironment and are manipulated to aid in cancer progression via CD47-SIRPα signaling. Thus, we have leveraged lipid-based nanoparticles (LNPs) to design a therapy uniquely situated to home to phagocytic macrophages expressing the SIRPα protein in metastatic OvCa. CD47-SIRPα presence was evaluated in patient histological sections using immunohistochemistry. 3D tumor spheroids generated on a hanging drop array with OVCAR3 high-grade serous OvCa and THP-1-derived macrophages created a representative model of cellular interactions involved in metastatic OvCa. Microfluidic techniques were employed to generate LNPs encapsulating SIRPα siRNA (siSIRPα) to affect the CD47-SIRPα signaling between the OvCa and macrophages. siSIRPα LNPs were characterized for optimal size, charge, and encapsulation efficiency. Uptake of the siSIRPα LNPs by macrophages was assessed by Incucyte. Following 48 h of 25 nM siSIRPα treatment, OvCa/macrophage heterospheroids were evaluated for SIRPα knockdown, platinum chemoresistance, and invasiveness. OvCa patient tumors and in vitro heterospheroids expressed CD47 and SIRPα. Macrophages in OvCa spheroids increased carboplatin resistance and invasion, indicating a more malignant phenotype. We observed successful LNP uptake by macrophages causing significant reduction in SIRPα gene and protein expressions and subsequent reversal of pro-tumoral alternative activation. Disrupting CD47-SIRPα interactions resulted in sensitizing OvCa/macrophage heterospheroids to platinum chemotherapy and reversal of cellular invasion outside of heterospheroids. Ultimately, our results strongly indicate the potential of using LNP-based nanoimmunotherapy to reduce malignant progression of ovarian cancer.

The Proteolytic Landscape of Ovarian Cancer: Applications in Nanomedicine.

Ovarian cancer (OvCa) is one of the leading causes of mortality globally with an overall 5-year survival of 47%. The predominant subtype of OvCa is epithelial carcinoma, which can be highly aggressive. This review launches with a summary of the clinical features of OvCa, including staging and current techniques for diagnosis and therapy. Further, the important role of proteases in OvCa progression and dissemination is described. Proteases contribute to tumor angiogenesis, remodeling of extracellular matrix, migration and invasion, major processes in OvCa pathology. Multiple proteases, such as metalloproteinases, trypsin, cathepsin and others, are overexpressed in the tumor tissue. Presence of these catabolic enzymes in OvCa tissue can be exploited for improving early diagnosis and therapeutic options in advanced cases. Nanomedicine, being on the interface of molecular and cellular scales, can be designed to be activated by proteases in the OvCa microenvironment. Various types of protease-enabled nanomedicines are described and the studies that focus on their diagnostic, therapeutic and theranostic potential are reviewed.

Laparoscopic Allograft Spacer Placement to Minimize Bowel Dose During Re-irradiation with Interstitial Brachytherapy.

In primary or re-irradiation of gynecologic malignancies, achieving optimal dosimetry with adjacent normal tissue becomes challenging. Surgical spacers are tissue-equivalent materials placed within the patient to protect organs at risk from long-term radiation effects and are commonly used in prostate cancer. We report the use of an allograft mesh to protect adhesed bowel from high-dose radiation for definitive treatment of recurrent endometrial cancer. An 88-year-old female was diagnosed with International Federation of Gynecology and Obstetrics (FIGO) stage II endometrial cancer after she developed urinary frequency, hesitancy, and hematuria. She underwent neoadjuvant chemoradiation, followed by laparoscopic hysterectomy with bilateral salpingo-oophorectomy and adjuvant vaginal cuff brachytherapy. She developed 1.8 cm bilateral vaginal cuff recurrence and was dispositioned for interstitial brachytherapy. An allograft mesh spacer was placed laparoscopically before repeat, high dose rate brachytherapy to protect nearby structures. Dose-escalation was achieved without compromising normal tissue constraints. The patient tolerated the procedure without evidence of long-term toxicity at one year. Multidisciplinary discussion may help identify patients who would benefit from spacer placement before select dose-escalated radiation therapy. Laparoscopic allograft mesh is one of many types of surgical spacers available for such patients.

