Lymph Node Metastasis In Breast Cancer Research Articles

P037 - The methylation of RASSF1A gene and axillary node metastases in breast cancer

P037 - The methylation of RASSF1A gene and axillary node metastases in breast cancer

Abstract P2-01-31: Validation of novel diagnostic kits using the semi-dry dot-blot method for detecting metastatic lymph nodes in breast cancer; distinguishing macrometastases and micrometastases

Abstract Background: The semi-dry dot-blot (SDB) method is a diagnostic procedure for detecting lymph node (LN) metastases. Metastases are confirmed by the presence of cytokeratin (CK) in lavage fluid of sectioned LNs that contain anti-pancytokeratin antibody, based on the theory that epithelial components such as CK are not found in normal LNs. We evaluated two novel SDB kits that use the newly developed anti-CK19 antibody for diagnosing LN metastases in breast cancer. Methods: We obtained 159 LNs dissected from 93 breast cancer patients from July 2013 to December 2015 at Nagasaki University Hospital, including 38 dissected axillary LNs and 121 sentinel LNs, sliced at 2-mm intervals and washed with phosphate-buffered saline. The suspended cells in the lavage fluid of sliced LNs were centrifuged and lysed to extract protein. This extracted protein was used with a low-power and a high-power kit to diagnose LN metastasis. The washed LNs were blindly diagnosed by pathologists using hematoxylin and eosin (H&E) stain. Diagnoses based on the kit were compared with their H&E counterparts. Results: Of the 159 LNs, 68 were assessed as positive and 91 as negative by permanent pathological examination with H&E. Sensitivity, specificity, and accuracy of the low-power kit for detecting LN metastases was 83.8%, 100%, and 93.1%, respectively. In 11 false-negative cases, there were nine micrometastases, producing a sensitivity of 96.4% for detecting macrometastases. Sensitivity, specificity, and accuracy of the high-power kit for detecting LN metastases was 92.6%, 92.3%, and 92.5%, respectively. Combining the low- and high-power kit results, sensitivity, specificity and accuracy for distinguishing macrometastases from micrometastases was 94.5%, 95.2%, and 95.0%, respectively. Diagnosis was achieved in approximately 20 min using the kits, at a cost of less than 25 USD. Conclusions: The kits in our study were accurate, quick, and cost-effective in diagnosing LN metastases without the loss of LN tissue. The kits' ability to distinguish macrometastases from micrometastases was excellent, which is important, clinically. Citation Format: Otsubo R, Hirakawa H, Oikawa M, Inamasu E, Baba M, Matsumoto M, Yano H, Kinoshita N, Abe K, Fukuoka J, Nagayasu T. Validation of novel diagnostic kits using the semi-dry dot-blot method for detecting metastatic lymph nodes in breast cancer; distinguishing macrometastases and micrometastases [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-01-31.

Intratumoral and peritumoral lymphatic vessel density both correlate with lymph node metastasis in breast cancer

The status of lymph node involvement is an important prognostic factor for breast cancer. However, the presence of intratumoral lymphatic vessels in primary tumor lesions and the relationship between lymphatic vessel density (LVD) and lymph node metastasis (LNM) have not been firmly established. Therefore, we performed a meta-analysis study to investigate these issues. According to the pre-established inclusion and exclusion criteria, 13 studies, involving 1029 breast cancer patients, were included in this study. Using immunohistochemical staining, intratumoral lymphatic vessels were detected in 40.07% of breast cancer patients (240/599), and peritumoral lymphatics were detected in 77.09% (397/515). All studies demonstrated that peritumoral LVD was higher than intratumoral LVD, with a pooled standard mean difference and 95% confidence interval (95% CI) of 1.75 (1.28 to 2.21). Both intratumoral LVD and peritumoral LVD positively correlated with LNM, with correlation coefficients of 0.14 (95% CI 0.05 to 0.23) and 0.31 (95% CI 0.13 to 0.49), respectively. In summary, our study reports the overall detection rate of intratumoral lymphatics and demonstrates the associations between intratumoral LVD, peritumoral LVD, and LNM in breast cancer. Additionally, controlled studies with a larger number of subjects are needed to establish these relationships.

