Expression Of Folate Receptor Alpha Research Articles

Correlating expression of NaPi2b and FRa in high grade serous ovarian cancer (HGSOC).

5545 Background: Biomarker driven therapies are increasingly being used for gynecologic cancers, with mirvetuximab soravtansine, a Folate Receptor alpha (FRa) targeting antibody drug conjugate (ADC) being a recent FDA approved agent for patients with FRa positive PROC. Uptifitamab rilsodotin (UpRi) is a late-stage, first in class NaPi2b-targeting ADC with a novel scaffold-linker-payload that enables high drug to antibody ratio and a controlled bystander effect. Approximately 29% of HGSOC patients have FRa positive tumors (Roche VENTANA FOLR1 (FOLR-2.1) RxDx Assay. USPI 2022); ~59% have NaPi2b positive tumors (Okeke et al, USCAP 2022). With the high unmet medical need in PROC and the evolving landscape of available treatments, understanding biomarker expression and correlation between FRa and NaPi2b is critical. Here we evaluate NaPi2b and FRa RNA expression correlation in HGSOC. Methods: Tumor samples (N=84) from the Ph1b UpRi expansion (EXP) cohort were analyzed via Nanostring (770 immune-focus genes from IO 360 panel + 30 customized ADC-related genes) to evaluate the association between NaPi2b and FRa expression. The cutoff for NaPi2b RNA positive or negative was based on approximately 60% of HGSOC patients having NaPi2b positive tumors (top 60% of samples based on expression deemed NaPi2b positive; bottom 40% NaPi2b negative) and FRa RNA cutoff assumed that ~30% of samples were FRa positive. To correlate the NaPi2b expression level between RNA and IHC, the N=84 samples with known expression levels via IHC were analyzed via Nanostring. Results: When evaluating NaPi2b and FRa RNA expression correlation, 21% (N=18) of samples had both NaPi2b positive and FRa positive expression. 38% (N=32) samples were NaPi2b positive and FRa negative. No statistically significant association was observed (Chi-squared test, p=0.129). NaPi2b RNA and IHC expression was evaluated; a statistically significant association was observed (Kappa statistic, p=0.001). Results are in the Table below. Conclusions: Based on this analysis of a limited sample size, there does not appear to be an association between FRa and NaPi2b expression, with the majority of NaPi2b positive samples not being FRa positive. Additionally, general NaPi2b prevalence and the correlation of expression between RNA and IHC suggest that NaPi2b may be a rational biomarker to integrate in RNA tumor panel testing. This research underscores the importance of early, comprehensive testing of all relevant biomarkers to guide therapy selection, and suggests that additional research is needed to evaluate the potential association between FRa and NaPi2b expression via IHC. [Table: see text]

Fallopian tubal histogenesis of ovarian endometriosis-A study of folate receptor-alpha expression.

Ovary is a common organ site involved by endometriosis. We previously found that fallopian tube may contribute to the histogenesis of ovarian endometriosis. The finding was novel and requires further studies. We addressed this issue by examining a differentially expressed gene folate receptor alpha (FOLR1) and its protein (FRA) in this study. A total of 144 tissue samples were studied. These included 32-paired tubal-endometrial-ovarian endometriosis samples (n = 96), 18 samples of ovarian endometriosis without corresponding fallopian tube or endometrium, and 30 ovarian tissue samples with ovarian surface epithelia but without endometriosis. Multiple comparisons among groups of ovarian endometriosis, normal fallopian tube and benign endometrium were performed. FOLR1 was highly expressed in the epithelia of fallopian tube and ovarian endometriosis, with paired endometrial samples showing a significantly lower level of expression. Similar differential studies for FRA protein were performed through Western blot and immunohistochemistry (IHC). The expression of folate receptor alpha at both mRNA and protein levels in the tissues (fallopian tube or ovarian endometriosis vs. the endometrium) were significantly different (p < 0.001). All ovarian surface mesothelial epithelia showed negative expression of FRA by IHC. The results further support that the fallopian tube may contribute to the development of ovarian endometriosis. Understanding the tubal contribution to ovarian endometriosis should ultimately contribute to ongoing investigative efforts aimed at identifying alternative ways to prevent and treat endometriosis. High level of FRA expression in the fallopian tube and endometriosis might be considered as potential tissue sites for targeted therapy.

