Triple-negative Patients Research Articles

11P Convoluted role of H19 long non-coding RNA in regulating ICAM-1 and PVR in breast cancer patients

Breast cancer (BC) is one of the major biomedical research priorities worldwide. Luminal B and triple negative BC patients represents the worst prognosis and highest incidence of recurrence. Among those patients, metastasis is the missing piece in the puzzle. Consequently, extensive research is urgently required to identify specific molecular targets responsible for luminal B and TNBC induced risk of metastasis. ICAM-1 (known as CD54) and PVR (known as CD155) are transmembrane glycoproteins that have been emerged as novel molecular targets that are markedly up-regulated in BC patients. Our group is recently focusing on unraveling the role of Long non-coding RNAs in regulating vital molecular targets in BC patients. LncRNAs regulating ICAM-1 and PVR is still a virgin field. Therefore, the aim of this study is to identify a lncRNA that could dually target ICAM-1 and PVR simultaneously. Forty BC patients were recruited. Bioinformatic analysis was performed to identify a lncRNA that could directly or indirectly target ICAM-1 and PVR. MDA-MB-231 and MCF-7 cells were cultured. Cells were transfected using Scrambled and H19 siRNAs by lipofection Technique. Total RNA was extracted using biozol from tissues and cell lines. Reverse transcription and qRT-PCR were performed. MTT, scratch and colony forming assays were performed. Student t-test was performed for statistical analysis. In silico, H19 lncRNA was found to indirectly target ICAM-1 and PVR through STAT3 axis. H19, ICAM-1 and PVR were found to be upregulated in BC patients. Upon patients stratification, H19 and PVR were found to be specifically upregulated in luminal B BC patients while ICAM-1 was found to be specifically upregulated in TNBC patients. Upon Knocking down of H19, transfection effeciency was evaluated and H19 siRNAs resulted in paradoxical impacts on ICAM-1 and PVR. Yet, H19 siRNAs resulted in a repression of cellular viability, colonogenicity and migration capacity. H19 inhibitory impact on the metastatic mediator ICAM-1 overrides its inductory effect on PVR in BC cells. Therefore, this study highlights H19 as an oncogenic lncRNA in BC and a novel molecular target for resistant luminal B and TNBC patients.

The role of immunotherapy in metastatic triple negative breast cancer: a narrative review of the current clinical trials

: Triple negative breast cancer is an aggressive subtype of breast cancer that lacks expression of estrogen and progesterone receptors, and does not overexpress the human epithelial growth factor 2 receptor. Triple negative breast cancer occurs in approximately 20% of breast cancer cases, and patients are more likely to present with an advanced stage, have higher relapse rates, and have a worse prognosis than other breast cancer types. There are no targeted therapies for the majority of patients with triple negative breast cancer. The mainstay of treatment remains chemotherapy and the historical median overall survival for metastatic patients is around one year. Immunotherapy has dramatically improved outcomes in other malignancies by harnessing the body’s immune system to attack cancer cells. Triple negative breast cancer is the most immunogenic type of breast cancer, with a large genetic mutational burden and a relatively high number of immune cells found in the cancer microenvironment compared to other breast cancer subtypes. While initial studies of single agent immunotherapy in metastatic triple negative breast cancer were promising, the survival outcomes have been disappointing. Combining systemic therapy with immunotherapy has become a recent focus of clinical trials, and is now the current standard of care for treatment naive metastatic triple negative patients that are programmed death-ligand 1 positive. The efficacy of combined chemoimmunotherapy for first-line treatment has yet to be replicated, and recent clinical data questions the true benefit. This review summarizes the clinical trials of single agent and combination strategies for immunotherapy in metastatic triple negative breast cancer. We also examine the potential role for biomarkers, and future direction of treatment.