Evaluation of Sentinel Lymph Node Intraoperative Touch Imprint Cytology in Breast Cancer Surgery

Background: One of the strongest prognostic factor for breast cancer is the regional lymph node status, which can be evaluated intraoperatively by sentinel lymph node biopsy. Many methods (e.g. frozen section, touch imprint cytology (TIC) and one-step nucleic-acid amplification) are available to detect metastatic cells in axillary lymph nodes. The aim of this study is to evaluate the feasibility of using TIC to detect metastatic cells in sentinel lymph nodes. Methods: This is a retrospective single center cohort analysis conducted on prospectively recorded data. The study included all patients admitted to the Department of General Surgery of Trieste University Hospital for invasive clinically node-negative breast cancer who underwent sentinel lymph node assessment by means of TIC. Results: Between January 2015 and December 2016, 343 patients (338 females and 5 males) underwent breast surgery and sentinel lymph node TIC. Patient’s median age was 66 (33-92) years. The sensitivity of TIC was 54% (95% C.I. 39% to 69%), whereas its specificity and accuracy were 99% (95% C.I. 98% to 100%) and 90%, respectively. The median time required to obtain the result was 20 (15-45) min. The overall cost per each TIC analysis was about 20.50€. Conclusion: Touch imprint cytology appears to be a fast, cost-effective and reliable technique to intraoperatively detect breast cancer lymph node metastasis.

Accuracy of Axillary Ultrasound in the Detection of Nodal Metastasis in Breast Cancer: Experience on 620 cases

Purpose: Axillary lymph node status is one of the most important prognostic factors in early-stage breast cancer. Sentinel lymph node biopsy is used to determine the status of axillary nodes. There is a subset of patients in whom preoperative identification of nodal metastases could lead directly to axillary dissection. Preoperative axillary ultrasonography is a generally available noninvasive technique for assessing nodal status. Materials and Methods: Based on retrospective data, we analyzed the sensitivity, specificity, positive and negative predictive values, and accuracy of preoperative ultrasonography performed at our institution on patients who underwent surgery for breast cancer from January 2009 to December 2010 (24 months). A total of 620 axillary ultrasonographic examinations were included, and results were compared with pathological exam. Results: Ultrasonography revealed unremarkable findings in 500/620 (80%) axillae. There were 368 true negatives, 91 true positives, 29 false positives and 132 false negatives. Sensitivity was 40.8% and specificity 92.7%. Preoperative ultrasonography had a positive predictive value of 75.8%, and a negative predictive value of 73.6%, with an accuracy of 74.0%. Conclusion: This sample from our institution represents one of the largest reported in the literature and shows that preoperative axillary ultrasound is a method with high specificity but relatively low sensitivity for detecting the presence of axillary metastasis.

Does the TNM classification of solitary internal mammary lymph node metastases in breast cancer still apply?

PurposeTNM classification of solitary internal mammary lymph node metastases (IMLNMs) in breast cancer varies depending on their method of detection: sentinel lymph node biopsy (pN1b) or clinical examination including radiological and/or physical examination (pN2b). This study aimed to evaluate whether there is a difference in prognosis between both groups.MethodsData of all patients diagnosed with primary invasive epithelial breast cancer between 2005 and 2008 were obtained from the Netherlands Cancer Registry. Patients with IMLNMs were divided in groups according to their pN1b and pN2b status. The main outcome measures disease-free survival (DFS) after 5 years and overall survival (OS) after 8 years were analyzed using Kaplan–Meier survival analysis. Cox regression analysis was used to determine independent predictors for DFS and OS.ResultsA total of 73 patients with pN1b status and 28 patients with pN2b status were included. DFS rate was 74.1% in the pN1b group compared to 85.0% in the pN2b group (p = 0.211). Regarding OS, 20.5% (pN1b) and 25.0% (pN2b) of the patients deceased within 8 years of follow-up (p = 0.589). In multivariable cox regression analysis, nodal status was not statistically significant for DFS (HR 0.29 [95% CI 0.04–2.33], p = 0.244) or OS (HR 1.04 [95% CI 0.37–2.89], p = 0.947).ConclusionsAlthough the TNM classification considers pN1b and pN2b to be distinct prognostic entities, we did not observe any prognostic differences between these groups. Therefore, solitary IMLNMs may be regarded as a single category in the future and revision of TNM classification should be considered.