Folate Decoration Supports the Targeting of Camptothecin Micelles against Activated Hepatic Stellate Cells and the Suppression of Fibrogenesis.

As the central cellular player in fibrogenesis, activated hepatic stellate cells (aHSCs) are the major target of antifibrotic nanomedicines. Based on our finding that activated HSCs increase the expression of folate receptor alpha (FRα), we tried to apply folic acid (FA) decoration to generate an active drug-targeting at aHSCs and suppress hepato-fibrogenesis. FA-conjugated poly(ethylene glycol)-poly(ε-caprolactone) copolymers (PEG-PCL) were synthesized and self-assembled into the spherical micelles that owned a uniform size distribution averaging at 60 nm, excellent hemo- and cyto-compatibility, and pH-sensitive stability. These FA-modified micelles were preferentially ingested by aHSCs as expected and accumulated more in acutely CCl4 injured mouse livers compared to nondecorated counterparts. Such an aHSC targetability facilitated the loaded medicinal camptothecin (CPT) to achieve a greater therapeutic efficacy and inhibition of MF phenotypic genes in aHSCs. Encouragingly, though free CPT and nontargeting CPT micelles produced negligible curative outcomes, FA-decorated CPT micelles yielded effectively remedial effects in chronically CCl4-induced fibrotic mice, as represented by a significant shrinkage of aHSC population, suppression of fibrogenesis, and recovery of liver structure and function, clearly indicating the success of the folate decoration-supported aHSC-targeted strategy for antifibrotic nanomedicines in fibrosis resolution.

Protein Expression of Folate Receptor Alpha in Adenoid Cystic Carcinoma of the Head and Neck.

PurposeFolate receptor alpha (FRα) is overexpressed in various cancer entities while expression in normal tissue is limited. Thus, FRα is an attractive target in cancer therapy. Currently, various therapeutic and diagnostic approaches are under investigation in clinical trials. The aim of this study was to assess the expression and clinical relevance of FRα in adenoid cystic carcinoma of the head and neck.Patients and MethodsIn this retrospective cohort study, 43 patients with adenoid cystic carcinoma (ACC) of the head and neck were included. FRα expression was analyzed in tumor tissue and tumor-free margin in a tissue microarray using immunohistochemical staining. Protein levels were correlated with clinical parameters.ResultsFRα staining was positive in 47% of ACC patients. The tumor-free margin was positive in 22%. Patients with positive tumor tissue showed positive margin staining in 55%. FRα expression was not associated with the clinical parameters (sex, age, staging, grading, perineural invasion, lymphovascular invasion).ConclusionFRα expression is common in ACC of the head and neck. Therefore, FRα should be further evaluated as a therapeutic target in ACC.

High expression of folate receptor alpha is associated with poor prognosis in patients with cervical cancer.

Folate receptor α (FRα) is a membrane protein expressed in various solid tumors but has limited expression in normal cells. Therefore, FRα is an attractive target for cancer treatment. This study aimed to investigate the relationship between FRα expression and the clinicopathological characteristics and survivals of cervical cancer. This retrospective study included patients with cervical cancer who underwent primary surgery between 2000 and 2020 at our institution. Immunohistochemical staining of FRα was performed using an anti-folate-binding protein/FBP antibody. FRα-positive staining was defined as ≥5% of tumor staining and FRα-high as ≥50% tumor staining with ≥2+ intensity. The association between FRα expression and survival was assessed using multivariate Cox regression analysis, adjusting for established prognostic factors. Overall, 123 patients were identified, and 140 tumor samples, including 17 paired primary and metastatic samples, were evaluated. As histological types, 67 patients had squamous cell carcinoma (SCC), and 56 patients had non-SCC. All primary tumors were FRα-positive. High FRα expression was observed in 25% of the cases and differed according to histology (SCC vs. non-SCC, 14.9% vs. 37.5%, p=0.004). FRα expression was significantly higher in metastatic tumors than in primary (170 [IQR, 140-205] vs. 125 [IQR, 110-150], p=0.0006). High FRα expression was significantly associated with worse overall survival (hazard ratio, 6.73; 95% confidence interval, 2.21-20.53; p=0.001). In cervical cancer, FRα expression was elevated in metastatic tumors and high expression was associated with a worse prognosis. Our study supports the development of FRα-targeted therapy for advanced cervical cancer.

