Indira Prabakaran

Tumoricidal activity of high-dose tumor necrosis factor-α is mediated by macrophage-derived nitric oxide burst and permanent blood flow shutdown

This study investigates the role of tumor nitric oxide (NO) and vascular regulation in tumor ulceration following high‐dose tumor necrosis factor‐α (TNF) treatment. Using TNF‐responsive (MethA) and nonresponsive (LL2) mouse tumors, tumor NO concentration was measured with an electrochemical sensor and tumor blood flow by Doppler ultrasound. Mice were also pretreated with a selective inducible nitric oxide synthase (iNOS) inhibitor, 1400 W. Tumors harvested from TNF‐treated mice were cryosectioned and immunostained for murine macrophages, or/and iNOS. MethA tumor‐bearing mice were depleted of macrophages. Pre‐ and post‐TNF tumor NO levels were measured continuously, and mice were followed for gross tumor response. In MethA tumors, TNF caused a 96% response rate, and tumor NO concentration doubled. Tumor blood flow decreased to 3% of baseline by 4 hr and was sustained at 24 hr and 10 days post‐TNF. Selective NO inhibition with 1400 W blocked NO rise and decreased response rate to 38%. MethA tumors showed tumor infiltration by macrophages post‐TNF and the pattern of macrophage immunostaining overlapped with iNOS immunostaining. Depletion of macrophages inhibited tumor NO increase and response to TNF. LL2 tumors had a 0% response rate to TNF and exhibited no change in NO concentration. Blood flow decreased to 2% of baseline by 4 hr, recovered to 56% by 24 hr and increased to 232% by 10 days. LL2 tumors showed no infiltration by macrophages post‐TNF. We conclude that TNF causes tumor infiltrating, macrophage‐derived iNOS‐mediated tumor NO rise and sustained tumor blood flow shutdown, resulting in tumor ulceration in the responsive tumor.

Mature CD83+ dendritic cells infected with recombinant gp 100 vaccinia virus stimulate potent antimelanoma T cells

BackgroundMature dendritics cells (DCs) are potent antigen-presenting cells that activate naive T lymphocytes and initiate cellular immune responses. The ability of CD83+ mature DCs infected with vaccinia virus encoding the gp 100 melanoma transgene (rV-gp 100) to stimulate an antimelanoma CD8+ T-cell response was investigated.MethodsMonocyte-derived immature or CD83+ mature DCs were infected with rV-gp100. The activation state of the DCs and the expression of gp 100 protein were evaluated by flow cytometry. The reactivity of antimelanoma CD8+ T cells was confirmed by measuring specific interferon γ secretion by using enzyme-linked immunosorbent assay in a mixed-tumor lymphocyte culture.ResultsBoth immature and CD83+ mature DCs expressed gp 100 protein when the DCs were infected with rV-gp 100. Calcium-signaling agents were required to induce maturation of both infected and nonifected immature DCs. Only rV-gp100-infected CD83+ DCs induced CD8+ T cells, after a single stimulation that recognized both peptide-pulsed target cells to multiple gp 100 epitopes and a melanoma cell line that endogenously expressed gp 100 antigen.ConclusionsCD83+ DCs transduced with rV-gp 100 are capable of generating a strong CD8+ T-cell response against melanoma tumor cells. Expression of melanoma antigens by mature DCs offers the potential advantage of presenting multiple endogenously processed T-cells epitopes and using multiple HLA restriction elements for antimelanoma vaccine therapy.

Rap2A Is Upregulated in Invasive Cells Dissected from Follicular Thyroid Cancer.