The relationship of lymphatic vessel density, lymphovascular invasion, and lymph node metastasis in breast cancer: a systematic review and meta-analysis.

Lymph node status is one of the key parameters used for determining the stage of breast cancer progression. The relationship of lymphatic vessel density (LVD), lymphovascular invasion (LVI), and lymph node metastasis (LNM) has not been clearly demonstrated yet. Databases of PubMed, Embase, and Web of Science were searched from inception up to 25 May 2016. Spearman correlation coefficient (r) and 95% confidence interval (CI) were used to determine the relationship within each group. Based on pre-established inclusion criteria, 28 studies involving 2920 breast cancer patients were included in this study. The r values of LVD-LVI, LVD-LNM, and LVI-LNM were 0.45 (95% CI: 0.31 to 0.57), 0.32 (95% CI: 0.23 to 0.40), and 0.24 (95% CI: 0.19 to 0.28), respectively. Compared with intratumoral LVD, peritumoral LVD showed more robust correlation with LVI (r = 0.53, 95% CI: 0.27 to 0.72) and LNM (r = 0.33, 95% CI: 0.18 to 0.46). The patients in LNM positive group presented with higher LVI detection rate of 45.85%, while in LNM negative group with detection rate of 23.85%. The results describe a triangle relationship between LVD, LVI, and LNM in breast cancer. Both LVD and LVI are indicated to be valuable predictors of LNM occurrence. Compared with intratumoral lymphatic vessels, peritumoral lymphatics might be the main disseminate route for breast tumor cells.

HER2 expression is a strong independent predictor of nodal metastasis in breast cancer

Identification of metastatic potential of breast cancer cells is necessary for proper management of this disease. This work aimed to estimate likelihood of axillary lymph node (ALN) involvement in breast cancer patients based on human epidermal growth factor receptor 2 (HER2) expression. Primary tumors of 317 breast cancer patients were evaluated for estrogen receptor (ER), progesterone receptor (PR) and HER2 expression by immunohistochemistry. The validity of these molecules to predict ALN metastasis was measured statistically and compared to predictive effect of other clinicopathological parameters. ER, PR and HER2 expression was detected in 75.7%, 73.2% and 19.9% of tumors, respectively. Although increased tumor size and grade, ER and PR negativity and HER2 positivity were strong indicators of ALN metastasis on univariate analyses, only tumor size and HER2 expression were independent predictors of ALN involvement on multivariate analysis. ROC curve showed a strong validity of the model using these two parameters to predict ALN status (AUC 0.86; p<0001). HER2-rich, luminal B and triple negative tumors had 6.87, 6.32 and 3.58 times increased risk of metastasis compared to luminal A tumors; respectively. HER2 expression in pT1 and pT2 tumors raised the risk of ALN metastasis by 7.7 and 7.6 times, respectively and grade 1 and 2 tumors that expressed HER2 were 16.0 and 7.8 times more likely to have ALN metastasis, respectively. To conclude, HER2 expression is associated with a significant rise of metastatic potential of breast cancer cells and could be a strong indicator of regional and distant metastasis of breast cancer.

Three-Dimensional Analysis of Patterns of Supraclavicular Nodal Metastases in Breast Cancer: Clinical Target Volume Covering Posterolateral Supraclavicular Fossa May Not Be Justified

Three-Dimensional Analysis of Patterns of Supraclavicular Nodal Metastases in Breast Cancer: Clinical Target Volume Covering Posterolateral Supraclavicular Fossa May Not Be Justified

One-step nucleic acid amplification (OSNA): where do we go with it?