High Expression of Folate Receptor Alpha (FOLR1) is Associated With Aggressive Tumor Behavior, Poor Response to Chemoradiotherapy, and Worse Survival in Rectal Cancer.

Objectives: Recently, molecular medicine targeting Folate Receptor Alpha (FOLR1), which mediates intracellular folate uptake and tumor cell proliferation, has been identified in several malignancies. However, the association between FOLR1 expression and rectal cancer remains unclear. Methods: Immunostaining of FOLR1 was performed on biopsy specimens from 172 rectal cancer patients undergoing preoperative chemoradiotherapy (CRT). FOLR1 expression was measured and divided into low (0+-2+) or high (3+-4+) level. Correlations between FOLR1 status and clinicopathologic features, tumor regression grade, disease-specific survival (DSS), local recurrence-free survival, and metastasis-free survival (MeFS) were analyzed, retrospectively. Results: High FOLR1 expression was significantly associated with advanced post-treatment tumor and nodal status (T3-4; N1-2, P = .001), vascular invasion (P = .042), perineural invasion (P = .012), and poor regression change after CRT (P = .001). In uni- and multi-variable survival analysis, FOLR1 overexpression remained a significant predictor of lower DSS (hazard ratio [HR], 2.328; 95% confidence interval [CI], 1.014-5.344; P = .046) and MeFS (HR, 2.177; 95% CI, 1.000-1.1286; P = .050). Conclusion: These results indicate that high FOLR1 status is associated with aggressive tumor behavior, poor response to CRT, and worse survival. Therefore, FOLR1 expression at initial biopsy may be useful in predicting outcomes and also be a target for the exploration of FOLR1-based therapeutic agents.

Folate Receptor Alpha Is Preferentially Expressed in the Carcinoma Component of Endometrial Carcinosarcomas: A Potential Target for Adjuvant Therapy.

Carcinosarcomas (CSs) of the endometrium are biphasic malignancies, composed of high-grade carcinomatous and sarcomatous components. Surgical stage and pathologic characteristics are the most important prognostic findings, with a 5-yr survival of 15% to 30% in advance stage disease. Folate receptor alpha (FRA) overexpression has been observed in endometrial carcinomas and not yet studied in CSs. This study evaluates semiquantitative expression of FRA in both carcinomatous and sarcomatous components of CSs on whole tissue sections. Immunohistochemistry for FRA expression was performed and extent and intensity of staining were recorded for each case for both histologic components. A total of 46 cases were stained for FRA. The majority of these (40/46, 87%) showed FRA staining at variable intensity in the carcinomatous component, stronger in serous carcinomas and high-grade endometrioid, while only a small subset of tumors demonstrated weak staining in the sarcomatous component (2/46, 4.35%). CS is known to be associated with poor prognosis and adjuvant therapy is recommended even in low stage disease. Serous and high-grade endometrioid carcinomas are the most common carcinomatous components of CSs and are known to show consistently high FRA expression. Folate plays a role in tumor cell migration and loss of cellular adhesion, which are key steps in epithelial-mesenchymal transition, the process by which CS develops from carcinoma cells. Our study shows expression of FRA in the carcinomatous component of almost all CS cases (87%), further favoring FRA as a target for adjuvant treatment. While expression of FRA in the sarcomatous component was rarely observed, the carcinomatous component being associated with metastatic potential underscores the importance of anti-FRA therapy for systemic disease control.

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 between the expression of folate receptor alpha (FRα) and clinicopathological features in patients with lung adenocarcinoma