Somatic mosaicism of an intragenic FANCB duplication in both fibroblast and peripheral blood cells observed in a Fanconi anemia patient leads to milder phenotype.

BACKGROUND: Fanconi anemia (FA) is a rare disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer. Patients harboring X-linked FANCB pathogenic variants usually present with severe congenital malformations resembling VACTERL syndrome with hydrocephalus. METHODS: We employed the diepoxybutane (DEB) test for FA diagnosis, arrayCGH for detection of duplication, targeted capture and next-gen sequencing for defining the duplication breakpoint, PacBio sequencing of full-length FANCB aberrant transcript, FANCD2 ubiquitination and foci formation assays for the evaluation of FANCB protein function by viral transduction of FANCB-null cells with lentiviral FANCB WT and mutant expression constructs, and droplet digital PCR for quantitation of the duplication in the genomic DNA and cDNA. RESULTS: We describe here an FA-B patient with a mild phenotype. The DEB diagnostic test for FA revealed somatic mosaicism. We identified a 9154 bp intragenic duplication in FANCB, covering the first coding exon 3 and the flanking regions. A four bp homology (GTAG) present at both ends of the breakpoint is consistent with microhomology-mediated duplication mechanism. The duplicated allele gives rise to an aberrant transcript containing exon 3 duplication, predicted to introduce a stop codon in FANCB protein (p.A319*). Duplication levels in the peripheral blood DNA declined from 93% to 7.9% in the span of eleven years. Moreover, the patient fibroblasts have shown 8% of wild-type (WT) allele and his carrier mother showed higher than expected levels of WT allele (79% vs. 50%) in peripheral blood, suggesting that the duplication was highly unstable. CONCLUSION: Unlike sequence point variants, intragenic duplications are difficult to precisely define, accurately quantify, and may be very unstable, challenging the proper diagnosis. The reversion of genomic duplication to the WT allele results in somatic mosaicism and may explain the relatively milder phenotype displayed by the FA-B patient described here.

A comprehensive approach to identification of pathogenic FANCA variants in Fanconi anemia patients and their families.

Fanconi anemia (FA) is a rare recessive DNA repair deficiency resulting from mutations in one of at least 22 genes. Two-thirds of FA families harbor mutations in FANCA. To genotype patients in the International Fanconi Anemia Registry (IFAR) we employed multiple methodologies, screening 216 families for FANCA mutations. We describe identification of 57 large deletions and 261 sequence variants, in 159 families. All but seven families harbored distinct combinations of two mutations demonstrating high heterogeneity. Pathogenicity of the 18 novel missense variants was analyzed functionally by determining the ability of the mutant cDNA to improve the survival of a FANCA-null cell line when treated with MMC. Overexpressed pathogenic missense variants were found to reside in the cytoplasm, and nonpathogenic in the nucleus. RNA analysis demonstrated that two variants (c.522G > C and c.1565A > G), predicted to encode missense variants, which were determined to be nonpathogenic by a functional assay, caused skipping of exons 5 and 16, respectively, and are most likely pathogenic. We report 48 novel FANCA sequence variants. Defining both variants in a large patient cohort is a major step toward cataloging all FANCA variants, and permitting studies of genotype-phenotype correlations.

Assessing the spectrum of germline variation in Fanconi anemia genes among patients with head and neck carcinoma before age 50.