The development of molecular biomarkers (BMs) of follicular thyroid carcinoma is aimed at advancing diagnosis of follicular neoplasm, as histological examination of those tumors does not lend itself to definitive diagnosis of carcinoma. We assessed the relative levels of expression of 6 genes: CCND2, PCSK2, PLAB, RAP2A, TSHR, and IGF-1R in archived thyroid tissue. The quantitative real-time PCR analysis revealed a significant change in 3 genes: PSCK2 (a 22.4-fold decrease, P = 2.81E − 2), PLAB (an 8.3-fold increase, P = 9.81E − 12), and RAP2A (a 6.3-fold increase, P = 9.13E − 10) in carcinoma compared with adenoma. Expression of PCSK2 was equally low, PLAB was equally high, whereas RAP2A expression was significantly higher (25.9-fold, P = 0.039) in microdissected carcinoma cells that have invaded through the thyroid capsule and entered blood vessels than in thyroid tumor cells growing under the capsule. Thus, RAP2A appeared as a unique and worthy of further evaluation candidate BM associated with invasion of thyroid follicular cells.

Vav2 protein overexpression marks and may predict the aggressive subtype of ductal carcinoma in situ.

BackgroundA subset of patients with ductal carcinoma in situ (DCIS) will develop invasive breast cancer (IBC). To date, there are no effective predictive biomarkers for identifying this subset with worse prognosis whose lesions are essentially indistinguishable histologically from those with favorable outcomes. We hypothesized that measurable parameters that discriminate DCIS from DCIS with concurrent invasion may serve as diagnostic biomarkers (BM) of progressive cancer in situ (CIS).ResultsUsing a novel imaging-based method of tissue testing, we measured the relative expression levels of three candidate BM proteins specifically implicated in IBC progression - the insulin-like growth factor I receptor (IGF-IR), Ras-related protein 1 (Rap1), and Vav2 oncoprotein. Protein profiles were compared in 42 histologically normal mammary epithelial samples, 71 CIS (35 without/36 with invasion either on diagnostic biopsy or final surgical excision), and 98 IBC of known estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status. The levels of the IGF-IR and Rap1 protein expression were significantly elevated in ER-positive (ER+/PR+/-/HER2 –) DCIS relative to normal epithelium (P <0.0001). The IGF-IR protein expression was also significantly up regulated in HER2-positive (ER+/-/PR+/-/HER2+) DCIS relative to normal epithelium (P = 0.0002). IGF-IR and Rap1 protein expression levels were similar among DCIS patients without or with concurrent invasion. Vav2 upregulation in DCIS relative to normal group was not associated with steroid hormone receptor and HER2 status, but was associated with the presence of concurrent invasion, including microinvasion (invasive foci of less than 1 mm). DCIS with high Vav2 were more than twice as likely to progress to invasive cancers as DCIS with low Vav2 (odds ratio, 2.42; 95% CI, 1.26-4-65; P =0.008). Furthermore, a receiver operating characteristic curve analysis revealed moderate ability of Vav2 protein expression measurements in DCIS to predict the existence of invasion concurrent with DCIS (area under the curve, 0.71; 95% CI, 0.59- 0.84).ConclusionsOur novel findings hold promise for utilizing Vav2 protein as a predictive BM for differentiating progressive from non-progressive DCIS.

Abstract B24: The diagnostic power of direct mRNA profiling in breast cancer core biopsy