ObjectivesFolate receptor alpha (FRα) is expressed on the cell surface, mediates its intracellular transport via receptor-mediated endocytosis, and is involved in cell division. Whether FRα could be a potential therapeutic target in FRα-expressing cancers remains unknown. Here, we retrospectively investigated the correlations between tumor FRα expression in lung adenocarcinoma (LADC) and clinicopathological features. Materials and methodsFRα expression was evaluated using a tissue microarray (TMA) constructed from surgical specimens of LADC and compared with clinicopathological features including the EGFR mutation status and the expressions of PD-L1, PD-L2, PD-1, CD4, CD8, CD204, and αSMA. If the proportion of positively stained tumor cells was greater than or equal to 5%, the tumor was considered to show FRα expression; if the H-score was more than or equal to 60, the tumor was considered to show high FRα expression. ResultsOverall, 466 TMA cores created from 233 LADC patients were evaluated: FRα-positive expression (FRα-pos)/negative (FRα-neg), 222/11; FRα high expression (FRα-HE)/low (FRα-LE), 190/43. AnEGFR mutation was present in 53.2 % of the patients. The median H-score of FRα expression, FRα-pos rate, and FRα-HE rate for EGFR mutation/wild type were 159/104 (p = 0.0002), 97.6/92.7 % (p = 0.0773), and 88.7/73.4 % (p = 0.0026), respectively. The H-scores for FRα had mild correlations with the proportion of tumor cells with positive staining for PD-L1 (r=-0.2557, p < 0.0001), the number of CD8-positive cells per square millimeter (r=-0.1767, p = 0.0069), and the area with positive staining for αSMA (r = 0.2049, p = 0.0017). No correlations were seen between FRα expression and other cancer-immunity markers. ConclusionTumor FRα expression was significantly higher in LADCs withEGFR mutation than in those with wild-type EGFR. This study suggested that FRα expression was related to cancer and microenvironment-immunity markers such as PD-L1 expression, CD8 cells, and αSMA.

IgA Fc-folate conjugate activates and recruits neutrophils to directly target triple-negative breast cancer cells.

According to the American Cancer Society, 1 in 8 women in the U.S. will develop breast cancer, with triple-negative breast cancer (TNBC) comprising 15-20% of all breast cancer cases. TNBC is an aggressive subtype due to its high metastatic potential and lack of targeted therapy. Recently, folate receptor alpha (FRA) is found to be expressed on 80% of TNBC with high expression correlating with poor prognosis. In this study, we examined whether binding IgA Fc-folate molecules to FRA receptors on TNBC cells can elicit and induce neutrophils (PMNs), by binding their FcαR1 receptors, to destroy TNBC cells. FRA was analyzed on TNBC cells and binding assays were performed using 3H-folate. Fc-folate was synthesized by linking Fc fragments of IgA via amine groups to folate. Binding specificity and antibody-dependent cellular cytotoxicity (ADCC) potential of Fc-folate to FcαR1 were confirmed by measuring PMN adhesion and myeloperoxidase (MPO) release in a cell-based ELISA. Fc-folate binding to FRA-expressing TNBC cells inducing PMNs to destroy these cells was determined using 51Cr-release and calcein-labeling assays. Our results demonstrate expression of FRA on TNBC cells at levels consistent with folate binding. Fc-folate binds with high affinity to FRA compared to whole IgA-folate and induces MPO release from PMN when bound to FcαR1. Fc-folate inhibited binding of 3H-folate to TNBC cells and induced significant cell lysis of TNBC cells when incubated in the presence of PMNs. These findings support the hypothesis that an IgA Fc-folate conjugate can destroy TNBC cells by eliciting PMN-mediated ADCC.

Abstract 3983: Tumor-targeting with novel dual-targeted 6-substituted thieno[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis

Abstract Epithelial ovarian cancer (EOC) is the deadliest gynecologic cancer, with limited progress in targeted therapy or in combating drug resistance. Reflecting this, there is an urgent need to develop novel inhibitors with minimal toxicity. EOC is associated with increasing expression of folate receptor alpha (FRα) with increasing stage and grade of disease. Of particular interest are targeted therapeutics toward FRα which spare transport by the ubiquitously expressed reduced folate carrier (RFC). FRα is a viable target for tumor selectivity with limited expression in normal tissues, where it is only expressed on apical membranes. We previously described 6-substituted thieno[2,3-d]pyrimidine (6TP) benzoyl antifolates with bridge lengths of 2-8 carbons as potent and selective inhibitors of tumor cells including EOCs that express FRs. The most potent compound (AGF50) included a 4-carbon bridge and showed FRα selectivity and inhibition of de novo purine biosynthesis. We synthesized AGF50 analogs with various aryl side-chain modifications (e.g., thiophene, furan, pyridine) including 2'fluorine substitutions. Toward isogenic Chinese hamster ovary cell lines individually expressing FRα, RFC and the proton-coupled folate transporter (PCFT), the 6TP analogs were completely selective toward FRα over RFC and PCFT, manifested as inhibition of cell proliferation with nmolar IC50s. Activity of this series extended to KB human tumor cells and human EOC cell lines (e.g., IGROV1). Drug effects were abolished with excess folic acid, confirming FRα uptake. Adenosine completely protected cells from the inhibitory effects of 6TPs in vitro, suggesting inhibition of one-carbon transfers mediated by glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase), leading to synthesis of purine nucleotides. Protection from 6TPs by AICA was incomplete (in contrast to a “pure” GARFTase inhibitor AGF94), suggesting inhibition of AICARFTase. Whereas AGF94 potently inhibited GARFTase (&amp;gt;98%) in KB cells, reflected in [14C]glycine incorporation into [14C]formyl GAR, inhibition by the 6TPs was incomplete (75-90%) up to 1 μM drug. AICARFTase inhibition was confirmed by measuring accumulation of AICA ribonucleotide (ZMP), although this varied for different 6TP analogs. Our studies establish an important structure-activity relationship for novel FRα-targeted 6TPs with aromatic side-chain modifications and imply the therapeutic potential of targeting AICARFTase for EOC. Citation Format: Adrianne Wallace-Povirk, Nian Tong, Carrie O'Connor, Zhanjun Hou, Aleem Gangjee, Larry Matherly, Xilin Zhou. Tumor-targeting with novel dual-targeted 6-substituted thieno[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3983.