BACKGROUND: Patients with Fanconi anemia (FA) have an increased risk for head and neck squamous cell carcinoma (HNSCC). The authors sought to determine the prevalence of undiagnosed FA and FA carriers among patients with HNSCC as well as an age cutoff for FA genetic screening. METHODS: Germline DNA samples from 417 patients with HNSCC aged <50 years were screened for sequence variants by targeted next-generation sequencing of the entire length of 16 FA genes. RESULTS: The sequence revealed 194 FA gene variants in 185 patients (44%). The variant spectrum was comprised of 183 nonsynonymous point mutations, 9 indels, 1 large deletion, and 1 synonymous variant that was predicted to effect splicing. One hundred eight patients (26%) had at least 1 rare variant that was predicted to be damaging, and 57 (14%) had at least 1 rare variant that was predicted to be damaging and had been previously reported. Fifteen patients carried 2 rare variants or an X-linked variant in an FA gene. Overall, an age cutoff for FA screening was not identified among young patients with HNSCC, because there were no significant differences in mutation rates when patients were stratified by age, tumor site, ethnicity, smoking status, or human papillomavirus status. However, an increased burden, or mutation load, of FA gene variants was observed in carriers of the genes FA complementation group D2 (FANCD2), FANCE, and FANCL in the HNSCC patient cohort relative to the 1000 Genomes population. CONCLUSIONS: FA germline functional variants offer a novel area of study in HNSCC tumorigenesis. FANCE and FANCL, which are components of the core complex, are known to be responsible for the recruitment and ubiquitination, respectively, of FANCD2, a critical step in the FA DNA repair pathway. In the current cohort, the increased mutation load of FANCD2, FANCE, and FANCL variants among younger patients with HNSCC indicates the importance of the FA pathway in HNSCC. Cancer 2017;123:3943-54. © 2017 American Cancer Society.

Paternal or Maternal Uniparental Disomy of Chromosome 16 Resulting in Homozygosity of a Mutant Allele Causes Fanconi Anemia.

Fanconi anemia (FA) is a rare inherited disorder caused by pathogenic variants in one of 19 FANC genes. FA patients display congenital abnormalities, and develop bone marrow failure, and cancer susceptibility. We identified homozygous mutations in four FA patients and, in each case, only one parent carried the obligate mutant allele. FANCA and FANCP/SLX4 genes, both located on chromosome 16, were the affected recessive FA genes in three and one family respectively. Genotyping with short tandem repeat markers and SNP arrays revealed uniparental disomy (UPD) of the entire mutation-carrying chromosome 16 in all four patients. One FANCA patient had paternal UPD, whereas FA in the other three patients resulted from maternal UPD. These are the first reported cases of UPD as a cause of FA. UPD indicates a reduced risk of having another child with FA in the family and has implications in prenatal diagnosis.

Non-16/18 high-risk HPV infection predicts disease persistence and progression in women with an initial interpretation of LSIL.

BACKGROUND: The current management strategy for women with low-grade squamous intraepithelial lesions (LSILs) is inefficient and expensive because of the inability to identify patients at high risk for disease progression. The current study was designed to determine the genotypic patterns of human papillomavirus (HPV) associated with the persistence and progression of cervical lesions in women with an initial interpretation of LSIL. METHODS: One hundred sixty-seven women with LSIL on Papanicolaou tests collected between December 1, 2009 and March 30, 2011 were studied. HPV DNA was extracted from residual SurePath specimens, genotypes were determined with a DNA microarray containing 40 HPV genotype probes, and microarray data were confirmed by sequencing. Follow-up Papanicolaou tests and/or biopsies were performed within a 20- to 46-month period after the initial diagnosis. RESULTS: Ninety-seven of the 167 cases with follow-up results were included in the study. Compared with the women with a regressed cervical lesion, those with a persistent cervical lesion (PCL) were significantly more commonly infected with high-risk human papillomavirus (HR-HPV) genotypes (P < .01) and particularly with non-16/18 HR-HPV genotypes (P < .05). The PCL group also had a significantly higher average number of HR-HPV genotypes and non-16/18 HPV genotypes per specimen (P < .01). Infection with HPV-16/18 genotypes was not significantly associated with the persistence or progression of cervical lesions. CONCLUSIONS: Infection with non-16/18 HR-HPV genotypes but not with HPV-16/18 genotypes was a strong predictor of the persistence and progression of cervical disease upon follow-up. Genotyping solely for HPV-16/18 would miss the majority of patients with LSIL who progress to high-grade squamous intraepithelial lesions. Pooled HR-HPV tests provide a better predictive value than HPV-16/18 genotyping alone in guiding the clinical management of patients with LSIL.

Comprehensive analysis of pathogenic deletion variants in Fanconi anemia genes.

