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Background: Epithelial ovarian cancer (EOC) remains a significant challenge in gynecologic oncology, particularly in the context of platinum-resistant disease. Mirvetuximab soravtansine (MIRV), was approved after trials revealed favorable response and survival outcomes. MIRV targets folate receptor alpha (FRα), a cell-surface receptor that is overexpressed in EOC and has been associated with aggressive disease phenotypes., Methods: This retrospective study analyzed 425 patient samples tested for FRα using the VENTANA® FOLR1 RxDx immunohistochemical assay. The patient cohort included cases with high grade serous carcinoma predominantly, tested across various anatomical sites. Statistical analysis examined the correlation between FRα positivity and clinical parameters such as tumor site and histology., Results: FRα was highly expressed in 36.3 % of the cases, with a significant association between FRα positivity and high grade serous ovarian histology. Tumor samples from the ovary, fallopian tube, adnexa, and dominant pelvic masses showed higher FRα positivity compared to metastatic sites (positive rates of 44.4 % vs 32.5 %, p = 0.02), highlighting the potential influence of tumor origin on expression of FRα. Time between sample collection and testing did not impact FRα expression, with sample testing spread over a median of 19.5 months post-collection. Eight patients had more than one specimen tested, of which 3 (37.5 %) had discordant results when a subsequent specimen was tested., Conclusion: Our results highlight a need for standardized protocols for FRα testing to ensure accurate biomarker evaluation across varied clinical settings. The heterogeneity in FRα expression, influenced by tumor histology and anatomical origin, warrant further investigation to optimize therapeutic outcomes., Prior Presentation: Preliminary findings from this study were previously presented in poster format at the Society of Gynecologic Oncology 2024 Annual Metting. We confirm that the submission complies with the journal requirements., Competing Interests: Declaration of competing interest This manuscript was funded by Labcorp. Rebecca A. Previs is a current employee of Labcorp, owns Labcorp stock, and receives travel support from Labcorp. Kyle C. Strickland is a current employee of Labcorp, owns Labcorp stock, and consulting fees from Foundation Medicine, Almac Pharmaceuticals, OncoQuest Pharmaceuticals, and CanariaBio. Zachary Wallen is a current employee of Labcorp, owns Labcorp stock, and receives travel support from Labcorp. Heidi Ko is a current employee of Labcorp and owns Labcorp stock. Michelle Green is a current employee of Labcorp. Maureen Cooper was a former employee of Labcorp and owns Labcorp stock. Elizabeth Lyon is a current employee of Labcorp and owns Labcorp stock. Michael Biorn is a current employee of Labcorp and owns Labcorp stock. Jennifer Armetta is a current employee of Labcorp and owns Labcorp stock. Rennie Quarles is a current employee of Labcorp and owns Labcorp stock. Catherine H Watson has no disclosures. Kari Ring received speakers' bureau honoraria from Ethicon. Jonathan L. Klein is a current employee of Labcorp and owns Labcorp stock. Brian Caveney is a board member of the Personalized Medicine Coalition, a current employee of Labcorp and owns Labcorp stock. Eric A. Severson is a current employee of Labcorp and owns Labcorp stock. Shakti Ramkissoon is a current employee of Labcorp and owns Labcorp stock., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Mirvetuximab soravtansine-gynx (MIRV) is a novel antibody-drug conjugate targeting folate receptor alpha (FRα), which is overexpressed in epithelial ovarian cancer (EOC), with limited expression on normal tissues. This integrated safety summary sought to characterize the safety profile of MIRV monotherapy in participants with FRα-expressing recurrent EOC., Methods: Safety data were retrospectively analyzed from 4 clinical studies (phase 1 trial [NCT01609556], phase 3 FORWARD I [NCT02631876], phase 2 SORAYA [NCT04296890], phase 3 MIRASOL [NCT04209855]) that evaluated participants with FRα-expressing recurrent EOC who received ≥1 dose of MIRV 6 mg/kg adjusted ideal body weight every 3 weeks., Results: In this analysis of 682 participants, 94 % had platinum-resistant ovarian cancer (PROC). Blurred vision (43 %), nausea (41 %), diarrhea (39 %), and fatigue (35 %) were the most common treatment-emergent adverse events (TEAEs) and were primarily grade 1-2 in severity. Grade ≥ 3 TEAEs occurred in 48 % of participants, with the most common being keratopathy and blurred vision (5 % each). Most TEAEs were managed with supportive care and dose modifications, and only 12 % of participants experienced a TEAE leading to discontinuation (1 % due to ocular events). No corneal ulcerations or perforations have been reported. Median time to onset of blurred vision and keratopathy was 5.9 and 6.7 weeks, respectively. Most blurred vision events and keratopathy events resolved completely (71 % and 66 %, respectively) or partially (15 % and 14 %, respectively)., Conclusions: As demonstrated among 682 participants, the safety profile of MIRV is well tolerated and consists primarily of low-grade gastrointestinal, fatigue, headache, peripheral neuropathy, and resolvable ocular adverse events., Competing Interests: Declaration of competing interest KNM reports receiving consulting fees from and participating on a data safety monitoring board or advisory board for Aadi Bioscience, Aravive, Inc., Alkermes, AstraZeneca, Blueprint Medicines Corporation, Clovis Oncology, Inc., Caris Life Sciences, Inc., Eisai Co, Ltd., GSK, Genentech, Inc./Roche, Jiangsu Hengrui Pharmaceuticals Co, Ltd., ImmunoGen, Inc., InxMed, I-Mab, Iovance Biotherapeutics, Inc., Eli Lilly and Company, Mereo BioPharma Group PLC, Merck, Myriad Genetics, Inc., Mersana Therapeutics, Inc., Novartis, Novocure GmbH, OncXerna Therapeutics, Inc., Onconova Therapeutics, Inc., Panavance Therapeutics Inc., Tarveda Therapeutics, Inc., VBL Therapeutics, and Verastem, Inc.; research funding from PTC Therapeutics, Eli Lilly and Company, Clovis Oncology, Inc., Genentech, Inc., GSK, and Verastem, Inc.; royalties or licenses from UpToDate; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from AstraZeneca, Great Debates & Updates, GSK, ImmunoGen, Inc., Medscape, PRIME, and RTP; and support for attending meetings and/or travel from AstraZeneca; and associate directorship with GOG Partners and sits on director's board for The GOG Foundation, Inc. DL reports receiving research funding from AstraZeneca, Clovis Oncology, Inc., Corcept Therapeutics Incorporated, Genmab A/S, GSK, ImmunoGen, Inc., Incyte Corporation, MSD, Novartis, PharmaMar, Roche, Alkermes, pharmaand GmbH, and Seagen Inc.; consulting fees from AstraZeneca, Clovis Oncology, Inc., Genmab A/S, GSK, ImmunoGen, Inc., MSD, Novartis, PharmaMar, and Seagen Inc.; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from AstraZeneca, Clovis Oncology, Inc., Daiichi Sankyo, Genmab A/S, GSK, ImmunoGen, Inc., MSD, Novocure GmbH, and Seagen Inc.; support for attending meetings and/or travel from AstraZeneca, Menarini, GSK, and MSD; and participation on a data safety monitoring board or advisory board for AstraZeneca, Clovis Oncology, Inc., Corcept Therapeutics Incorporated, Genmab A/S, GSK, ImmunoGen, Inc., MSD, Oncoinvent, Novocure GmbH, Seagen Inc., Daiichi Sankyo, and Sutro Biopharma, Inc. and is member and president elect of MITO, board of directors of GCIG, and member of ENGOT. A Oa reports receiving support for attending meetings and/or travel from Roche, AstraZeneca, and PharmaMar and participation on a data safety monitoring board or advisory board for Agenus Inc., AstraZeneca, Clovis Oncology, Inc., Corcept Therapeutics Incorporated, Deciphera Pharmaceuticals, Inc., Daiichi Sankyo, Debiopharm, Eisai Europe Ltd., Exelixis, Inc., F. Hoffmann-La Roche, Genmab A/S, GlaxoSmithKline, ImmunoGen, Inc., ITeos Therapeutics, Merck Sharp & Dohme, Mersana Therapeutics, Inc., Myriad Genetics, Inc., Novocure GmbH, OncXerna Therapeutics, Inc., PharmaMar, Regeneron Pharmaceuticals, Inc., Shattuck Labs, Inc., Seagen Inc./Pfizer Inc., Sutro Biopharma, Inc., and Zentalis Pharmaceuticals, Inc. A Oz reports receiving institutional grants from AstraZeneca and Amgen Inc., consulting fees for participation on the Oncology Tumor Advisory Council with Bristol-Myers Squibb Company, participation on a data safety monitoring board or advisory board with AstraZeneca and GlaxoSmithKline (both uncompensated), participation on steering committees for AstraZeneca and GlaxoSmithKline (both uncompensated), participation as the principal investigator for investigator-initiated trials with AstraZeneca and GlaxoSmithKline, and participation on steering committees with AstraZeneca and GlaxoSmithKline (both uncompensated). NC reports receiving consulting fees from AstraZeneca, Clovis Oncology, Inc., Eisai Co, Ltd., GSK, ImmunoGen, Inc., Mersana Therapeutics, Inc., MSD Oncology, Pfizer Inc., Roche/Genentech, Inc., Pieris Pharmaceuticals, Inc., and Novocure GmbH; honoraria from AstraZeneca, Clovis Oncology, Inc., Eisai Co, Ltd., GSK, ImmunoGen, Inc., MSD Oncology, Novartis, and Roche/Genentech, Inc.; and participation on a data safety monitoring board or advisory board for AstraZeneca, Clovis Oncology, Inc., Eisai Co, Ltd., GSK, ImmunoGen, Inc., Mersana Therapeutics, Inc., MSD Oncology, Pfizer Inc., Roche/Genentech, Inc., and Novocure GmbH, and an immediate family member is an employee of Sarepta. TVG reports receiving consulting fees from AbbVie Inc., AstraZeneca, BioNTech SE, Cancer Communications and Consultancy Ltd., Eisai Co, Ltd., GSK, ImmunoGen, Inc., Incyte Corporation, Karyopharm Therapeutics Inc., MSD/Merck, OncXerna Therapeutics, Inc., Seagen Inc., Tubulis GmbH, and Zentalis Pharmaceuticals, Inc.; travel, accommodations, or expenses from AstraZeneca, PharmaMar, MSD/Merck, and ImmunoGen, Inc.; funding for academic research from AstraZeneca, Amgen Inc., and Roche; and chair of the Belgium and Luxembourg Gynaecological Oncology Group (unremunerated). All payments were institutional. DMO reports receiving medical writing support, support for clinical trials at his institution, and personal fees for consulting and advisory boards outside of the submitted work from ImmunoGen, Inc.; grants or contracts from AbbVie Inc., Advaxis Inc., Agenus Inc., Alkermes, Aravive, Inc., Arcus Biosciences, Inc., AstraZeneca, BeiGene USA Inc., Boston Biomedical, Inc., Bristol-Myers Squibb Company, Clovis Oncology, Inc., Deciphera Pharmaceuticals, Inc., Eisai Co, Ltd., EMD Serono Inc., Exelixis, Inc., Genentech, Inc., Genmab A/S, GlaxoSmithKline, GOG Foundation, Hoffmann-La Roche Ltd., Incyte Corporation, Iovance Biotherapeutics, Inc., Karyopharm Therapeutics Inc., Leap Therapeutics, Inc., Ludwig Institute for Cancer Research, Merck & Co, Inc., Merck Sharp & Dohme Corp, Mersana Therapeutics, Inc., National Cancer Institute, Novartis, Novocure GmbH, NRG Oncology, OncoC4, Inc., Pfizer Inc., Precision Therapeutics Inc., Prelude Therapeutics Incorporated, Regeneron Pharmaceuticals, Inc., RTOG Foundation, Rubius Therapeutics, Seattle Genetics, Inc. (Seagen Inc), Sutro Biopharma, Inc., SWOG, Tesaro Inc., and Verastem, Inc.; receiving consulting fees from AbbVie Inc., Adaptimmune, Agenus Inc., Arquer Diagnostics Ltd., Arcus Biosciences, Inc., AstraZeneca, Atossa Therapeutics, Inc., Boston Biomedical, Inc., Cardiff Oncology, Inc., Celcuity, Inc., Clovis Oncology, Inc., Corcept Therapeutics Incorporated, Duality Biologics Co Ltd., Eisai Co, Ltd., Elevar Therapeutics, Inc., Exelixis, Inc., Genentech, Inc., Genelux Corporation, GlaxoSmithKline, GOG Foundation, Hoffmann-La Roche Ltd., ImmunoGen, Inc., Imvax, Inc., InterVenn Biosciences, InxMed, Iovance Biotherapeutics, Inc., Janssen, Jazz Pharmaceuticals, Inc., Laekna Therapeutics, Leap Therapeutics, Inc., Luzsana Biotechnology, Inc., Merck & Co, Inc., Merck Sharp & Dohme Corp, Mersana Therapeutics, Inc., Myriad Genetics, Inc., Novartis, Novocure GmbH, OncoC4 Inc., Onconova Therapeutics, Inc., Regeneron Pharmaceuticals, Inc., RepImmune, R-Pharm US, LLC, Roche Diagnostics Corporation, Seattle Genetics, Inc. (Seagen Inc), Sorrento Therapeutics, Inc., Sutro Biopharma, Inc., Tarveda Therapeutics, Inc., Toray, Trillium Therapeutics, Umoja Biopharma, Inc., Verastem, Inc., VBL Therapeutics, Vincerx Pharma, Inc., Xencor, Inc., and Zentalis Pharmaceuticals, Inc.; participation on a data safety monitoring board for Watermark Research Partners; and a leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid from GOG Foundation Board. SB reports institutional funding for academic research form AstraZeneca and GSK; consulting fees from AstraZeneca, Epsilogen Ltd., GlaxoSmithKline, ImmunoGen, Inc., Merck Sharp & Dohme, Mersana Therapeutics, Inc., Novartis, OncXerna Therapeutics, Inc., Seagen Inc., Shattuck Labs, Inc., and Regeneron Pharmaceuticals, Inc.; honoraria for advisory boards from AstraZeneca, Epsilogen Ltd., GlaxoSmithKline, ImmunoGen, Inc., Mersana Therapeutics, Inc., MSD, Myriad Genetics, Inc., Novartis, OncXerna Therapeutics, Inc., Regeneron Pharmaceuticals, Inc., Seagen Inc., and Shattuck Labs, Inc.; and honoraria for lectures from Amgen Inc., Pfizer Inc., AstraZeneca, ImmunoGen, Inc., GSK, Clovis Oncology, Inc., Mersana Therapeutics, Inc., Novocure GmbH, Merck Sharp & Dohme, Roche, and Takeda Pharmaceutical Company Limited. CGM reports receiving honoraria from Janssen Oncology and travel, accommodations, or expenses from Ipsen Pharma, Pfizer Inc., and Bayer AG. PH reports receiving honoraria from Amgen Inc., AstraZeneca, Roche, Clovis Oncology, Inc., Stryker, MSD Oncology, Zai Lab Limited, SOTIO Biotech, Eisai Co, Ltd., GlaxoSmithKline, Exscientia plc, Mersana Therapeutics, Inc., ImmunoGen, Inc., Miltenyi Biotec, and Daiichi Sankyo; participation on an advisory board for AstraZeneca, Roche, MSD, GlaxoSmithKline, Clovis Oncology, Inc., ImmunoGen, Inc., Novartis, Eisai Co, Ltd., and Miltenyi Biotec; and institutional research funding from AstraZeneca, Roche, Genmab A/S, GlaxoSmithKline, ImmunoGen, Inc., Clovis Oncology, Inc., Seagen Inc., Novartis, DFG, the European Union, and DKH Holding Ltd. GEK reports receiving institutional grants or contracts from Eli Lilly and Company and Merck; consulting fees from GOG Foundation and ImmunoGen, Inc.; payment or honoraria for speakers bureaus from AstraZeneca, ImmunoGen, Inc., and Merck; payment for expert testimony from Foundation Medicine, Inc.; and participation on a data safety monitoring board or advisory board for Repare Therapeutics, Inc. and TORL BioTherapeutics LLC. PP reports consulting fees from AstraZeneca (institution) and MSD (personal); payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from PharmaMar (personal); payment for expert testimony from MSD (institution); support for attending meetings and/or travel from AstraZeneca, Eisai Co, Ltd., GSK, OSE Immunotherapeutics, and PharmaMar; and participation on a data safety monitoring board or advisory board for MSD and Roche. MM and YW report employment at ImmunoGen, Inc. at the time of analysis and reporting. RLC reports receiving consulting fees from AstraZeneca, GSK, ImmunoGen, Inc., Novocure GmbH, OncXerna Therapeutics, Inc., Onconova Therapeutics, Inc., Epsilogen Ltd., Pfizer Inc., Merck, Alkermes, Gradalis, Inc., Agenus Inc., Mersana Therapeutics, Inc., Karyopharm Therapeutics Inc., Deciphera Pharmaceuticals, Inc., Roche/Genentech, Inc., and Genelux Corporation; grants from Clovis Oncology, Inc., AbbVie Inc., AstraZeneca, ImmunoGen, Inc., Seagen Inc., and Merck; participation in independent data monitoring committees for Eisai Co, Ltd. and VBL Therapeutics; and board membership with the GOG Foundation. MB reports nothing to disclose. UAM reports participation on a data safety monitoring board for Alkermes and Symphogen A/S and participation on an advisory board for Allarity Therapeutics, Inc., NextCure, Inc., ProfoundBio, Inc., Agenus Inc., Eisai Co, Ltd., ImmunoGen, Inc., Novartis, Boehringer Ingelheim, Eli Lilly and Company, Tango Therapeutics, Inc., Ovarian Cancer Research Alliance, and GSK., (Copyright © 2024. Published by Elsevier Inc.)