Isolation of Circulating Tumor Cells from Multiple Epithelial Cancers with ApoStream(®) for Detecting (or Monitoring) the Expression of Folate Receptor Alpha.

This study describes our efforts to further the field of noninvasive diagnostics, specifically in the area of liquid biopsies in oncology. We employed laser scanning cytometry using highly selective antibodies to interrogate circulating tumor cells (CTCs) that were isolated using ApoStream® technology to identify folate receptor alpha (FRα)-positive cells. We demonstrate that FRα+ CTCs can be isolated from patients with metastatic cancers, including NSCLC adenocarcinoma, breast cancer, and ovarian cancer, whereas squamous cell lung cancer and normal healthy controls were devoid of FRα+ CTCs. We believe that the developed methodology will have applications in both the diagnosis and the monitoring of FRα-expressing cancers. Folate receptor alpha (FRα) expression may have utility as a potential diagnostic and therapeutic target in solid tumors. As tissue samples are not always available for patient screening, this study evaluated a noninvasive assay in CTCs from blood samples to detect FRα expression. The presence of FRα+ CTCs enriched using ApoStream® and detected using laser capture cytometry was evaluated in blood samples from cancer patients [NSCLC adenocarcinoma (n = 14), breast cancer (n = 20), ovarian cancer (n = 6), and squamous lung cancer patients (n = 6)] and healthy subjects (n = 20). The data demonstrated that FRα+ CTCs were detected in blood from NSCLC adenocarcinoma, breast, and ovarian cancer patients, whereas squamous cell lung cancer patients and normal healthy controls lacked FRα+ CTCs as previously known. We demonstrate that CTCs captured using ApoStream® can be used to detect FRα+ CTCs and may have clinical utility as a real-time liquid biopsy for assessing FRα levels in cancer patients.

Serum folate receptor alpha as a biomarker for ovarian cancer: Implications for diagnosis, prognosis and predicting its local tumor expression.

Folate receptor alpha (FRA) is a GPI-anchored glycoprotein and encoded by the FOLR1 gene. High expression of FRA is observed in specific malignant tumors of epithelial origin, including ovarian cancer, but exhibits very limited normal tissue expression, making it as an attractive target for the ovarian cancer therapy. FRA is known to shed from the cell surface into the circulation which allows for its measurement in the serum of patients. Recently, methods to detect the soluble form of FRA have been developed and serum FRA (sFRA) is considered a highly promising biomarker for ovarian cancer. We prospectively investigated the levels of sFRA in patients clinically suspected of having malignant ovarian tumors. A total of 231 patients were enrolled in this study and analyzed for sFRA as well as tumor expression of FRA by immunohistochemistry. High sFRA was predominantly observed in epithelial ovarian cancer patients, but not in patients with benign or borderline gynecological disease or metastatic ovarian tumors from advanced colorectal cancers. Levels of sFRA were highly correlated to clinical stage, tumor grade and histological type and demonstrated superior accuracy for the detection of ovarian cancer than did serum CA125. High sFRA was significantly associated with shorter progression-free survival in both early and advanced ovarian cancer patients. Finally, tumor FRA expression status was strongly correlated with sFRA levels. Taken together, these data suggest that sFRA might be a useful noninvasive serum biomarkers for future clinical trials assessing FRA-targeted therapy.