Fanconi anemia (FA) is a rare recessive disease resulting from mutations in one of at least 16 different genes. Mutation types and phenotypic manifestations of FA are highly heterogeneous and influence the clinical management of the disease. We analyzed 202 FA families for large deletions, using high-resolution comparative genome hybridization arrays, single-nucleotide polymorphism arrays, and DNA sequencing. We found pathogenic deletions in 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. We find 35% of FA families carry large deletions, accounting for 18% of all FA pathogenic variants. Cloning and sequencing across the deletion breakpoints revealed that 52 FANCA deletion ends, and one FANCC deletion end extended beyond the gene boundaries, potentially affecting neighboring genes with phenotypic consequences. Seventy-five percent of the FANCA deletions are Alu-Alu mediated, predominantly by AluY elements, and appear to be caused by nonallelic homologous recombination. Individual Alu hotspots were identified. Defining the haplotypes of four FANCA deletions shared by multiple families revealed that three share a common ancestry. Knowing the exact molecular changes that lead to the disease may be critical for a better understanding of the FA phenotype, and to gain insight into the mechanisms driving these pathogenic deletion variants.

Massively parallel sequencing, aCGH, and RNA-Seq technologies provide a comprehensive molecular diagnosis of Fanconi anemia.

Current methods for detecting mutations in Fanconi anemia (FA)-suspected patients are inefficient and often miss mutations. We have applied recent advances in DNA sequencing and genomic capture to the diagnosis of FA. Specifically, we used custom molecular inversion probes or TruSeq-enrichment oligos to capture and sequence FA and related genes, including introns, from 27 samples from the International Fanconi Anemia Registry at The Rockefeller University. DNA sequencing was complemented with custom array comparative genomic hybridization (aCGH) and RNA sequencing (RNA-seq) analysis. aCGH identified deletions/duplications in 4 different FA genes. RNA-seq analysis revealed lack of allele specific expression associated with a deletion and splicing defects caused by missense, synonymous, and deep-in-intron variants. The combination of TruSeq-targeted capture, aCGH, and RNA-seq enabled us to identify the complementation group and biallelic germline mutations in all 27 families: FANCA (7), FANCB (3), FANCC (3), FANCD1 (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (2), and FANCL (3). FANCC mutations are often the cause of FA in patients of Ashkenazi Jewish (AJ) ancestry, and we identified 2 novel FANCC mutations in 2 patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of FA.

Incidental medical information in whole-exome sequencing.

Genomic technologies, such as whole-exome sequencing, are a powerful tool in genetic research. Such testing yields a great deal of incidental medical information, or medical information not related to the primary research target. We describe the management of incidental medical information derived from whole-exome sequencing in the research context. We performed whole-exome sequencing on a monozygotic twin pair in which only 1 child was affected with congenital anomalies and applied an institutional review board-approved algorithm to determine what genetic information would be returned. Whole-exome sequencing identified 79525 genetic variants in the twins. Here, we focus on novel variants. After filtering artifacts and excluding known single nucleotide polymorphisms and variants not predicted to be pathogenic, the twins had 32 novel variants in 32 genes that were felt to be likely to be associated with human disease. Eighteen of these novel variants were associated with recessive disease and 18 were associated with dominantly manifesting conditions (variants in some genes were potentially associated with both recessive and dominant conditions), but only 1 variant ultimately met our institutional review board-approved criteria for return of information to the research participants.

Multidrug resistance-linked gene signature predicts overall survival of patients with primary ovarian serous carcinoma.

PURPOSE: This study assesses the ability of multidrug resistance (MDR)-associated gene expression patterns to predict survival in patients with newly diagnosed carcinoma of the ovary. The scope of this research differs substantially from that of previous reports, as a very large set of genes was evaluated whose expression has been shown to affect response to chemotherapy. EXPERIMENTAL DESIGN: We applied a customized TaqMan low density array, a highly sensitive and specific assay, to study the expression profiles of 380 MDR-linked genes in 80 tumor specimens collected at initial surgery to debulk primary serous carcinoma. The RNA expression profiles of these drug resistance genes were correlated with clinical outcomes. RESULTS: Leave-one-out cross-validation was used to estimate the ability of MDR gene expression to predict survival. Although gene expression alone does not predict overall survival (OS; P = 0.06), four covariates (age, stage, CA125 level, and surgical debulking) do (P = 0.03). When gene expression was added to the covariates, we found an 11-gene signature that provides a major improvement in OS prediction (log-rank statistic P < 0.003). The predictive power of this 11-gene signature was confirmed by dividing high- and low-risk patient groups, as defined by their clinical covariates, into four specific risk groups on the basis of expression levels. CONCLUSION: This study reveals an 11-gene signature that allows a more precise prognosis for patients with serous cancer of the ovary treated with carboplatin- and paclitaxel-based therapy. These 11 new targets offer opportunities for new therapies to improve clinical outcome in ovarian cancer.