Objective: Mirvetuximab soravtansine may be a potentially effective therapeutic option for ovarian low-grade serous carcinoma (LGSC), but the prevalence of folate receptor alpha (FRα) overexpression in this tumor type is unknown. We sought to characterize FRα expression in LGSC and its association with clinical and molecular features., Methods: FRα immunohistochemistry was performed on a tissue microarray comprised of 89 LGSCs and 42 ovarian serous borderline tumors (SBTs). Clinical tumor-normal panel-based sequencing was performed on 78 LGSCs. Associations between FRα-high status and clinicopathologic characteristics and survival outcomes were examined., Results: Of 89 LGSCs, 36 (40%) were FRα-high (≥75% of viable tumor cells exhibiting moderate-to-strong membranous expression). Of 9 patients with LGSC and samples from different timepoints, 4 (44%) had discordant results, with conversion from FRα-negative to FRα-high in 3 (33%) cases. There was no association between FRα-high status with age, race, or progression-free/overall survival. A MAPK pathway genetic alteration, most commonly involving KRAS (n = 23), was present in 45 (58%) LGSCs. Those lacking MAPK pathway alterations were more likely to be FRα-high compared to MAPK-altered LGSCs (61% vs 20%, p < 0.001). In SBTs, FRα-high expression was associated with high-risk (micropapillary) histology and/or subsequent LGSC recurrence compared to conventional SBTs without malignant recurrence (53% vs 9%, p = 0.008)., Conclusions: Future studies of FRα-directed therapy in patients with LGSC are warranted. Discordant FRα status at recurrence suggests potential benefit for retesting. A biomarker-driven approach to direct treatment selection in LGSC is recommended. As high FRα expression is more common amongst tumors lacking MAPK pathway genetic alterations, FRα testing to determine eligibility for mirvetuximab soravtansine therapy is particularly recommended for this subgroup., Competing Interests: Declaration of competing interest Y.L. Liu reports research funding from AstraZeneca, GSK, and Repare Therapeutics, outside the submitted work. A. Iasonos reports consulting fees from Mylan. D.S. Chi reports personal fees from Apyx Medical, Verthermia Inc., Biom ‘Up, and AstraZeneca, as well as recent or current stock/options ownership of Apyx Medical, Verthemia, Intuitive Surgical, Inc., TransEnterix, Inc., Doximity, Moderna, and BioNTech SE. C. Aghajanian reports grant support paid to the institution from Abbvie, Clovis, Genentech, and AstraZeneca; consulting fees from Eisai/Merck, Mersana Therapeutics, Roche/Genentech, Abbvie, AstraZeneca/Merck, and Repare Therapeutics; advisory board participation for Blueprint Medicine; and unpaid participation on the Board of Directors of the GOG Foundation and NRG Oncology. R.E. O'Cearbhaill reports personal fees from Tesaro/GSK, Regeneron, R-PHARM, Seattle Genetics, Fresenius Kabi, Gynecologic Oncology Foundation, Bayer, Curio, Miltenyi, 2seventybio and Immunogen, and other from Hitech Health, all outside the submitted work. She is a non-compensated steering committee member for the PRIMA, Moonstone (Tesaro/GSK), and DUO-O (AstraZeneca) studies and a non-compensated advisor for Carina Biotech. Her institute receives funding for clinical research from Bayer/Celgene/Juno, Tesaro/GSK, Merck, Ludwig Cancer Institute, Abbvie/StemCentrx, Regeneron, TCR2 Therapeutics, Atara Biotherapeutics, MarkerTherapeutics, Syndax Pharmaceuticals, Genmab/Seagen Therapeutics, Sellas Therapeutics, Genentech, Kite Pharma, Acrivon, Lyell Immunopharma, and Gynecologic Oncology Foundation. K. Long Roche reports travel support from Intuitive Surgical. R.N. Grisham reports honoraria from GSK, AstraZeneca, Natera, Springworks, Corcept, MJH, and PER, outside the submitted work. M.H. Chui reports consulting fees from Verastem Oncology, outside the submitted work. The other authors have no conflicts of interest to report., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Folate receptor alpha (FRα) has been reported to be expressed in up to 80% of triple-negative breast cancers (TNBC) with limited expression in normal tissues, making it a promising therapeutic target. Mirvetuximab soravtansine (mirvetuximab-s) is an antibody drug conjugate which has shown promise in the treatment of FRα-positive solid tumors in early phase clinical trials. Herein, are the results of the first prospective phase II trial evaluating mirvetuximab-s in metastatic TNBC. Patients with advanced, FRα-positive TNBC were enrolled on this study. Mirvetuximab-s was administered at a dose of 6.0 mg/kg every 3 weeks. 96 patients with advanced TNBC consented for screening. FRα staining was performed on tumor tissue obtained from 80 patients. The rate of FRα positivity by immunohistochemistry was 10.0% (8/80). Two patients were treated on study, with best overall responses of stable disease in one and progressive disease in the other. Adverse events were consistent with earlier studies. The study was terminated early due to the low rate of FRα positivity in the screened patient population and lack of disease response in the two patients treated. The observed rate of FRα positivity was considerably lower than previously reported and none of the patients had a partial or complete response. Treatment with mirvetuximab-s should only be further explored in TNBC if an alternate biomarker strategy is developed for patient selection on the basis of additional preclinical data.