The one-step nucleic acid amplification (OSNA) assay was initially developed for the intraoperative assessment of sentinel lymph node metastases in breast cancer. This assay measures cytokeratin 19 (CK19) mRNA copy number and is widely used in hospitals. The results of the IBCSG 23-01, ACOSOG Z0011, and AMAROS trials demonstrated that no further axillary dissection is required for patients with sentinel lymph nodes that tested positive for cancer, which has led to a decreasing trend in the need for intraoperative assessment of lymph nodes. Here, I review studies relevant to OSNA and discuss perspectives on future applications of OSNA in cancer surgery. The studies reviewed were identified by carrying out a search on PubMed for all articles pertaining to OSNA and published prior to the end of June 2016 using the keywords "OSNA" or "one-step nucleic acid amplification" in the title or abstract. Method comparison studies between OSNA and pathological assessment for the detection of lymph node metastasis in breast cancer revealed that in a pooled assessment OSNA had a high specificity (94.8%), high concordant rate (93.8%), and a negative predictive value (97.6%). Similar results have been found for gastric, colorectal, and lung cancers in multicenter studies. These results demonstrate that OSNA can serve as an alternative method to pathological assessment for examining lymph node metastasis. Multicenter prospective studies with a large sample size are needed to definitively reveal the superiority of OSNA over pathological assessment to predict prognosis. Technical refinements to improve the assay are essential to its further development as a new standard for testing in place of pathological examination.

Abstract 56: Suppression of 6-Phosphofructo-2-Kinase (PFKFB3) for the treatment of breast cancer

Abstract Glucose uptake by tumors is stimulated by estradiol (E2) administration in estrogen receptor positive (ER+) breast cancer patients. Notably, this E2-induced metabolic flare is predictive of the clinical effectiveness of anti-estrogens and, therefore, downstream metabolic regulators of E2 are expected to have utility as anti-breast cancer agents. Although the stimulation of glucose metabolism by E2 has been demonstrated, relatively little is known about the precise downstream effectors required for E2 to stimulate glucose metabolism in breast cancer. We have demonstrated that the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of the glycolytic flux, is elevated in breast cancer lymph node metastases and that exposure of ER+ human MCF-7 and T-47D breast cancer cells to E2 causes a rapid increase in 14C-glucose uptake and glycolysis that is coincident with an induction of PFKFB3 mRNA (via ER binding to its promoter), protein expression and the intracellular concentration of its product, fructose-2,6-bisphosphate (F26BP). Importantly, we also found that selective inhibition of PFKFB3 expression and activity using siRNA or a PFKFB3 inhibitor, PFK158, markedly reduces the E2-mediated increase in F26BP, 14C-glucose uptake and glycolysis. In the current study, we sought to determine if co-administration of PFK158, with ICI 182,780 (fulvestrant), would offer a greater anti-tumor effect. In unpublished results, we demonstrated that the combination of the anti-estrogen, fulvestrant, with PFK158 results in greater regressions of MCF-7 xenografts than either drug alone in athymic BALB/c mice. In addition to regulating glucose metabolism, PFKFB3 has been found to be a regulator of the cell cycle via cyclin dependent kinases. We postulated that the combination of PFK158 with the newly FDA-approved CDK4/6 inhibitor palbociclib may result in a synergistic decrease in tumor cell growth. We found that exposure of breast cancer cells to palbociclib and PFK158 causes a synergistic increase in cell cycle arrest and apoptotic cell death. In addition, we observed a synergistic decrease in phospho-retinoblastoma protein (Rb), a key downstream target of CDK4/6. Importantly, we found that the combination of PFK158 and palbociclib did not cause an increase in cell cycle arrest or apoptosis in normal human mammary epithelial cells. Taken together, these data indicate that PFKFB3 inhibitors such as PFK158 may have clinical utility for the treatment of ER+ breast cancers when combined with anti-estrogen agents and/or CDK4/6 inhibitors. Citation Format: Yoannis Imbert-Fernandez, Amy Clem, Brian Clem, Gilles Tapolsky, Sucheta Telang, Jason Chesney. Suppression of 6-Phosphofructo-2-Kinase (PFKFB3) for the treatment of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 56.