Phase 1-2 study of docetaxel plus aflibercept in patients with recurrent ovarian, primary peritoneal, or fallopian tube cancer.

BACKGROUND: Biologically targeted therapies have been postulated as a viable strategy to improve outcomes for women with ovarian cancer. We assessed the safety, tolerance, pharmacokinetics, relevant circulating and image-derived biomarkers, and clinical activity of combination aflibercept and docetaxel in this population. METHODS: For the phase 1 (pharmacokinetic) study, eligible patients had measurable, recurrent or persistent epithelial ovarian, primary peritoneal, or fallopian tube carcinoma with a maximum of two prior chemotherapy regimens. Aflibercept was administered intravenously over three dose levels (2, 4, or 6 mg/kg; one dose every 21 days) to identify the maximum tolerated dose for the phase 2 study. Pharmacokinetics were assessed and dynamic imaging was done during a lead-in phase with single-agent aflibercept (cycle 0) and during combination therapy with intravenous docetaxel (75 mg/m(2)). Eligibility for the phase 2 study was the same as for phase 1. Patients were enrolled in a two-stage design and given aflibercept 6 mg/kg intravenously and docetaxel 75 mg/m(2) intravenously, every 3 weeks. The primary endpoint was objective response rate (ORR) as assessed by Response Evaluation Criteria in Solid Tumors version 1.0. The trial has completed enrolment and all patients are now off study. The trial is registered at ClinicalTrials.gov, number NCT00436501. FINDINGS: From the phase 1 study, the recommended phase 2 doses of aflibercept and docetaxel were found to be 6 mg/kg and 75 mg/m(2), respectively. Log-linear pharmacokinetics (for unbound aflibercept) were observed for the three dose levels. No dose-limiting toxicities were noted. 46 evaluable patients were enrolled in the phase 2 trial; 33 were platinum resistant (15 refractory) and 13 were platinum sensitive. The confirmed ORR was 54% (25 of 46; 11 patients had a complete response and 14 had a partial response). Grade 3-4 toxicities observed in more than two patients (5%) were: neutropenia in 37 patients (80%); leucopenia in 25 patients (54%); fatigue in 23 patients (50%); dyspnoea in ten patients (22%); and stomatitis in three patients (7%). Adverse events specifically associated with aflibercept were grade 1-2 hypertension in five patients (11%), and grade 2 proteinuria in one patient (2%). INTERPRETATION: Combination aflibercept plus docetaxel can be safely administered at the dose and schedule reported here, and is associated with substantial antitumour activity. These findings suggest that further clinical development of this combination in ovarian cancer is warranted. FUNDING: US National Cancer Institute, US Department of Defense, Sanofi-Aventis, Gynecologic Cancer Foundation, Marcus Foundation, and the Commonwealth Foundation.

De novo deletion of chromosome 20q13.33 in a patient with tracheo-esophageal fistula, cardiac defects and genitourinary anomalies implicates GTPBP5 as a candidate gene.

BACKGROUND: Tracheo-esophageal fistula (TEF) with/or without esophageal atresia (EA) is a common congenital malformation that is often accompanied by other anomalies. The causes of this condition are thought to be heterogeneous but are overall not well understood. CASE REPORT: We identified a patient with a TEF/EA, as well as cardiac and genitourinary anomalies, who was found to have a 0.7 Mb de novo deletion of chromosome 20q13.33. One gene within the deleted interval, GTPBP5, is of particular interest as a candidate gene. CONCLUSIONS: GTPBP5 bears further study as a cause of TEF/EA accompanied by other malformations.

Analysis of FOXF1 and the FOX gene cluster in patients with VACTERL association.