Folate Receptor Alpha (FRA) is a membrane protein expressed on the apical surface of epithelial cells. Its expression in increased in certain tumors, where it can serve as a target for therapy. Triple Negative Breast Carcinoma (TNBC) are a heterogeneous group of tumors, with limited therapy options and poor prognosis. We aimed to study the expression of FRA in TNBC. Tissue microarrays were prepared from archived paraffin blocks of 300 TNBC resection specimens. Staining for FRA immunohistochemistry was carried out using the clone 26B3.F2. Clinical and pathologic details of the patients were obtained from the electronic medical records. Chi square test was performed for correlation of clinicopathological features with FRA expression. Kaplan Meir and Cox Regression analysis were carried out to study the Disease Free Survival (DFS) and Overall Survival (OS). FRA showed positivity in 43% (129/300) of TNBCs in our study. In univariate analysis, TNBC expressing FRA had a significantly better OS compared to FRA negative tumors (p - 0.035). Also, FRA positive tumors showed a trend towards longer DFS, though this was not statistically significant. In multivariate analysis however, FRA expression did not emerge as a significant factor. To conclude, TNBCs in our study showed FRA expression and though this did not emerge as an important prognostic factor, it can represent a therapeutic target for future clinical trials., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Folate receptor alpha (FRα) is a membrane-bound protein with a high affinity for folate, which is necessary for the biosynthesis of amino acids and nucleotide bases. It has been shown to be a potential prognostic and therapeutic target, primarily in lung and ovarian cancer, as well as in breast cancer. The aim of this study was to examine FRα expression in a cohort of patients with triple negative breast cancer (TNBC), in correlation with clinicopathological parameters and prognostic factors. By using polyclonal FRα antibody on archival paraffin blocks immunohistochemistry was performed. To evaluate the expression of FRα, H-score was used, which marks both the proportion of stained cells and the intensity of staining. Statistical analysis correlating FRα expression with clinicopathologic parameters and clinical outcome were performed. FRα was expressed in most of the patients (85%). Significant correlation of expression and histologic grade (Mann Whitney U test, P = 0,03) and type of tumor (P = 0,02), was found. It was noticed that with higher Ki-67 proliferation index values, H-score has lower values (r = -0,284, P = 0,006). Multivariant regression analysis (Cox regression, Stepwise method) showed H-score as a significant predictor for the risk of disease recurrence (OR = 1,005, P = 0,04). No correlation between FRα expression and overall survival (OS) and disease-free survival (DFS) was found. In conclusion, FRα is highly expressed in TNBC, and, given the correlation with clinicopathological parameters, subpopulation of patients could be identified that could be potential targets for new therapeutic perspectives in the treatment of this breast cancer subtype., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Meningiomas are well-encapsulated benign brain tumors and surgical resection is often curative. Nevertheless, this is not always possible due to the difficulty of identifying residual disease intraoperatively. We hypothesized that meningiomas overexpress folate receptor alpha (FRα), allowing intraoperative molecular imaging by targeting FRα with a near-infrared (NIR) dye., Objective: To determine FRα expression in both human and canine meningioma cohorts to prepare for future clinical studies. Present a case study of a meningioma resection with intraoperative NIR fluorescence imaging., Methods: Tissue samples of 27 human meningioma specimens and 7 canine meningioma specimens were immunohistochemically stained for FRα along with normal dura, skeletal muscle, and kidney tissue. We then enrolled a patient with a pituitary adenoma and tuberculum sella meningioma in a clinical trial in which the patient received an infusion of folate-linked, NIR fluorescent dye prior to surgery., Results: In the cohort of human meningiomas, 9 WHO grade I, 12 grade II, and 6 grade III tumors were identified. Eighty-nine percent of WHO grade I, 67% of grade II, and 50% of grade III tumors overexpressed FRα. In the 7 canine meningioma samples, 100% stained positively for FRα. Both human and canine normal dura from autopsy samples demonstrated no evidence of FRα overexpression. In the case study, the meningioma demonstrated a high NIR signal-to-background-ratio of 4.0 and demonstrated strong FRα immunohistochemistry staining., Conclusion: This study directly demonstrates FRα overexpression in both human and canine meningiomas. We also demonstrate superb intraoperative imaging of a meningioma using a FRα-targeting dye., (Copyright © 2018 by the Congress of Neurological Surgeons.)