Evaluation of the Metasin assay for intraoperative assessment of sentinel lymph node metastases in breast cancer

AimsSentinel lymph node (SLN) biopsy is the preferred surgical technique for staging the axilla in clinically node-negative breast cancer. Accurate intraoperative staging allows for the immediate performance of an axillary...

435 - Impact of Aspirin on factors associated with breast cancer lymph node metastasis

435 - Impact of Aspirin on factors associated with breast cancer lymph node metastasis

Diagnostic Performance of Indocyanine Green-Guided Sentinel Lymph Node Biopsy in Breast Cancer: A Meta-Analysis.

BackgroundThe diagnostic performance of indocyanine green (ICG) fluorescence-guided sentinel lymph node biopsy (SLNB) for the presence of metastases in breast cancer remains unclear.ObjectiveWe performed a meta-analysis to investigate the diagnostic performance of ICG-guided SLNB.MethodsEligible studies were identified from searches of the databases PubMed and EMBASE up to September 2015. Studies that reported the detection rate of ICG fluorescence-guided SLNB with full axillary lymph node dissection and histological or immunohistochemical examinations were included. A meta-analysis was performed to generate pooled detection rate, sensitivity, specificity, false negative rate, diagnostic odds ratio (DOR) and a summary receiver operator characteristic curve (SROC).ResultsNineteen published studies were included to generate a pooled detection rate, comprising 2594 patients. The pooled detection rate was 0.98 (95% confidence interval [CI], 0.96–0.99). Six studies finally met the criteria for meta-analysis, which yielded a pooled sensitivity of 0.92 (95% CI, 0.85–0.96), specificity 1 (95% CI, 0.97–1), and DOR 311.47 (95% CI, 84.11–1153.39). The area under the SROC was 0.9758. No publication bias was found.ConclusionICG fluorescence-guided SLNB is viable for detection of lymph node metastases in breast cancer. Large-scale randomized multi-center trials are necessary to confirm our results.

AKR1B10 promotes breast cancer metastasis through integrin α5/δ-catenin mediated FAK/Src/Rac1 signaling pathway

Aldo-keto reductase 1B10 (AKR1B10) is not expressed in normal breast, but upregulated in primary and metastatic breast cancers, being a negative prognostic factor. This study characterized the molecular mechanisms of AKR1B10-promoted breast cancer metastasis. Ectopic expression of AKR1B10 in breast cancer cells MCF-7 and MDA-MB-231 or siRNA-mediated silencing in BT-20 cells affected cell adhesion, migration and invasion in cell culture, and metastasis to the lung in the nude mice through upregulation of integrin α5 and δ-catenin. Silencing of integrin α5 or δ-catenin eradicated the cell adhesion and migration enhanced by AKR1B10, both of which acted synergistically. In these cells, the integrin α5 mediated focal adhesion kinase (FAK) signaling pathway was activated by AKR1B10, which, along with δ-catenin, stimulated Rac1-mediated cell migration and movement. In human primary and lymph node metastatic breast cancer, AKR1B10, integrin α5 and δ-catenin were correlatively upregulated with r=0.645 (p<0.0001) and r=0.796 (p<0.0001), respectively. These data suggest that AKR1B10 promotes breast cancer metastasis through activation of the integrin α5 and δ-catenin mediated FAK/Src/Rac1 signaling pathway.