VACTERL association, a relatively common condition with an incidence of approximately 1 in 20,000 -35,000 births, is a non-random association of birth defects that includes vertebral defects (V), anal atresia (A), cardiac defects (C), tracheo-esophageal fistula (TE), renal anomalies (R) and limb malformations (L). Although the etiology is unknown in the majority of patients, there is evidence that it is causally heterogeneous. Several studies have shown evidence for inheritance in VACTERL, implying a role for genetic loci. Recently, patients with component features of VACTERL and a lethal developmental pulmonary disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), were found to harbor deletions or mutations affecting FOXF1 and the FOX gene cluster on chromosome 16q24. We investigated this gene through direct sequencing and high-density SNP microarray in 12 patients with VACTERL association but without ACD/MPV. Our mutational analysis of FOXF1 showed normal sequences and no genomic imbalances affecting the FOX gene cluster on chromosome 16q24 in the studied patients. Possible explanations for these results include the etiologic and clinical heterogeneity of VACTERL association, the possibility that mutations affecting this gene may occur only in more severely affected individuals, and insufficient study sample size.

Distinctive DNA methylation patterns of cell-free plasma DNA in women with malignant ovarian tumors.

OBJECTIVE: Epithelial ovarian carcinoma (OvCa) is rarely detected early, and it is also difficult to determine whether an adnexal mass is benign or malignant. Previously, we noted differences in methylation patterns of cell-free plasma DNA (cfpDNA) in women without disease compared to patients with OvCa. In this work, we investigated whether methylation patterns of cfpDNA can differentiate between benign and malignant tumors. METHODS: Methylation patterns in cfpDNA were determined in three cohorts (30 samples each) using a microarray-based assay (MethDet 56). Principal component analysis, supervised clustering, linear discrimination analysis, and 25 rounds of 5-fold cross-validation were used to determine informative genes and assess the sensitivity and specificity of differentiating between OvCa vs. healthy control (HC), benign ovarian disease (mostly serous cystadenoma, BOD) vs. HC, and OvCa vs. BOD samples. RESULTS: Differential methylation of three promoters (RASSF1A, CALCA, and EP300) differentiated between OvCa vs. HC with a sensitivity of 90.0% and a specificity of 86.7%. Three different promoters (BRCA1, CALCA, and CDKN1C) were informative for differentiating between BOD vs. HC, with a sensitivity of 90.0% and a specificity of 76.7%. Finally, two promoters (RASSF1A and PGR-PROX) were informative for differentiating between OvCa vs. BOD, with a sensitivity of 80.0% and a specificity of 73.3%. CONCLUSIONS: This proof-of-principle data show that differential methylation of promoters in cfpDNA may be a useful biomarker to differentiate between certain benign and malignant ovarian tumors.

Clinical and biological impact of EphA2 overexpression and angiogenesis in endometrial cancer.

OBJECTIVE: EphA2 overexpression predicts poor prognosis in endometrial cancer. To explore mechanisms for this association and assess its potential as therapeutic target, the relationship of EphA2 expression to markers of angiogenesis was examined using patient samples and an orthotopic mouse model of uterine cancer. EXPERIMENTAL DESIGN: Expression of EphA2, estrogen receptor (ER), progesterone receptor (PR), Ki-67, vascular endothelial growth factor (VEGF) and microvessel density (MVD) was evaluated using immunohistochemistry in 85 endometrioid endometrial adenocarcinomas (EEC) by two independent investigators. Results were correlated with clinicopathological characteristics. The effect of EphA2- agonist monoclonal antibody EA5, alone or in combination with docetaxel was studied in vitro and in vivo. Samples were analyzed for markers of angiogenesis, proliferation and apoptosis. RESULTS: Of 85 EEC samples, EphA2 was overexpressed in 47% of tumors and was significantly associated with high VEGF expression (p=0.001) and high MVD counts (p=0.02). High EphA2 expression, high VEGF expression and high MVD counts were significantly associated with shorter disease-specific survival. EA5 led to decrease in EphA2 expression and phosphorylation in vitro. In the murine model, while EA5 (33-88%) and docetaxel (23-55%) individually led to tumor inhibition over controls, combination therapy had the greatest efficacy (78-92%, p.