Introduction: Treatment of advanced stage ovarian carcinoma is challenging, and despite surgical treatment and chemotherapy, the 5-year survival rate is estimated around 30%. Early recurrence and resistance to platinum-based chemotherapy are associated with poor prognosis and limited response to available second-line chemotherapy. The relative incidence of endocervical adenocarcinoma (EAC) compared with squamous cell carcinoma is increasing. Although the first-line treatment modality for early stage EAC is surgical resection, for locally advanced disease chemoradiation or neoadjuvant chemotherapy is used. Recently, folate along with its receptor alpha (FRA) has been studied as a potential target in gynecologic malignancy. The objective of this study was to elucidate FRA expression in chemotherapy resistant ovarian cancer and primary EAC., Methods: FRA expression was evaluated in tissue samples in an epithelial ovarian tumor microarray and 2 study groups: platinum resistant ovarian cancer and primary EAC. Staining intensity was analyzed with a semiquantitative staining algorithm., Results: FRA expression was positive in 32 of 40 (80%) ovarian tumors in the control group. In the platinum resistant ovarian cancer group, FRA was expressed in all 30 samples with moderate to strong staining. None of the EAC samples stained positive for FRA expression., Conclusions: FRA expression occurs frequently in epithelial ovarian cancer. Our data supports that FRA expressions are maintained after chemotherapy treatment. Folate targeted therapies may be most useful in patients with chemotherapy resistant disease based on high levels of FRA expression in these tumors. There is likely no benefit to folate therapy as an adjuvant treatment in EAC.

Human folate receptors (hFRα and hFRβ) are membrane proteins anchored to the cell surface by glycosylphosphatidylinositol. They play an important role in cell growth by taking up folate for de novo synthesis of purines and methylation of DNA, lipids, and proteins. Thus, controlling folate uptake through hFRs may lead to the development of anti-cancer drugs. Development of hFRs-targeting drug requires a large amount of hFRs. However, it is difficult to prepare active forms of hFRs from prokaryotic cells because of their high content of cysteine residues that form disulfide bonds. Here, we prepared active forms of hFRα and hFRβ from inclusion bodies of Escherichia coli. The crucial steps in our preparation were intensive washing of the inclusion bodies to remove impurities derived from E. coli and gradual dropping of solubilized hFRs into refolding buffers to correctly reform disulfide bonds. The binding activity of prepared hFRs to folate was confirmed by biolayer interferometry measurements. Finally, we successfully prepared the active form of 2.52 mg hFRα and 2.4 mg hFRβ from 10 g of E. coli cell bodies., (Copyright © 2018. Published by Elsevier Inc.)

OBJECTIVE Pituitary adenomas account for approximately 10% of intracranial tumors and have an estimated prevalence of 15%-20% in the general US population. Resection is the primary treatment for pituitary adenomas, and the transsphenoidal approach remains the most common. The greatest challenge with pituitary adenomas is that 20% of patients develop tumor recurrence. Current approaches to reduce recurrence, such as intraoperative MRI, are costly, associated with high false-positive rates, and not recommended. Pituitary adenomas are known to overexpress folate receptor alpha (FRα), and it was hypothesized that OTL38, a folate analog conjugated to a near-infrared (NIR) fluorescent dye, could provide real-time intraoperative visual contrast of the tumor versus the surrounding nonneoplastic tissues. The preliminary results of this novel clinical trial are presented. METHODS Nineteen adult patients who presented with pituitary adenoma were enrolled. Patients were infused with OTL38 2-4 hours prior to surgery. A 4-mm endoscope with both visible and NIR light capabilities was used to visualize the pituitary adenoma and its margins in real time during surgery. The signal-to-background ratio (SBR) was recorded for each tumor and surrounding tissues at various endoscope-to-sella distances. Immunohistochemical analysis was performed to assess the FRα expression levels in all specimens and classify patients as having either high or low FRα expression. RESULTS Data from 15 patients (4 with null cell adenomas, 1 clinically silent gonadotroph, 1 totally silent somatotroph, 5 with a corticotroph, 3 with somatotrophs, and 1 somatocorticotroph) were analyzed in this preliminary analysis. Four patients were excluded for technical considerations. Intraoperative NIR imaging delineated the main tumors in all 15 patients with an average SBR of 1.9 ± 0.70. The FRα expression level of the adenomas and endoscope-to-sella distance had statistically significant impacts on the fluorescent SBRs. Additional considerations included adenoma functional status and time from OTL38 injection. SBRs were 3.0 ± 0.29 for tumors with high FRα expression (n = 3) and 1.6 ± 0.43 for tumors with low FRα expression (n = 12; p < 0.05). In 3 patients with immunohistochemistry-confirmed FRα overexpression (2 patients with null cell adenoma and 1 patient with clinically silent gonadotroph), intraoperative NIR imaging demonstrated perfect classification of the tumor margins with 100% sensitivity and 100% specificity. In addition, for these 3 patients, intraoperative residual fluorescence predicted postoperative MRI results with perfect concordance. CONCLUSIONS Pituitary adenomas and their margins can be intraoperatively visualized with the preoperative injection of OTL38, a folate analog conjugated to NIR dye. Tumor-to-background contrast is most pronounced in adenomas that overexpress FRα. Intraoperative SBR at the appropriate endoscope-to-sella distance can predict adenoma FRα expression status in real time. This work suggests that for adenomas with high FRα expression, it may be possible to identify margins and to predict postoperative MRI findings.

Purpose: To characterize folate receptor alpha (FRα) expression in archival and fresh biopsy tumor samples from relapsed ovarian cancer patients., Methods: Patients with ovarian tumors amenable to biopsy were eligible to enroll. Eligibility included a minimum requirement of FRα positivity in archival tumor samples (≥25% of cells with ≥2+ staining intensity). Patients received mirvetuximab soravtansine at 6mg/kg once every 3weeks. Core needle biopsies were collected before and after treatment and FRα levels assessed by immunohistochemistry. Descriptive statistics were used to summarize the association between receptor expression and response., Results: Twenty-seven heavily pre-treated patients were enrolled. Six individuals (22%) did not have evaluable pre-treatment biopsies due to insufficient tumor cells. The concordance of FRα expression in archival and biopsy tissues was 71%, and no major shifts in receptor expression were seen in matched pre- and post-treatment biopsy samples. Adverse events were generally mild (≤grade 2) with keratopathy (48%), fatigue (44%), diarrhea, and blurred vision (each 37%) being the most common treatment-related toxicities. The confirmed objective response rate (ORR) was 22%, including two complete responses and four partial responses. Superior efficacy measures were observed in the subset of patients with the highest FRα levels (ORR, 31%; progression-free survival, 5.4months)., Conclusion: Concordance of FRα expression in biopsy versus archival tumor samples suggests that archival tissue can reliably identify patients with receptor-positive tumors and is appropriate for patient selection in mirvetuximab soravtansine clinical trials. Regardless of the tissue source analyzed, higher FRα expression was associated with greater antitumor activity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) has one of the poorest prognosis among malignancies. Thus, the identification of markers useful in developing innovative diagnostic and therapeutic methods is an imperative need. Folate receptor alpha (FRα) has been associated with prognosis in several cancers and has served as a target of novel anti-tumor therapies. However, FRα expression in PDAC and its correlation with the clinical course of the disease has not been thoroughly investigated. In this study, we analyzed FRα expression in 140 PDAC specimens and 7 PDAC cell lines in order to define the significance of FRα expression in PDAC and its potential role as a target for immunotherapy. Immunohistochemical analysis demonstrated that FRα expression intensity was low, intermediate and high in 22(16%), 73(52%) and 45(32%) PDACs, respectively. The staining was located in both membrane and cytoplasm in most cases (123, 88%). Lower FRα expression was associated with cigarette smoking (p<0.001), alcohol consumption (p<0.001), and lymphovascular invasion (p=0.002). Additionally, lower FRα expression was associated with poor overall survival (5-year overall survival: low 13%, intermediate 31%, high 33%; p=0.006). FRα expression (HR=0.61; p=0.03) and Charlson Comorbidity Index (HR=1.16; p=0.01) emerged as independent predictors of survival. The analysis by flow cytometry of 7 PDAC cell lines (AsPC-1, Capan-2, MIA PaCa-2, PANC-1, PDAC2, PDAC3, and PDAC5) demonstrated the highest expression of FRα on the PDAC3 cell line (45%). Therefore, a higher FRα expression is predictive of a favorable prognosis in PDAC and FRα may represent a promising target for novel treatments, including immunotherapy.