Relationship between the expression of folate receptor alpha in pituitary adenomas and part of the clinical indicators

Objective To study the expression and significance of folate receptor (FR) α in pituitary adenomas in order to provide a theoretical basis for targeting the diagnosis and treatment of pituitary adenomas. Methods RT-PCR, Western blot and immunohistochemistry were used to detect mRNA and protein expression levels of FRα in pituitary adenomas and normal pituitary tissue, and the relationship between the FRα expression and part of the clinical indicators were studied. Results Real-time quantitative polymerase chain reaction (RT-PCR) showed that the mRNA expression level of FRα in nonfunctioning (NF) pituitary adenomas was significantly higher than that of pituitary adenomas with endocrine function and normal pituitary tissue (P 0.05). Conclusions The expression of FRα in NF pituitary adenomas was significantly higher than that in pituitary adenomas with endocrine function and normal pituitary tissue, and it might be associated with the invasiveness of pituitary adenomas. This provided a certain research foundation for NF adenomas in future molecular targeted diagnosis and treatment. Key words: Folic acid; Pituitary neoplasms; Neoplasm invasiveness; Molecular targeted therapy

Abstract P2-11-19: Expression of folate receptor alpha in ductal carcinoma in situ of the breast and comparison with that of invasive ductal carcinoma

Abstract Background: We have previously shown that the expression of Folate receptor alpha (FRA) is associated with high grade ER-/PR- or triple negative (TN) invasive ductal carcinoma of the breast. The aim of the current study is to investigate the expression pattern of FRA in a cohort of ductal carcinoma in situ (DCIS), analyze its relationship with different clinicopathological features and expression of several key biomarkers, and compare these findings to our previous data on invasive ductal carcinoma (IDC). Methods: Ninety-eight cases of DCIS of the breast diagnosed between 1997 and 2008 in our institution were identified, reviewed and 6 blocks of tissue microassays (TMA) were constructed. The association between the expression of FRA (≥10% designed as positive expression) and the clinicopathologic features, the expression of ER, PR, HER2 and Ki-67, and the clinical outcome were evaluated, and these results were compared to those from IDC (399/430 cases with informative data). Results: Among the 98 cases of DCIS in TMA, 88 cases were informative. The expression rate of FRA was 21% for DCIS, less than the 29% seen in IDC. Expression of FRA was associated with high nuclear grades (42%), though less frequently than with IDC (52%). FRA expression was also associated with ER negativity (46%), PR negativity (33%) and high Ki-67 expression (50%) in DCIS, although significantly less than in IDC (75%, 63%, and 56%, respectively for negative ER and PR, and high Ki-67). Also, only 50% of ER/PR negative, TN and basal-like DCIS expressed FRA, significantly lower than that of IDC (74%, 80% and 80%, respectively). Due to the limited cases of DCIS, we did not demonstrate a significant difference in disease-free survival between FRA positive and negative tumors (p-value = 0.1792), although we did observe that IDC with FRA expression was significantly associated with a worse disease-free survival (p-value = 0.04). Comparison between FRA expression between DCIS and IDC FRA≥10% in DCISFRA≥10% in IDCp-value 88 cases399 cases Nuclear 342%52%0.0185ER negative46%75%0.0442PR negative33%63%0.0214HER2 3+31%37%0.7003ER-/PR-50%74%0.0973Triple Negative50%80%0.1622Basal-Like50%80%0.1739 Conclusion: Our data demonstrates that FRA expression is associated with high nuclear grade, ER/PR negative and TN DCIS, though at a significantly lower rate than was observed in IDC, suggesting a possible role for this receptor in early breast carcinogenesis. There are differences in the rates of FRA expression between DCIS and IDC, suggesting that FRA may be one of the key molecules involved in progression of DCIS to IDC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-19.