Molecular Subtype May Be More Associated With Prognosis and Chemotherapy Benefit Than Tumor Size in T1N0 Breast Cancer Patients: An Analysis of 2,168 Patients for Possible De-Escalation Treatment

PurposeBreast cancer (BC) patients with T1N0 tumors have relatively favorable clinical outcomes. However, it remains unclear whether molecular subtypes can aide in prognostic prediction for such small, nodal-negative BC cases and guide decision-making about escalating or de-escalating treatments.Patients and MethodsT1N0 BC patients diagnosed between 2009 and 2017 were included and classified into three subgroups according to receptor status: 1) hormonal receptor (HR)+/human epidermal growth factor receptor-2 (HER2)−; 2) HER2+; and 3) triple negative (TN) (HR−/HER2−). Patients’ characteristics and relapse events were reviewed. Kaplan–Meier analysis and Cox regression were used to assess the iDFS and BCSS. The effects of risk factors and adjuvant treatment benefits were evaluated by calculating hazard ratios (HRs) for invasive disease-free survival (iDFS) and breast cancer-specific survival (BCSS) with Cox proportional hazards models.ResultsIn total, 2,168 patients (1,435 HR+/HER2−, 427 HER2+, 306 TN) were enrolled. The 5-year iDFS rates were 93.6, 92.7, and 90.6% for HR+/HER2−, HER2+, and TN patients, respectively (P = 0.039). Multivariate analysis demonstrated that molecular subtype (P = 0.043), but not tumor size (P = 0.805), was independently associated with iDFS in T1N0 BC. TN patients [HRs = 1.77, 95% confidence interval (CI) = 1.11–2.84, P = 0.018] had a higher recurrence risk than HR+/HER2− patients. Adjuvant chemotherapy benefit was not demonstrated in all T1N0 patients but interacted with molecular subtype status. TN (adjusted HRs = 2.31, 95% CI = 0.68–7.54) and HER2+ (adjusted HRs = 2.26, 95% CI = 0.95–5.63) patients receiving chemotherapy had superior iDFS rates. Regarding BCSS, molecular subtype tended to be related to outcome (P = 0.053) and associated with chemotherapy benefit (P = 0.005).ConclusionMolecular subtype was more associated with disease outcome and chemotherapy benefit than tumor size in T1N0 BC patients, indicating that it may guide possible clinical de-escalating therapy in T1N0 BC.

Abstract PD4-05: Axillary recurrence is a rare event in node-positive patients. treated with sentinel node biopsy alone after neoadjuvant chemotherapy: Results of a prospective study

Abstract Background: Four prospective multi-institutional trials have demonstrated that clinically node-positive patients (cN1) who receive neoadjuvant therapy (NAC) and convert to cN0 can be reliably staged with sentinel lymph node biopsy (SLNB) with false-negative rates (FNRs) of < 10%, when ≥ 3 SLNs are retrieved. Since study patients all had axillary lymph node dissection (ALND), the rate of axillary recurrence after SLNB alone is unknown. Of concern is the possibility that residual chemotherapy-resistant axillary disease could lead to higher recurrence rates than seen in the primary surgery setting for cN0 patients where SLN FNRs of 5-10% result in axillary recurrence in < 1% of cases. Here we report regional recurrence rates in a prospectively defined cohort of cN1 patients receiving NAC, followed by a negative SLNB using a standardized technique, and no further axillary surgery. Methods: From 06/2014 to 02/2019, patients with cT1-3 biopsy-proven cN1 breast cancer who received NAC and converted to cN0 by physical exam were prospectively managed with SLNB with dual tracer mapping and omission of ALND if ≥ 3 SLNs were pathologically negative. Nodes were not routinely clipped, and retrieval of clipped metastatic nodes was not required. Pathologically negative SLNs were defined as the absence of any metastases including isolated tumor cells. Results: Of 610 cN1 patients treated with NAC, 555 (91%) converted to cN0 and had SLNB; 234 (42%) had ≥ 3 negative SLNs and were treated with SLNB alone. Median patient age was 49 years and median tumor size at presentation was 3 cm; 61% were HER2+ and 18% triple negative. Most (91%) received doxorubicin-based NAC and 88% received adjuvant radiotherapy (RT), with 80% (n = 164) of RT patients receiving nodal RT (Table). At a median follow-up of 35 months, there was only 1 (0.4%) axillary recurrence for the entire cohort, synchronous with a breast recurrence, in a patient who refused RT. Among patients who received RT (n = 205), there were no axillary recurrences. The 4-year rate of distant recurrence for all patients was 6.1% (95% CI, 3.4-10.7%) and 4-year overall survival was 93.9% (95% CI, 87.6-97.1%). Conclusion: In cN1 patients treated with NAC, rates of axillary recurrence in patients with ≥ 3 pathologically negative SLNs treated with SLNB alone were low, without routine nodal clipping. Although further follow-up is needed, multiple studies have shown that nodal recurrence is an early event, particularly in HER2+ and triple negative patients, who comprised the majority of the population. Our findings support omitting ALND in cN1 patients after NAC when the SLNs are negative using an optimal SLNB technique. Table. Patient PopulationOverall cohort (n = 234)Age, years (median, IQR)49 (40, 58)Tumor size at presentation, cm (median, IQR)3.0 (2.2, 5.0)Number SLNs retrieved (median, IQR)4 (3, 5)Palpable nodes at presentation (n, %)179 (76%)HistologyDuctal211 (90%)Lobular and mixed7 (3%)Micropapillary and mixed10 (4%)Other3 (1%)Occult3 (1%)DifferentiationWell1 (0.5%)Moderate36 (15%)Poor196 (84%)Unknown1 (0.5%)Receptor StatusHR+/HER2-47 (20%)HR+/HER2+80 (34%)HR-/HER2+64 (27%)HR-/HER2-43 (18%)Breast SurgeryBCS118 (50%)Mastectomy116 (50%)Breast pCR¥Yes161 (70%)No70 (30%)NAC regimenAC-T197 (84%)AC-T + carbo15 (6.4%)TC8 (3.4%)Other14 (6%)Neoadjuvant anti-HER2 treatmentHP (dual-therapy)144 (100%)Adjuvant RTYes205 (88%)No*29 (12%)¥3 patients had occult primary breast cancer and were not included in breast pCR calculation; *6/29 patients who did not receive RT enrolled in NSABP B-51 Citation Format: Andrea V Barrio, Giacomo Montagna, Anita Mamtani, Varadan Sevilimedu, Hiram S Cody, III, Mahmoud El-Tamer, Mary L Gemignani, Alexandra S Heerdt, Tracy-Ann Moo, Melissa Pilewskie, George Plitas, Lisa Sclafani, Kimberly J Van Zee, Monica Morrow. Axillary recurrence is a rare event in node-positive patients. treated with sentinel node biopsy alone after neoadjuvant chemotherapy: Results of a prospective study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD4-05.