Aim: Folic acid (FA)-decorated polyamidoamine dendrimer G4 (G4-FA) was synthesized and studied for targeted delivery of genes to head and neck cancer cells expressing high levels of folate receptors (FRs)., Methods: Cellular uptake, targeting specificity, cytocompatibility and transfection efficiency were evaluated., Results: G4-FA competes with free FA for the same binding site. G4-FA facilitates the cellular uptake of DNA plasmids in a FR-dependent manner and selectively delivers plasmids to FR-high cells, leading to enhanced gene expression., Conclusion: G4-FA is a suitable vector to deliver genes selectively to head and neck cancer cells. The fundamental understandings of G4-FA as a vector and its encouraging transfection results for head and neck cancer cells provided support for its further testing in vivo., Competing Interests: Financial & competing interests disclosure This work was supported, in part, by the National Science Foundation (CAREER award CBET0954957), and National Institutes of Health (R01EY024072 and R01DE024381). Confocal microscopy was performed at the VCU Department of Anatomy & Neurobiology Microscopy Facility, supported, in part, by funding from NIH-NINDS Center Core Grant P30NS047463 and, in part, by funding from NIH-NCI Cancer Center Support Grant P30CA016059. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

The folate receptor (FR) is overexpressed on the vascular side of cancerous cells including those of the breast, ovaries, testes, and cervix. We hypothesized that a folate-conjugated immunoglobulin (F-IgG) would bind to the FR that is overexpressed on melanoma tumor cells to target these cells for lysis by natural killer (NK) cells. Folate receptor expression was confirmed in the Mel-39 (human melanoma) cell line by flow cytometry and immunoblot analysis using KB (human oral epithelial) and F01 (human melanoma) as a positive and a negative control, respectively. FR-positive and FR-negative cell lines were treated with F-IgG or control immunoglobulin G in the presence or absence of cytokines to determine NK cell ability to lyse FR-positive cell lines. NK cell activation was significantly upregulated and lysis of Mel 39 tumor cells increased following treatment with F-IgG compared with control immunoglobulin G at all effector : target (E : T) ratios (P<0.01). This trend further increased by NK cell stimulation with the activating cytokine interleukin-12. NK cell production of cytokines such as interferon-gamma, macrophage inflammatory protein 1α, and regulated on activation normal T-cell expressed and secreted (RANTES) was also significantly increased in response to costimulation with interleukin-12 stimulation and F-IgG-coated Mel 39 target cells compared with controls (P<0.01). In contrast, F-IgG did not bind to the FR-negative cell line F01 and had no significant effect on NK cell lysis or cytokine production. This research indicates the potential use of F-IgG for its ability to induce an immune response from NK cells against FR-positive melanoma tumor cells, which can be further increased by the addition of cytokines.

This work reports a graphene oxide-based nanovehicle with conjugation of pegylated folate for targeted delivery of anticancer drugs and fluorescein-labeled peptide for therapeutic self-monitoring in vitro and in vivo. The nanovehicle could absorb hydrophobic and aromatic drug molecules with high loading capacity and efficiency of more than 1.7 mg mg(-1) and 90%, respectively. MTT and flow cytometric assays demonstrated that the drug-loaded nanovehicle could specifically transport and release the drugs into the folate receptor high-expressed cancer cells, which ensured a high therapeutic efficiency to cancer cells and prevented the injury to normal cells. Moreover, confocal fluorescence imaging confirmed that the drug-induced cancer cell death could be visualized with the light-up fluorescence of fluorescein activated by caspase-3. The targeted delivery of drug and self-evaluation of therapeutic efficacy were further successfully realized by living imaging in tumor-bearing mice, which broaden the applications of this theranostic system in vivo and may offer new opportunities for precise cancer treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Thoroughly investigation of folate receptor (FR) expression related to targeting drug delivery in tumor cells has been intensively pursued in recent years. Herein, a simple and versatile strategy for determination of FR expression based on targeted imaging of tumor cells with fluorescent nano-conjugates was developed. The fluorescent nano-conjugates were composed of poly 2-vinyl-4,4-dimethyl azlactone (PVDMA) as the linker, folic acid as the targeting unit and amino-Rhodamine B as the fluorescent ligand. Owing to possessing dimethyl azlactone groups in polymer framework, PVDMA could easily reacted with amines or alcohols, and form water soluble materials. Fluorescent imaging studies indicated that the prepared nano-conjugates could specifically target tumor cells and monitor the over expressing of FR. Moreover, the FR expression up-regulation in HeLa cells through medicines regulation has been further explored. This new protocol opens an effective way through synthesis and design of novel fluorescent nano-conjugates for FR expression investigation in tumor cells via targeted imaging, showing great potential in drug delivery mechanism study and cancer therapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Folate receptor alpha (FRα) is known to be upregulated in a variety of cancers, including non-small cell lung cancer (NSCLC) and breast cancer. To ensure reliable implementation of diagnostic- and therapeutic agents, concordance of FRα expression between biopsy, primary tumor and metastases is important. Using immunohistochemistry (Mab 26B3.F2) these concordances were investigated in 60 NSCLC and 40 breast cancer patients. False positivity of FRα expression on breast and lung cancer biopsies was limited to less than 5%. In NSCLC, FRα expression was shown in 21/34 adenocarcinomas and 4/26 squamous cell carcinomas (SCC). Concordance of FRα expression between biopsy and primary tumor was achieved in respectively 83% and 91% of adenocarcinomas and SCCs. Approximately 80% of all local and distant metastases of NSCLC patients showed concordant FRα expression as their corresponding primary tumor. In breast cancer, FRα positivity was shown in 12/40 biopsies, 20/40 lumpectomies and 6/20 LN metastases, with concordance of 68% between biopsy and primary tumor and 60% between primary tumor and LN metastases. In conclusion, this study shows high concordance rates of FRα expression between biopsies and metastases compared to primary NSCLC and breast cancers, underscoring the applicability of FRα-targeted agents in these patients.

Background: Nanomedicine is a very promising field and nanomedical drugs have recently been used as therapeutic agents against cancer. In a previous study, we showed that Nanoceria (NCe), nanoparticles of cerium oxide, significantly inhibited production of reactive oxygen species, cell migration and invasion of ovarian cancer cells in vitro, without affecting cell proliferation and significantly reduced tumor growth in an ovarian cancer xenograft nude model. Increased expression of folate receptor-α, an isoform of membrane-bound folate receptors, has been described in ovarian cancer. To enable NCe to specifically target ovarian cancer cells, we conjugated nanoceria to folic acid (NCe-FA). Our aim was to investigate the pre-clinical efficacy of NCe-FA alone and in combination with Cisplatin., Methods: Ovarian cancer cell lines were treated with NCe or NCe-FA. Cell viability was assessed by MTT and colony forming units. In vivo studies were carried in A2780 generated mouse xenografts treated with 0.1 mg/Kg NCe, 0.1 mg/Kg; NCe-FA and cisplatinum, 4 mg/Kg by intra-peritoneal injections. Tumor weights and burden scores were determined. Immunohistochemistry and toxicity assays were used to evaluate treatment effects., Results: We show that folic acid conjugation of NCe increased the cellular NCe internalization and inhibited cell proliferation. Mice treated with NCe-FA had a lower tumor burden compared to NCe, without any vital organ toxicity. Combination of NCe-FA with cisplatinum decreased the tumor burden more significantly. Moreover, NCe-FA was also effective in reducing proliferation and angiogenesis in the xenograft mouse model., Conclusion: Thus, specific targeting of ovarian cancer cells by NCe-FA holds great potential as an effective therapeutic alone or in combination with standard chemotherapy.