Background: Worldwide, biopsy is the gold standard for pathological assessment of cancer. Routinely, immunohistochemistry (IHC) can be performed to evaluate various tumor markers on serial sections of formalin-fixed paraffin-embedded (FFPE) tissue. Comprehensive molecular analysis of biopsy is limited by the microscopic size of samples and the scarcity of quantitative tools for FFPE tissue examination. Development of the new tools for molecular analysis of biopsy is needed to stratify, as early as possible, a patient’s individual risk for tumor aggressiveness and sensitivity to therapy to avoid potential over- and under treatment. Objective: To assess the feasibility of direct mRNA expression profiling of all three classic clinical markers of cancer progression - estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in routinely collected FFPE core needle biopsy (CNB) of the breast. Methods: We obtained tissue from the Department of Pathology and Laboratory Medicine at the University of Pennsylvania, the University of Pennsylvania Tumor Tissue and Biospecimen Bank and the Cooperative Human Tissue Network. Steroid hormone receptor and HER2 status, determined by FDA-approved methods, was obtained from the surgical pathology reports. For direct (i.e. without amplification) mRNA profiling, we ran the Panomics/Affymetrix QuantiGene Plex 2.0 assay on Luminex according to manufacturer’s protocol. After the removal of low-abundance housekeeping genes expressing samples, the relative activity of target genes, ERS1, PGR, ERBB2 encoding ER alpha, PR and HER2 was detected. mRNA profiling was completed in 107 breast samples: 6 clinically normal (N) cases from reduction mammoplasty, 17 ductal carcinoma in situ ( DCIS) on CNB , 42 invasive ductal carcinoma (IDC) and 42 non-malignant matched tissue from surgical excisions. Groups were compared using one-way analysis of variance (ANOVA). Results: A pair-wise comparison among tissue groups revealed 3-fold higher expression levels of ERS1 in ER+ DCIS on CNB (P=. 055), and statistically significantly higher expression levels of ERS1 in the ER+/PR+ (P= .021) and ER+/PR- (P= .004) subgroups of IDC than in the N group. Remarkably, compared with N tissue, ERS1 levels were already decreased in non-malignant tissue adjacent to ER-/PR-/HER2- subgroup of IDC and were further significantly decreased in ER-/PR-/HER2- IDC itself (P= .023). Compared to the N group, significant decreases in PGR levels were found only in ER-/PR-/HER2- subgroup of IDC (P= .003), whereas significant increases in ERBB2 levels were found in both HER2+ DCIS on CNB (P= . 037) and HER2+IDC in excisions (P= .005). Conclusions: Our study, for the first time, proved the feasibility of using FFPE CNB as an RNA source for direct mRNA expression profiling. These findings support the notion that IHC evaluation of ER, PR, and HER2 status in the CNB DCIS may be complemented by concurrent detection of ESR1/PGR/ERBB2 mRNA levels. Citation Format: Indira Prabakaran, Paul J. Zhang, Marina Guvakova. The diagnostic power of direct mRNA profiling in breast cancer core biopsy. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B24.

Abstract 5283: Vav2 oncoprotein up regulation may predict the aggressive subtype of ductal carcinoma in situ

Background: A subset of patients with ductal carcinoma in situ (DCIS) will develop invasive breast cancer (IBC). To date, there are no effective predictive biomarkers for identifying this subset with worse prognosis whose lesions are essentially indistinguishable histologically from those with favorable outcomes. We hypothesized that measurable parameters that discriminate DCIS from DCIS with concurrent invasion may serve as diagnostic biomarkers (BM) of progressive cancer in situ (CIS). Methods: Using a novel imaging-based method of tissue testing, we measured the relative expression levels of three candidate BM proteins specifically implicated in IBC progression - the insulin-like growth factor I receptor (IGF-IR), Ras-related protein 1 (Rap1), and Vav2 oncoprotein. Protein profiles were compared in 42 histologically normal mammary epithelial samples, 71 CIS (35 without/36 with invasion either on diagnostic biopsy or final surgical excision), and 98 IBC of known estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status. Groups were compared using one-way analysis of variance (ANOVA) followed by pair-wise t-tests with Tukey9s correction for multiple testing. Receiver operating characteristic (ROC) curves were constructed to evaluate how well each BM can predict tumor type. Results: The levels of the IGF-IR and Rap1 protein expression were significantly elevated in ER-positive (ER+/PR+/-/HER2 -) DCIS relative to normal epithelium (P Conclusions: This study describes the use of a novel imaging method for archival tissue testing, which may inform the status of protein BM in archived tissue and may help to stratify a women9s individual risk for tumor invasiveness to avoid potential over- or under-treatment. Despite the apparent limitation of our study DCIS cohort size, our novel findings hold promise for utilizing Vav2 as a predictive BM of progressive DCIS and a target for cancer therapy. Citation Format: Marina A. Guvakova, Yun Qing Jiang, Indira Prabakaran, Fei Wan, Nandita Mitra, Paul J. Zhang, Douglas L. Fraker. Vav2 oncoprotein up regulation may predict the aggressive subtype of ductal carcinoma in situ. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5283. doi:10.1158/1538-7445.AM2015-5283

Abstract B40: The quest for biomarkers of progressive DCIS using archived tissue.