Predictive factors for non-sentinel node metastases in breast cancer: Preliminary results of a risk model

Predictive factors for non-sentinel node metastases in breast cancer: Preliminary results of a risk model

Evaluación clínica, ecográfica y citológica de la axila en el cáncer de mama

ObjectiveTo evaluate the efficacy of axillary ultrasound and ultrasound-guided fine-needle aspiration cytology for the diagnosis of lymph node metastases in breast cancer. Material and methodsThis study included 340 consecutive patients with breast cancer. In all patients, the ipsolateral axilla was examined with ultrasound. Ultrasound-guided fine-needle aspiration cytology was performed in suspicious-looking lymph nodes. In all patients, the final pathologic result was obtained by sentinel node biopsy or axillary clearance. ResultsThe following values were obtained: sensitivity 80%, specificity 100%, positive predictive value 100%, negative predictive value 91%, false positives 0%, false negatives 9%.In patients with a clinically negative axilla and ultrasound-guided fine-needle aspiration cytology there were more than 2 metastatic lymph nodes in 63% of tumors measuring up to 20mm in diameter and in 83% of tumours larger than 20mm.In tumours measuring up to 20mm, there was only one metastatic lymph node in 92% of patients with a false negative result in the axillary ultrasound and in only 2 metastatic lymph nodes in 8%. ConclusionAxillary ultrasound and ultrasound-guided fine needle aspiration cytology have a high precision for axillary staging, offering useful information for treatment planning and allowing precise selection of breast cancer patients with a low tumour volume in the axilla.

Abstract P3-01-22: Sentinel lymph node metastases in breast cancer: A contributor to distant metastases?

Abstract Breast cancer metastasis remains a major cause of mortality in patients. Although significant progress has been made in understanding the mechanisms of this complex process, the findings have yet to be translated into improved survival rates in patients with metastatic disease. The presence of lymph node metastasis in most breast cancer patients is associated with tumor aggressiveness, poorer prognosis and often results in the need for systemic therapy. However, whether tumor cells in the lymph node exit and contribute to distant metastases remains controversial. To track the fate of tumor cells that metastasized to the lymph node, we engineered breast tumor cell lines to express Dendra2, a photo-convertible protein that fluoresces green in the native state and on light activation converts to red fluorescence. Using a novel chronic lymph node window that allows time-lapse imaging over a period of 10 days, we were able to track the movement of cancer cells in the dynamic microenvironment using high- resolution multi-photon microscopy. Our studies show that tumor cells entering the lymph node through the afferent lymphatic vessel proliferate in the sub-capsular sinus and later begin to invade the lymph node parenchyma. Importantly, by photo-conversion of tumor cells in the lymph node, we are able to track the tumor cells that escaped the lymph node and entered the blood circulation. The circulating tumor cells that transited through the lymph node were viable and proliferated in vitro. Our data indicate that metastatic breast cancer cells can exit the node and potentially colonize distant organs. Citation Format: Pereira ER, Kedrin D, Jones D, Beech E, Taghian A, Padera TP. Sentinel lymph node metastases in breast cancer: A contributor to distant metastases?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-01-22.

Abstract P3-01-18: Validation of a novel diagnostic kit using the semi-dry dot-blot method for detecting metastatic lymph nodes in breast cancer