Metabolism of homocysteine and other sulfur amino acids is closely associated with metabolism of folates. In this study, we analyzed the possible role of folates and sulfur amino acids in the development of features of the metabolic syndrome in the BXH/HXB recombinant inbred strains derived from the spontaneously hypertensive rat (SHR) and Brown Norway progenitors. We mapped a quantitative trait locus for cysteine concentrations to a region of chromosome 1 that contains a cis-acting expression quantitative trait locus regulating mRNA levels of folate receptor 1 (Folr1) in the kidney. Sequence analysis revealed a deletion variant in the Folr1 promoter region of the SHR. Transfection studies demonstrated that the SHR-promoter region of Folr1 is less effective in driving luciferase reporter gene expression than the Brown Norway promoter region of Folr1. Results in the SHR.BN-chr.1 congenic strain confirmed that the SHR variant in Folr1 cosegregates with markedly reduced renal expression of Folr1 and renal folate reabsorption, decreased serum levels of folate, increased serum levels of cysteine and homocysteine, increased adiposity, ectopic fat accumulation in liver and muscle, reduced muscle insulin sensitivity, and increased blood pressure. Transgenic rescue experiments performed by expressing a Folr1 transgene in the SHR ameliorated most of the metabolic disturbances. These findings are consistent with the hypothesis that inherited variation in the expression of Folr1 in the kidney influences the development of the metabolic syndrome and constitutes a previously unrecognized genetic mechanism that may contribute to increased risk for diabetes mellitus and cardiovascular disease., (© 2015 American Heart Association, Inc.)

Nanoparticles are widely recognized as a vehicle for tumor-targeted therapies. There are many factors that can influence the uptake of nanoparticles, such as the size of the nanoparticles, and/or their shape, elasticity, surface charge and even the cell cycle phase. However, the influence of the cell cycle on the active targeting of a drug delivery system has been unknown until now. In this study, we initially investigated the folate receptor α (FR-α) expression in different phases of HeLa cells by flow cytometric and immunocytochemical methods. The results obtained showed that FR-α expression was cell cycle-dependent, i.e. the S cells' folate receptor expression was the highest as the cell progressed through its cycle. Then, we used folate modified poly(L-amino acid) micelles (FA-PM) as an example to investigate the influence of the cell cycle on the active targeting drug delivery system. The results obtained indicated that the uptake of FA-PM by cells was influenced by the cell cycle phase, and the S cells took up the greatest number of folate conjugated nanoparticles. Our findings suggest that future studies on ligand-mediated active targeting preparations should consider the cell cycle, especially when this system is used for a cell cycle-specific drug.

Folate has a central role in the cell metabolism. This study aims to explore the DNA methylation pattern of the folate transporter genes FOLR1, PCFT, and RFC1 as well as the corresponding protein expressions in colorectal cancer (CRC) tissue and adjacent non-cancerous mucosa (ANCM). Our results showed statistically significant differences in the DNA-methylated fraction of all three genes at several gene regions; we identified three differentially methylated CpG sites in the FOLR1 gene, five CpG sites in the PCFT gene, and six CpG sites in the RFC1 gene. There was a pronounced expression of the FRα and RFC proteins in both the CRC and ANCM tissues, though the expression was attenuated in cancer compared to the paired ANCM tissues. The PCFT protein was undetectable or expressed at a very low level in both tissue types. Higher methylated fractions of the CpG sites 3-5 in the RFC1 gene were associated with a lower protein expression, suggestive of epigenetic regulation by DNA methylation of the RFC1 gene in the colorectal cancer. Our results did not show any association between the RFC and FRα protein expression and tumor stage, TNM classification, or tumor location. In conclusion, this is the first study to simultaneously evaluate both DNA methylation and protein expression of all three folate transporter genes, FOLR1, PCFT, and RFC1, in colorectal cancer. The results encourage further investigation into the possible prognostic implications of folate transporter expression and DNA methylation.

Background: Folate receptor alpha (FOLR1/FRA) is expressed in a number of epithelial cancers and in particular epithelial ovarian cancer (EOC), especially of the serous histotype. Recent studies have shown that EOC originates from the fallopian tube fimbriae rather than from epithelial cells lining the ovary. We have previously shown by immunohistochemistry a strong correlation between FRA expression in EOC and normal and fallopian adenocarcinoma. Folate receptor beta (FOLR2/FRB) has been described to be expressed by macrophages both in inflammatory disorders and certain epithelial cancers. Given the high sequence identity of these two folate receptor family members we sought to investigate the architectural and cell-specific expression of these two receptors in gynecologic tissues., Methods: RNA scope, a novel chromogenic in situ hybridization assay tool, was used to examine expression of the alpha (FOLR1) and beta (FOLR2) isoforms of folate receptor relative to each other as well as to the macrophage markers CD11b and CD68, in samples of normal fallopian tube and fallopian adenocarcinoma as well as normal ovary and EOC., Results: We demonstrated expression of both FOLR1 and FOLR2 in EOC, normal fallopian tube and fallopian adenocarcinoma tissue while very little expression of either marker was observed in normal ovary. Furthermore, FOLR2 was shown to be expressed almost exclusively in macrophages, of both the M1 and M2 lineages, as determined by co-expression of CD11b and/or CD68, with little or no expression in epithelial cells., Conclusions: These findings further substantiate the hypothesis that the cell of origin of EOC is tubal epithelium and that the beta isoform of folate receptor is primarily restricted to macrophages. Further, macrophages expressing FOLR2 may represent tumor associated or infiltrating macrophages (TAMs) in epithelial cancers.

Farletuzumab is a humanized monoclonal antibody against folate receptor α (FRA). The purpose of the study is to assess safety and tolerability, the pharmacokinetic (PK) profile, and preliminary antitumor effect. Patients with ovarian cancer (OC) or FRA-expressing solid tumors who are resistant to standard treatments were eligible for the study. After single-dose administration for PK assessment, farletuzumab was administered by intravenous injection, repeating every week until disease progression. Dose-limiting toxicities (DLTs) were defined as grade 4 hematological and grade 3/4 nonhematological toxicities. Dose escalation was planned in 4 cohorts (50, 100, 200, and 400 mg/m(2)). Fourteen patients with OC and two patients with gastric cancer (GC) received farletuzumab infusion. Neither DLTs nor grade 3/4 toxicities were reported in all cohorts. Major adverse events, including grade 1/2 infusion related reaction (15 patients, 93.8%), headache (seven patients, 43.8%), and nausea and decreased appetite (five patients each, 31.3%), were observed and medically managed. AUC and Cmax increased dose-dependently and linear PK profiles were observed. No tumor shrinkage was recorded, but long-term disease stabilization for 25 and 20 months was observed in one patient with clear cell OC (100 mg/m(2)) and one patient with GC (400 mg/m(2)), respectively. No cumulative toxicity occurred in any patient. Farletuzumab was well tolerated in Japanese patients with a similar PK profile as compared with the US population. Long-term disease stabilization was observed in a subpopulation of clear cell OC and GC; both of them were resistant and progressive after standard chemotherapies (ClinicalTrials.gov Identifier: NCT01049061).

Aim: Human pituitary adenomas account for 10% of intracranial tumours and occur in about 20% of the population. They cause hypopituitarism or the compression of adjacent regional structures. However, little is known about the molecular pathogenesis that contributes to the development of these tumours., Material and Methods: In this study, we investigated the relationship between the expression level of folate receptor alpha and some clinical factors (endocrine, age, gender, repeated operation or not, mean diameter of tumour and invasiveness) of pituitary adenomas. Realtime fluorescent quantitative reverse transcriptase polymerase chain reaction was used to determine the expression of folate receptor alpha mRNA in pituitary adenomas and normal pituitaries. Folate receptor alpha protein levels were quantified using Western blot analysis and Immunohistochemistry., Results: We found folate receptor alpha mRNA and protein were significantly upregulated in clinically nonfunctional pituitary adenomas compared to functional tumours. For nonfunctional tumours, folate receptor alpha expression was much higher in the invasive group than in non-invasive group., Conclusion: These results suggest that the overexpression of folate receptor alpha mRNA and protein by nonfunctional pituitary adenomas may facilitate the growth of these tumours. Potentially, this finding could be exploited to develop innovative molecular targeted diagnosis and treatment for human nonfunctional pituitary adenomas.