Abstract P2-11-20: Evaluation of folate receptor alpha expression in breast cancer by immunohistochemistry: Analysis with three different cut-off points

Abstract Background: Folate receptor alpha (FRA) is a newly recognized molecule that is selectively expressed in certain types of solid tumors, and several targeted therapies against FRA have shown promise in clinical trials for treating FRA-expressing ovarian and lung cancer. FRA has been shown to be selectively expressed in ER/PR negative and triple negative (TN) breast cancer; tumor types which would benefit from FRA targeted therapy. With these FRA-targeted therapies becoming available, correctly identifying eligible patients is very important. Currently, the cut off points for FAR expression by immunochemical (IHC) analysis have not been uniform. The commonly used cut points for expression that have been reported included 5%, 10% and the M score (using both staining intensity (3+, 2+, and 1+) and percentage of cells 0-100% (X, Y, Z for each staining intensity) in the equation (M-score = [3X+2Y+1Z]/ 6). The aim of the current study is to investigate these 3 most frequently used cut off points for FRA IHC analysis, in an effort to identify the one that is the most clinically relevant. Methods: 430 cases of infiltrating ductal carcinoma diagnosed between 1997 and 2008 in our institution were identified, reviewed, and 25 blocks of tissue microassays (TMA) were constructed. The association between the expression of FRA by IHC analysis, the clinicopathologic features, expression of ER, PR, HER2 and Ki-67, and the clinical outcome of these tumors were evaluated. Three cut off points (≥5%, ≥10% and M score of ≥10) were compared. 5% and 10% were scored as percent of membrane/cytoplasmic staining of the tumor cells regardless of the staining intensity while the M score takes into consideration both percentage and intensity of the staining of tumor cells.Results: Among the 430 cases studied, 399 cases had informative data from IHC analysis. The positive rates for FRA were 33%, 29% and 17%, respectively, for 5%, 10% and M score 10 as the positive cut off. All three cut off points were associated with significantly higher tumor grades and higher nodal status; they were also associated significantly with ER negativity, PR negativity or high proliferation rates (Ki-67≥15%) (P-values &amp;lt;0.0001 in all), and ER-/PR-, TN, or Basal-like tumors (p-values &amp;lt;0.0001 in all). Also, they were all significantly associated with worse disease-free survival with the p-values of 0.027, 0.026, and 0.009, for 5%, 10% and M score 10, respectively. FRA positive rates with three different cut off points FRA ≥5% (%)FRA ≥10% (%)FRA M-score ≥10 (%)Age &amp;gt;50 (year)32%28%17%Tumor size &amp;lt;2 (cm)28%26%16%Histologic grade 351%47%32%LN status &amp;gt;041%34%20%ER negative77%74%56%PR negative66%63%46%HER2 3+48%37%26%Ki67&amp;gt;15%60%56%41%ER-/PR-77%74%54%Triple Negative82%80%60%Basal-like82%80%59% Conclusion: Our data demonstrates that all three cut off points (5%, 10% and M score 10) could effectively separate breast tumors into subgroups where FRA expression correlated with specific pathologic features and a worse disease-free survival compared with FRA negative tumors. Using an M score of &amp;gt;10 as the cut off for FRA may be most specific method for determining the prognostic significance of FRA expression in breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-20.

Significance of Folate Receptor Alpha and Thymidylate Synthase Protein Expression in Patients with Non–Small-Cell Lung Cancer Treated with Pemetrexed

Folate receptor alpha (FRA) regulates cellular uptake of folates and antifolates. Information about FRA protein expression in metastatic non-small-cell lung cancer (NSCLC) is limited. We investigated FRA as a biomarker for pemetrexed-based chemotherapy and compared it with thymidylate synthase (TS), the main target of pemetrexed. Pretreatment tumor specimens from 207 patients with advanced NSCLC were assessed for FRA and TS protein expression by immunohistochemistry using the H-score (range, 0-300) and correlated to patients' clinicopathological data, radiographic response, progression-free survival (PFS), and overall survival (OS). Low total (cytoplasmic and nuclear) TS protein expression (H-score < 210) was associated with improved PFS (median: 5.6 versus 3.5 months; hazard ratio [HR] = 0.6379, p = 0.0131) and prolonged OS (median: 22.5 versus 11.5 months; HR = 0.5680,p = 0.0107). An association between lower TS levels and response to pemetrexed-based therapy was found-mean H-score 187 ± 5, median 180 for responders versus mean H-score 201 ± 4, median 210, for non-responders, p = 0.0244. High intracellular FRA expression (H-score ≥110) was associated with prolonged OS (28.9 versus 11.7 months, HR = 0.5316, p = 0.0040) and a trend for association with PFS (5.6 versus 4.1 months, HR = 0.7395, p = 0.0801) was noted. Membranous FRA expression was seen in 83% of patients, moreover, high membranous expression (H-score ≥20) was associated with improved PFS (5.6 versus 3.7 months, HR = 0.6445, p = 0.0306) and OS (22.1 versus 11.5 months, HR = 0.5378, p = 0.0131). A large number of NSCLC patients have high expression of FRA and/or a low level of TS expression. Expression levels of FRA and TS were associated with clinical benefit from pemetrexed therapy.