Abstract PS16-26: The prognostic role of MMP 9 in early breast cancer

Abstract Background: Matrix metalloproteinase 9 (MMP9) is involved in the extracellular matrix degradation during physiological and pathological conditions including tumorigenesis. This translational study was aimed to evaluate the prognostic role of the intratumoral MMP9 expression and correlate it with presence of CTCs in early breast cancer. Methods: A total of 318 primary breast cancer (PBC) patients were enrolled into this study. Surgical specimens were processed by the tissue microarray method and subjected to immunohistochemistry using the MMP9 monoclonal antibody. The MMP9 expression was evaluated in tumor cell as well as in tumor associated stroma. Quantitative real-time polymerase chain reaction -based assays was applied for identification of CTCs. Results: Significantly increased expression of MMP9 was found in breast cancer cells when compared to tumor associated stroma. A positive correlation was determined between MMP9 expression and hormone positive status as well as low proliferation index of analysed breast cancer tumour cells. Additionally, in tumor associated stroma was confirmed only the association with hormone receptor status. The univariate survival analysis of whole tested population detected no prognostic role of MMP9 expression neither in tumor cells (HR = 0.96, 95% CI 0.58-1.59, P = 0.864) nor in tumor associated stroma (HR = 1.29, 95% CI 0.60-2.78, P = 0.547). However, the subgroup of in hormone receptor negative and triple negative patients with absence of MMP9 expression in tumor cells and stroma had significantly better disease-free survival (DFS) (HR = 0.33, 95% CI 0.12-0.93, P = 0.025, and (HR = 0.14, 95% CI 0.00-4.81, P = 0.002, respectively) and (HR = 0.17, 95% CI 0.05-0.57, P = 0.003); (HR = 0.12, 95% CI 0.00-4.89, P = 0.001) compared with patients with presence of MMP9. Moreover, while tumor MMP9 was prognostic in CTC_EMT positive subgroup (HR = 0.40, 95% CI 0.16-0.95, P = 0.047), absence of stromal MMP9 had protective role in CTC_EP positive patients (HR = 0.18, 95% CI 0.01-2.75, P = 0.053). Conclusions: Our data suggest that the increased expression of MMP9 in PBC was related with favorable tumor characteristics. However, it´s prognostic value was limited only to hormone receptor negative, triple negative, CTC_EMT and CTC_EP positive subgroups. Therefore, we can suppose that evaluating of MMP9 tumor expression could help identify patients with increased risk of disease recurrence in these subgroups of patients. Citation Format: Michal Mego, Zuzana Cierna, Marian Karaba, Gabriel Minarik, Juraj Benca, Tatiana Sedlackova, Ivana Mrvova, Daniel Pindak, Katarina Rejlekova, Jozef Mardiak, Katarina Kalavska. The prognostic role of MMP 9 in early breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-26.