Background: Folate receptor 1 (FOLR1) is expressed in the majority of ovarian carcinomas (OvCa), making it an attractive target for therapy. However, clinical trials testing anti-FOLR1 therapies in OvCa show mixed results and require better understanding of the prognostic relevance of FOLR1 expression. We conducted a large study evaluating FOLR1 expression with survival in different histological types of OvCa., Methods: Tissue microarrays composed of tumour samples from 2801 patients in the Ovarian Tumour Tissue Analysis (OTTA) consortium were assessed for FOLR1 expression by centralised immunohistochemistry. We estimated associations for overall (OS) and progression-free (PFS) survival using adjusted Cox regression models. High-grade serous ovarian carcinomas (HGSC) from The Cancer Genome Atlas (TCGA) were evaluated independently for association between FOLR1 mRNA upregulation and survival., Results: FOLR1 expression ranged from 76% in HGSC to 11% in mucinous carcinomas in OTTA. For HGSC, the association between FOLR1 expression and OS changed significantly during the years following diagnosis in OTTA (Pinteraction=0.01, N=1422) and TCGA (Pinteraction=0.01, N=485). In OTTA, particularly for FIGO stage I/II tumours, patients with FOLR1-positive HGSC showed increased OS during the first 2 years only (hazard ratio=0.44, 95% confidence interval=0.20-0.96) and patients with FOLR1-positive clear cell carcinomas (CCC) showed decreased PFS independent of follow-up time (HR=1.89, 95% CI=1.10-3.25, N=259). In TCGA, FOLR1 mRNA upregulation in HGSC was also associated with increased OS during the first 2 years following diagnosis irrespective of tumour stage (HR: 0.48, 95% CI: 0.25-0.94)., Conclusions: FOLR1-positive HGSC tumours were associated with an increased OS in the first 2 years following diagnosis. Patients with FOLR1-negative, poor prognosis HGSC would be unlikely to benefit from anti-FOLR1 therapies. In contrast, a decreased PFS interval was observed for FOLR1-positive CCC. The clinical efficacy of FOLR1-targeted interventions should therefore be evaluated according to histology, stage and time following diagnosis.

We describe a modification and post-functionalization technique for a donor-acceptor-donor type monomer; 6-(4,7-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-2H-benzo[d][1,2, 3]triazol-2-yl)hexan-1-amine. Folic acid was attached to the fluorescent structure. The conjugation was confirmed via NMR and Fourier transform infrared analyses. Cytotoxicity was investigated and the comparison of association of targeted monomeric structures in tumor cells was monitored via fluorescence microscopy., (© 2014 American Institute of Chemical Engineers.)

Folate receptor alpha (FRα) mediates folate uptake by endocytosis, and while folate is essential to DNA methylation and synthesis and may have an important role in proliferating cells. FRα is known to be expressed in rapidly proliferating cells, including many cancer cell lines, but there has been no systematic assessment of expression in cervical cancer cell lines. The aim of the present study was to evaluate the effects of FRα on proliferation and apoptosis of cervical cells and correlation mechanism. In this study, we investigated the biological function of FRα in Hela cells using RNA interference. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK8) assay, while cell cycling and apoptosis were assessed by flow cytometry, mRNA levels by real time- PCR and protein levels of FRα, c-Fos and c-Jun by Western blotting. The results revealed that FRα was highly expressed in Hela cells and its silencing with a small interfering RNA (siRNA) inhibited cell proliferation and induced cell apoptosis, arresting the cell cycle in G0/G1 stages while decreasing the proportion in S and G2/M stages, and suppressed the expression levels of c-Fos and c-Jun. In conclusion, the results of this study indicated that FRα down-regulation might be capable of suppressing cervical cancer cell proliferation and promoting apoptosis. It suggested that FRα might be a novel therapeutic target for cervical cancer.

Objective: Based on its expression profile, folate receptor alpha (FRA) is an attractive candidate for targeted diagnostics and therapeutics. However, applicability of these agents in residual or recurrent disease could be influenced by chemotherapy. We evaluated whether chemotherapy modified FRA expression in non-mucinous epithelial ovarian (EOC) and endometrial carcinoma (EC)., Methods: FRA staining was evaluated by immunohistochemistry, using MAb 26B3, in 81 patients (41 EOCs and 40 ECs) and 17 control tissues (5 benign ovarian cysts, 5 normal ovarian, and 7 normal endometrial tissues). Chemotherapy effect was evaluated in 42 patients (30 paired samples at primary and interval debulking surgery and 12 from primary and recurrent disease). FRA expression was assessed using a semi-quantitative staining algorithm, the M-score (range 0-50)., Results: Median difference in M-score between tumor and control samples was 27.5 for EOC (95% CI 10.0 to 45.0) and 6.7 for EC (95% CI -6.7 to 21.7). Paired samples from both primary and interval debulking surgery did not differ in FRA expression in EOC (median difference of M-score between paired samples of 0.0 [95% CI -2.6 to 2.6]). Recurrent EOC tumors reflected FRA status at diagnosis (median difference of M-score between paired samples of 3.3 [95% CI -7.0 to 13.6])., Conclusions: This study shows no significant difference in FRA expression after chemotherapy, strengthening the rationale for FRA targeted diagnostics and therapeutics in FRA expressing tumors, whether newly diagnosed or at recurrence., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Through this research a lentiviral vector expressing the gene of folate-binding protein-1 (FOLR1) was constructed and the corrsponding expression products were identified. Firstly, full-length of the FORL1 gene was amplified by PCR and cloned into the plasmid pWPI. Then it was further confirmed by PCR and sequencing. Secondly, after the recombinant pWPI and its helper plasmid co-transfected the virus packaging 293T cells, SKOV3 cells were infected with the virus particles and sorted by flow cytometry. Thirdly, the FOLR1 gene was detected by RT-PCR and its protein expression was detected by Western blot. Finally, the recombinant expression vector was successfully constructed, and lentiviruses were successfully packaged by the 293T cells. A great quantity of green fluorescent cells could be seen after the SKOV3 cells were effectively infected with the lentiviruses carrying the FOLR1 gene. The sorting could be done and detected by cytometrying the FORL1 gene and its stable expression by the two methods above, which laid experimental foundation for exploring its biological function in ovarian cancers.

Folate conjugated amphiphilic polymeric micelles have attracted much attention for active targeted delivery of drugs in folate receptor α (FR-α) positive tumors. However, the efficacy improvement of targeted delivery folate-based nanovehicles was limited by the abundance of FR-α on the surface of tumor cells. Recently, it was found that FR-α expression of Hela cells could be up-regulated by modulators such as dexamethasone, which open a new avenue to enhance the efficiency of targeted delivery from the biological view. In this study, folate-conjugated or plain lipid-core micelles loaded with fluorescent coumarin 6 or mitoxantrone (MTN) were prepared by self-assembly. In addition, FOLR1 mRNA and cell surface FR-α levels were evaluated in Hela cells with dexamethasone treatment. The endocytosis of folate-conjugated or plain micelles loaded with coumarin 6 was examined with confocal microscopy and flow cytometry, which displayed that folate-conjugated micelles can be internalized more efficiently in dexamethasone-treated Hela cells than in normal Hela cells. Moreover, the antitumor activity of folate-conjugated micellar MTN was also improved through up-regulation of FR-α. Therefore, FR-α up-regulation using modulators has great potential to improve the therapeutic efficacy of folate-conjugated nanovehicles in some FR-α positive tumors via receptor-mediated endocytosis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

An immunohistochemical evaluation for folate receptor-α (FRA) was undertaken to evaluate expression in gynecologic malignancies involving ovary, endometrium, and the fallopian tube. Commercial tissue microarrays were assessed using an optimized manual immunohistochemical method using MAb 26B3, a newly described monoclonal antibody. A positive result was defined as ≥5% of the sample demonstrating membranous staining. A semiquantitative staining algorithm, defined as the M-score, was used to analyze staining intensity between sample histotypes. MAb 26B3 showed uniform membranous staining and high levels of expression of FRA in ovarian, fallopian tube, and endometrial cancers. All serous ovarian cancers analyzed (70) were positive for FRA expression and no relationship to stage or grade was found. However, a significant difference for FRA expression, between serous and mucinous ovarian carcinomas, was demonstrated (P=0.014). In addition, approximately 90% of all endometrial adenocarcinomas were positive for FRA expression but, unlike ovarian serous carcinomas, a statistically significant relationship to grade was observed (P=0.0029). Although normal ovary is completely devoid of FRA immunoreactivity, normal fallopian tube and cortical serous/tubal inclusion cysts demonstrated uniform and intense FRA staining of columnar epithelium supporting the hypothesis that serous ovarian carcinoma is similar to the tubal epithelium. The data presented further support the hypothesis that FRA expression in gynecologic tumors is due to the cell of origin normally expressing this receptor. This is possibly due to an associated growth advantage, rather than the process of tumorigenesis resulting in aberrant expression of FRA per se.