Abstract Background: The semi-dry dot-blot (SDB) method is a diagnostic procedure for detecting lymph node (LN) metastases. The metastases are visualized by the presence of cytokeratin (CK) with lavage fluid of sectioned LNs by anti-pancytokeratin antibody, based on the theory that epithelial components such as CK are not found in normal LNs. We previously reported 93.3% sensitivity, 96.9% specificity, and 96.6% accuracy for this method in detecting metastasis in sentinel LNs, compared with permanent pathological diagnosis in breast cancer. In this study, we evaluated a novel kit that applies the SDB method using the newly developed anti-CK19 antibody for diagnosing LN metastases in breast cancer. Methods: We obtained 141 LNs dissected from 81 breast cancer patients from July 2013 to April 2015 at Nagasaki University Hospital and the Japanese Red Cross Nagasaki Genbaku Hospital, including 33 dissected axillary LNs and 108 sentinel LNs, which were sliced at 2-mm intervals and washed with phosphate-buffered saline. The suspended cells in the lavage fluid of sliced LNs were centrifuged to collect the cell pellet and lysed with lysis buffer to extract protein. This extracted protein was used with the kit to diagnose LN metastasis. The washed LNs were blindly diagnosed by pathologists using hematoxylin and eosin (H&amp;E) stain. Diagnoses based on the kit were compared with their H&amp;E counterparts. Results: Of the 141 LNs, 57 were assessed as positive and 84 as negative by permanent pathological examination with H&amp;E. Use of the kit resulted in correct diagnoses in 46 of the 57 pathologically positive cases and all of pathologically negative cases. Sensitivity, specificity and accuracy of the kit in detecting LN metastases were 80.7% (95% confidence interval [95% CI]: 75.6–80.7%), 100% (95% CI: 96.5–100%), and 92.2% (95% CI: 88.1–92.2%), respectively. In 11 false-negative cases, there were 9 micrometastases; therefore, sensitivity was 95.5% (95% CI: 90.1–95.5%) in cases of macrometastases. Diagnosis was achieved in approximately 20 min using the kit, reducing the diagnostic time by half compared with the original SDB method. The cost of this kit was within 8 USD, and we are currently developing an improved kit for the detection of smaller metastases. Conclusions: The kit in our study is accurate, quick, and cost-effective in diagnosing LN metastases without the loss of LN tissue. Its sensitivity in detecting macrometastases is excellent, which is important in clinical practice. Citation Format: Otsubo R, Hirakawa H, Oikawa M, Shibata K, Tanaka A, Matsumoto M, Yano H, Nagayasu T. Validation of a novel diagnostic kit using the semi-dry dot-blot method for detecting metastatic lymph nodes in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-01-18.

Abstract A84: Regulation of 6-phosphofructo-2-kinase (PFKFB3) by estradiol and implications for the treatment of ER+ metastatic breast cancer

Abstract Estradiol (E2) administered to estrogen receptor positive (ER+) breast cancer patients stimulates glucose uptake by tumors. This E2-induced metabolic flare is predictive of the clinical effectiveness of anti-estrogens and downstream metabolic regulators of E2 are expected to have utility as targets for the development of anti-breast cancer agents. While the stimulation of glucose metabolism by E2 has been demonstrated, relatively little is known about the precise downstream effectors required for E2 to stimulate glucose metabolism in breast cancer. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) control glycolytic flux via their product, fructose-2,6-bisphosphate (F26BP), which activates 6-phosphofructo-1-kinase (PFK-1). We recently demonstrated that PFKFB3 expression is elevated in breast cancer lymph node metastases and that exposure of human MCF-7 and T-47D breast cancer cells to E2 causes a rapid increase in 14C-glucose uptake and glycolysis that is coincident with an induction of PFKFB3 mRNA (via ER binding to its promoter), protein expression and the intracellular concentration of its product, F26BP. Importantly, we also found that selective inhibition of PFKFB3 expression and activity using siRNA or a PFKFB3 inhibitor, PFK158, markedly reduces the E2-mediated increase in F26BP, 14C-glucose uptake and glycolysis. In the current study, we sought to determine if co-administration of PFK158, with ICI 182,780 (fulvestrant), would offer a greater anti-tumor effect. In unpublished results, we demonstrated that the combination of the anti-estrogen, fulvestrant, with PFK158 results in greater regressions of MCF-7 xenografts than either drug alone in athymic BALB/C mice. In addition to regulating glucose metabolism, PFKFB3 has been found to be a regulator of the cell cycle via cyclin dependent kinases. We postulated that the combination of PFK158 with the newly FDA-approved CDK4/6 inhibitor palbociclib may result in a synergistic increase in apoptosis. We found that PFK158 markedly increased apoptosis caused by palbociclib in MCF-7 breast cancer cells but not in normal human mammary epithelial cells. Taken together, these data indicate that PFKFB3 inhibitors such as PFK158 may have clinical utility for the treatment of ER+ breast cancers when combined with anti-estrogen agents and/or CDK4/6 inhibitors. Citation Format: Yoannis Imbert-Fernandez, Brian Clem, Gilles Tapolsky, Jason Chesney. Regulation of 6-phosphofructo-2-kinase (PFKFB3) by estradiol and implications for the treatment of ER+ metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A84.