Folate receptor alpha expression in low-grade serous ovarian cancer: Exploring new therapeutic possibilities.

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.

A phase II study of Mirvetuximab Soravtansine in triple-negative breast cancer.

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.

Triple Negative Breast Cancer: Expression of Folate Receptor Alpha in Indian population.

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.

Correlation of folate receptor alpha expression with clinicopathological parameters and outcome in triple negative breast cancer.

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.

Escherichia coli expression, purification, and refolding of human folate receptor α (hFRα) and ß (hFRß).

Human folate receptors (hFRα and hFRß) 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 hFRß 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 hFRß from 10 g of E. coli cell bodies.

Characterization of folate receptor alpha (FRα) expression in archival tumor and biopsy samples from relapsed epithelial ovarian cancer patients: A phase I expansion study of the FRα-targeting antibody-drug conjugate mirvetuximab soravtansine.

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.

Folic acid tagged nanoceria as a novel therapeutic agent in ovarian cancer.

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.

A phase I study of farletuzumab, a humanized anti-folate receptor α monoclonal antibody, in patients with solid tumors.

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).

DNA methylation and expression of the folate transporter genes in colorectal cancer.

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.

Evidence for a time-dependent association between FOLR1 expression and survival from ovarian carcinoma: implications for clinical testing. An Ovarian Tumour Tissue Analysis consortium study.

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.

Folate receptor alpha (FRA) expression remains unchanged in epithelial ovarian and endometrial cancer after chemotherapy.

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.

Expression of folate receptor-α (FRA) in gynecologic malignancies and its relationship to the tumor type.

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.

[Construction and identification of lentiviral vector carrying FOLR1 gene].

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.

Incrimination of heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) as a candidate sensor of physiological folate deficiency.

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.

Folate receptor-α expression in resectable hepatic colorectal cancer metastases: patterns and significance.

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.

Targeted dendrimer chemotherapy in an animal model for head and neck squamous cell carcinoma.

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.

Folate Receptor Overexpression in Human and Canine Meningiomas-Immunohistochemistry and Case Report of Intraoperative Molecular Imaging.

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.

Folate receptor overexpression can be visualized in real time during pituitary adenoma endoscopic transsphenoidal surgery with near-infrared imaging.

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.

Folate Receptor Alpha Expression in Platinum Resistant/Refractory Ovarian Carcinomas and Primary Endocervical Adenocarcinomas.

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.

Expression status of folate receptor alpha is a predictor of survival in pancreatic ductal adenocarcinoma.

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.