Purpose: The overall objective of this study was to investigate the differential expression of folate receptor-alpha (FR-α), sodium-dependent multivitamin transporter (SMVT), and amino acid transporter [B ((0, +))] in retinoblastoma (Y-79) and retinal pigment epithelial (ARPE-19) cells., Methods: Polymerase chain reaction (PCR) analysis was performed to confirm the existence of FR-α, SMVT, and B ((0, +)) in Y-79 and ARPE-19 cell lines. Quantitative real-time PCR was also performed to determine the relative expression of FR-α, SMVT, and B ((0, +)) at mRNA level in these cell lines. Quantitative uptake of [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] Arginine was studied in Y-79 and ARPE-19 cells. Further, saturation kinetics of [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] Arginine was performed in the presence of various concentrations of respective cold substrates to determine the kinetic parameters (K(m) and V(max)) in Y-79 and ARPE-19 cells., Results: PCR analysis had confirmed the existence of FR-α, SMVT, and B ((0, +)) in Y-79 and ARPE-19 cells. Quantitative real-time PCR analysis had shown significantly higher expression of FR-α, SMVT, and B ((0, +)) mRNA levels in Y-79 cells compared with ARPE-19 cells. Quantitative uptake of [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] Arginine was found to be significantly higher in Y-79 cells relative to ARPE-19 cells. [(3)H] Folic acid uptake process followed saturation kinetics with apparent K(m) of 8.29 nM and V(max) of 393.47 fmol/min/mg protein in Y-79 cells and K(m) of 80.55 nM and V(max) of 491.86 fmol/min/mg protein in ARPE-19 cells. [(3)H] Biotin uptake process also displayed saturation kinetics with K(m) of 8.53 μM and V(max) of 14.12 pmol/min/mg protein in Y-79 cells and K(m) of 138.25 μM and V(max) of 38.85 pmol/min/mg protein in ARPE-19 cells. [(14)C] Arginine uptake process followed saturation kinetics with K(m) of 16.77 μM and V(max) of 348.27 pmol/min/mg protein in Y-79 cells and K(m) of 52.03 μM and V(max) of 379.21 pmol/min/mg protein in ARPE-19 cells., Conclusions: This work demonstrated for the first time the higher expression and affinity of FR-α, SMVT, and B ((0, +)) mRNA levels in retinoblastoma (Y-79) cells compared with retinal pigment epithelial (ARPE-19) cells.

Introduction: The mechanism of pathogenesis of adenomas pituitary is still unknown; differences between pituitary cells of different origin are observed. Identification of genes specific to pituitary adenomas should give better understanding of differences in their response to therapy, especially to radiotherapy. The aim of our study was to independently validate differences in the expression of FOLR1, BAG1, LAPTM4B between functioning (FA) and non-functioning (NFA) pituitary adenomas reported by microarray-based studies., Material and Methods: Analysis of gene expression was performed by real-time quantitative PCR (QPCR) in 76 pituitary adenomas, 25 functioning and 51 non-functioning ones. The expression of the examined genes was normalized to the reference index, obtained by calculation of the geometric mean of reference genes expression: GUS-B, B2M, ACTB, EIF3S10, UBE2D2 and ATP6V1E., Results: Two genes showed significant differences in expression between non-functioning adenomas and functioning ones (FA) (FOLR1 32.4 x greater p = 0.022, BAG1 2.2 x lower p = 0.0002). The expression of LAPTM4B (1.1 x lower) was only insignificantly changed. The expression of FOLR1 in all tumours (functioning and non-functioning) was higher in older patients (over 50 years of age) (p = 0.018). Expression of BAG1 was significantly lower in older patients (p = 0.015). In a subgroup of pure non-functioning adenomas there was a higher expression of FOLR1 in older patients (p = 0.006). Analysis of expression profiles and invasiveness of tumours did not reveal any significant differences both in non-functioning and functioning tumours., Conclusions: Among pituitary adenomas, the highest level of expression FOLR1 is seen in NFA which are negative by immunohistochemistry to all pituitary hormones while GH-producing adenomas are the only class of pituitary tumours where FOLR1 expression is virtually absent. For BAG1 we confirm a significantly higher expression in functioning (both PRL and GH producing) adenomas than non-functioning ones, while LAPTM4B does not exhibit any expression changes between different classes of pituitary tumours.

The mechanism underlying the sensing of varying degrees of physiological folate deficiency, prior to adaptive optimization of cellular folate uptake through the translational up-regulation of folate receptors (FR) is unclear. Because homocysteine, which accumulates intracellularly during folate deficiency, stimulated interactions between heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) and an 18-base FR-α mRNA cis-element that led to increased FR biosynthesis and net up-regulation of FR at cell surfaces, hnRNP-E1 was a plausible candidate sensor of folate deficiency. Accordingly, using purified components, we evaluated the physiological basis whereby L-homocysteine triggered these RNA-protein interactions to stimulate FR biosynthesis. L-homocysteine induced a concentration-dependent increase in RNA-protein binding affinity throughout the range of physiological folate deficiency, which correlated with a proportionate increase in translation of FR in vitro and in cultured human cells. Targeted reduction of newly synthesized hnRNP-E1 proteins by siRNA to hnRNP-E1 mRNA reduced both constitutive and L-homocysteine-induced rates of FR biosynthesis. Furthermore, L-homocysteine covalently bound hnRNP-E1 via multiple protein-cysteine-S-S-homocysteine mixed disulfide bonds within K-homology domains known to interact with mRNA. These data suggest that a concentration-dependent, sequential disruption of critical cysteine-S-S-cysteine bonds by covalently bound L-homocysteine progressively unmasks an underlying RNA-binding pocket in hnRNP-E1 to optimize interaction with FR-α mRNA cis-element preparatory to FR up-regulation. Collectively, such data incriminate hnRNP-E1 as a physiologically relevant, sensitive, cellular sensor of folate deficiency. Because diverse mammalian and viral mRNAs also interact with this RNA-binding domain with functional consequences to their protein expression, homocysteinylated hnRNP-E1 also appears well positioned to orchestrate a novel, nutrition-sensitive (homocysteine-responsive), posttranscriptional RNA operon in folate-deficient cells.

Purpose: Nanoparticle drug delivery offers a potential solution in the treatment of cancer. Using a heterotopic tumor model for head and neck squamous cell carcinoma (HNSCC), tumors of variable folate binding protein-alpha (FBP-α) have been treated to delineate receptor necessity as well as efficacy and toxicity of folate targeted chemotherapy., Materials and Methods: University of Michigan Squamous Cell Carcinoma (UM-SCC) and American Type Culture Collection (ATCC) cell lines were screened using quantitative real-time polymerase chain reaction for FBP-α expression. Acetylated generation 5 dendrimers conjugated to the targeting moiety folic acid and the therapeutic moiety methotrexate were fabricated and administered to severe combined immunodeficiency (SCID) CB-17 mice inoculated with UM-SCC-1, UM-SCC-17B, and UM-SCC-22B cancer cells. Mice were injected with targeted therapy, free methotrexate, or saline control and monitored for drug efficacy and toxicity., Results: Targeted therapy was effective relative to receptor level expression. Targeted therapy could be delivered in molar doses 3 times that of free drug. The treatment of a high folate expression tumor cell population was noted to have increased efficacy over saline (P < .01) and free methotrexate (P = .03) as well as decreased systemic toxicity., Conclusions: This report represents the first translation of dendrimer-based chemotherapy to HNSCC and underscores its effectiveness as an antitumor agent in human cancer cell lines with lower levels of FBP-α than the in vitro and in vivo models previously reported., (Copyright © 2011 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Folate receptor alpha (FRα), encoded by folate receptor 1 (adult) gene, has emerged as a cancer biomarker and potential therapeutic target. In addition, its expression in tumors may offer prognostic information. The aim of this study was to assess the prognostic value of FRα expression and other common molecular markers in resected liver metastases from colorectal cancer. To maximize potential biological differences, we selected two groups of patients with markedly different outcomes as study subjects. Immunohistochemical analysis of FRα expression and other common markers (thymidylate synthase, p53, p27, BCL2, ki67, MLH1, MSH2 and MGMT) on tissue microarrays was carried out on samples from 160 patients; 56 patients survived at least 10 years following liver resection, and 104 died within 2 years of surgery. These markers were evaluated and compared with standard clinical predictors of outcome including a previously validated clinical risk score. Our results showed that in addition to known clinical risk factors, FRα positivity was significantly associated with the early death group (32% compared with 13%; P=0.03). None of the other common molecular markers were differentially expressed between the two groups. On multivariate analysis, clinical risk score, margin status and FRα expression were independently associated with outcome. Specific multivariate comparisons confirmed that FRα expression was associated with outcome independent of the clinical risk score and margin. These data demonstrate that FRα expression is present in a subset of resected hepatic colorectal cancer metastases, and this marker is independently associated with survival after hepatic resection. The prognostic value of FRα expression and the utility of FRα-targeted therapies in stage-IV colorectal cancer patients deserve further exploration.