Background: Presently, an indolent ductal carcinoma in situ (DCIS) cannot be distinguished from a progressive tumor, making the appropriate treatment of DCIS patients a major clinical dilemma. Here, we tested if measuring protein levels in archived tissue could facilitate the development of biomarkers (BMs) accurately discriminating progressive from non-invasive DCIS. We theorized that BMs of progressive lesion are measurable parameters that distinguish pure DCIS from DCIS with concurrent invasive breast cancer (IBC). To test the hypothesis, we studied the insulin-like growth factor I receptor (IGF-IR), Ras oncogene-like protein 1 (Rap1), and oncoprotein Vav2 implicated in the progression of IBC. Methods: We developed and applied a new quantitative imaging-based uniplex (IBU) method to measure in-tissue protein expression. The quantification was performed on a continuous scale, and multiple repeated measurements of relative (rather than absolute) intensity of immunohistochemical staining took into account fluctuations of background. Protein profiling was completed in 211 breast samples: 42 histologically normal, 71 cancer in situ (with/without associated invasion), and 98 IBC. Groups were compared using one-way analysis of variance (ANOVA) followed by pair-wise t-tests with Tukey9s correction for multiple testing. Receiver operating characteristic (ROC) curves were constructed to evaluate how well each BM can predict tumor type. Results: A pair-wise comparison among tissue groups revealed statistically significantly higher levels of IGF-IR and Rap1 in the in situ and IBC groups than in the normal group (P Conclusions: The application of our IBU method identified previously undetected changes in IGF-IR, Rap1, and Vav2 that co-evolve with IBC progression. Vav2 may be a valuable BM for the diagnosis of progressive DCIS. Improvement in earlier detection of DCIS with aggressive phenotype would go a long way in advancing efforts towards prevention of IBC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B40. Citation Format: Marina A. Guvakova, YunQing Jiang, Indira Prabakaran, Fei Wan, Nandita Mitra, Douglas L. Fraker, Paul J. Zhang. The quest for biomarkers of progressive DCIS using archived tissue. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B40.

Abstract C23: The quest for biomarkers of progressive DCIS using archived tissue

Background: Presently, an indolent ductal carcinoma in situ (DCIS) cannot be distinguished from a progressive tumor, making the appropriate treatment of DCIS patients a major clinical dilemma. Here, we tested if measuring protein levels in archived tissue could facilitate the development of biomarkers (BMs) accurately discriminating progressive from non-invasive DCIS. We theorized that BMs of progressive lesion are measurable parameters that distinguish pure DCIS from DCIS with concurrent invasive breast cancer (IBC). To test the hypothesis, we studied the expression of the insulin-like growth factor I receptor (IGF-IR), Ras oncogene –like protein 1 (Rap1), and oncoprotein Vav2 implicated in the progression of IBC. Methods: We developed and applied a new quantitative imaging-based uniplex (IBU) method to measure in-tissue protein expression. The quantification was performed on a continuous scale, and multiple repeated measurements of relative (rather than absolute) intensity of immunohistochemical staining took into account fluctuations of background. Protein profiling was completed in 211 breast samples: 42 histologically normal, 71 cancer in situ (with/without associated invasion), and 98 IBC. Groups were compared using one-way analysis of variance (ANOVA) followed by pair-wise t-tests with Tukey9s correction for multiple testing. Receiver operating characteristic (ROC) curves were constructed to evaluate how well each BM can predict tumor type. Results: A pair-wise comparison among tissue groups revealed statistically significantly higher levels of IGF-IR and Rap1 in the in situ and IBC groups than in the normal group (P Conclusions: The application of our IBU method identified previously undetected changes in IGF-IR, Rap1, and Vav2 that co-evolve with IBC progression. Vav2 may be a valuable BM for the diagnosis of progressive DCIS. Improvement in earlier detection of DCIS with aggressive phenotype would go a long way in advancing efforts towards prevention of IBC. Citation Format: Marina A. Guvakova, YunQing Jiang, Indira Prabakaran, Fei Wan, Nandita Mitra, Douglas L. Fraker, Paul J. Zhang. The quest for biomarkers of progressive DCIS using archived tissue. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C23.