Folate receptor alpha (FRA) expression in breast cancer: identification of a new molecular subtype and association with triple negative disease

Given that several targeted therapies directed towards folate receptor alpha (FRA) are in late stage clinical development, the sensitive and robust detection of FRA in tissues is of paramount importance relative to patient selection, prognosis and prediction. In the present study we undertook an immunohistochemical evaluation of expression of FRA in breast cancer samples using formalin-fixed, paraffin-embedded (FFPE) tissues, primarily invasive ductal carcinomas, using a newly described monoclonal antibody, 26B3. Samples assessed included both tissue microarrays (TMA) and whole tissue sections from archival tissue blocks. Normal breast shows a highly restricted expression of FRA to luminal membrane staining of secretory ductal cells, consistent with FRA secretion into milk. In early stage (stages I-III) invasive ductal carcinomas, FRA staining was observed in approximately 30% of all samples, independent of molecular subtype (estrogen receptor (ER)/progesterone receptor (PR)/human epidermal growth factor receptor type 2 (Her2)). However, FRA expression was shown to associate with ER/PR negative tumors relative to ER/PR positive tumors (p = 0.012) and perhaps more importantly, with triple negative breast cancers (TNBC; p < 0.0001). FRA immunoreactivity was also shown to be retained in stage IV metastatic breast cancer samples from diverse anatomic sites including lymph node and bone. In metastatic breast cancer, FRA was shown to be expressed in 86% of TNBC patients. Taken together, these data suggest that FRA expressing breast cancer represents a novel molecular subtype and, further, may represent a new therapeutic target for this devastating disease.

Folate receptor alpha expression in lung cancer: diagnostic and prognostic significance.

With the advent of targeted therapies directed towards folate receptor alpha, with several such agents in late stage clinical development, the sensitive and robust detection of folate receptor alpha in tissues is of importance relative to patient selection and perhaps prognosis and prediction of response. The goal of the present study was to evaluate the expression of folate receptor alpha in non-small cell lung cancer specimens to determine its frequency of expression and its potential for prognosis. The distribution of folate receptor alpha expression in normal tissues as well as its expression and relationship to non-small cell lung cancer subtypes was assessed by immunohistochemistry using tissue microarrays and fine needle aspirates and an optimized manual staining method using the recently developed monoclonal antibody 26B3. The association between folate receptor alpha expression and clinical outcome was also evaluated on a tissue microarray created from formalin fixed paraffin embedded specimens from patients with surgically resected lung adenocarcinoma. Folate receptor alpha expression was shown to have a high discriminatory capacity for lung adenocarcinomas versus squamous cell carcinomas. While 74% of adenocarcinomas were positive for folate receptor alpha expression, our results found that only 13% of squamous cell carcinomas were FRA positive (p<0.0001). In patients with adenocarcinoma that underwent surgical resection, increased folate receptor alpha expression was associated with improved overall survival (Hazard Ratio 0.39, 95% CI 0.18-0.85). These data demonstrate the diagnostic relevance of folate receptor alpha expression in non-small cell lung cancer as determined by immunohistochemistry and suggest that determination of folate receptor alpha expression provides prognostic information in patients with lung adenocarcinoma.

Erratum to: The effect of chemotherapy on expression of folate receptor-alpha in ovarian cancer

Erratum to: Cell Oncol. (2011) DOI 10.1007/s13402-011-0052-6 The affiliations of the authors of the above mentioned article, published in Cellular Oncology, volume 35, issue 1, February 2012, pp. 9–18, should read: L. M. A. Crane . M. van Oosten . G. M. van Dam Department of Surgery, Division of Surgical Oncology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands H. J. G. Arts . A. G. J. van der Zee Department of Obstetrics and Gynaecology, Division of Gynaecologic Oncology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands P. S. Low Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA J. Bart (*Corresponding author) Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, P.O. Box 30.001, 9700 RB Groningen, the Netherlands