Abstract GS4-08: Clinical utility of repeated circulating tumor cell (CTC) enumeration as early treatment monitoring tool in metastatic breast cancer (MBC) - a global pooled analysis with individual patient data

Abstract Background: Several studies suggest clinical utility of serial circulating tumor cell (CTC) enumeration as a means of assessing response status in metastatic breast cancer (MBC). The aim of this study is to conduct a comprehensive pooled analysis comprising globally available data to further define and explore the role of CTC enumeration as a tool for early treatment monitoring in patients with MBC with a focus on the predictive power in different breast cancer subtypes and clinical settings. Methods: In a global effort, peer-reviewed published studies with data on repeated CTC assessments (CellSearch® technology; Menarini Silicon Biosystems; Bologna, Italy) in MBC patients were screened and investigators were asked to provide individual patient data for this pooled analysis. 2761 cases from 32 data sets with data on both baseline and one follow up CTC assessments were included in the analysis (median time interval between the two CTC assessments 35 days). Data were analyzed using log rank tests and Cox regressions to evaluate the association between serial CTC enumeration results and overall survival (OS) in the full patient cohort and defined subgroups. Results: 588 (21.3%) patients had no CTCs at both time points (neg/neg), 236 (8.5%) patients were CTC negative at baseline and CTC positive at follow up (neg/pos), 712 (25.8%) patients converted from CTC positive at baseline to CTC negative (pos/neg), and 1225 (44.4%) patients had at least one CTC at both time points (pos/pos). Log rank tests showed significant differences in OS between these four CTC change groups (p < 0.0001 for all pairwise comparisons except for the comparison between neg/pos and pos/neg, p = 0.015). Median OS for the neg/neg, neg/pos, pos/neg and pos/pos group was 45.6, 26.1, 34.6, and 17.6 months, respectively. Hazard ratios (HR) (reference group neg/neg) were 1.38 (95% CI 1.16 - 1.64) for the pos/neg group, 1.78 (95% CI 1.43 - 2.22) for the neg/pos group, and 3.06 (95% CI 2.63 - 3.56) for the pos/pos group. Results were similar if a cutoff of 5 CTCs was used for CTC positivity (pos/neg group: HR 1.43, 95% CI 1.25 - 1.63; neg/pos group: HR 2.39, 95% CI 1.91 - 2.99; pos/pos group: HR 3.54, 95% CI 3.12 - 4.02). In total, 2586 patients could be assigned to different tumor subtypes based on known hormone receptor (ER) and HER2 status of the primary tumor: 1513 (58.5%) patients had a luminal-like tumor (ER positive, HER2 negative), 682 (26.4%) patients had a HER2-positive tumor, and 391 (15.1%) patients had a triple-negative tumor. In patients with luminal-like tumors, the hazard ratios were 1.67 (95% CI 1.29 - 2.17), 2.01 (95% CI 1.45 - 2.77), and 3.87 (95% CI 3.09 - 4.83) for the pos/neg, neg/pos, and pos/pos group, respectively. In patients with HER2-positive tumors, the neg/pos group (HR 1.68, 95% CI 1.12 - 2.53) and the pos/pos group (HR 2.11, 95% CI 1.58 - 2.83) showed significantly worse OS compared to the neg/neg group, while in triple-negative patients, the pos/pos group had a significantly shorter OS compared to the neg/neg group (HR 2.99, 95% CI 2.11 - 4.24). The results will be up-dated by inclusion of additional large data sets (CALGB 40502, CALGB 40503, COMET, SWOG S0500, TBCRC 001) for the analysis to be presented at SABCS 2020. Conclusion: This large pooled analysis confirms that at a median of 35 days after treatment initiation, follow-up CTC assessments strongly predict overall survival. These results suggest potential clinical utility of CTC monitoring as early response marker in MBC, especially in luminal-like tumors. Citation Format: Wolfgang J Janni, Tracy C. Yab, Daniel F. Hayes, Massimo Cristofanilli, Francois-Clement Bidard, Michail Ignatiadis, Meredith M. Regan, Catherine Alix-Panabières, William E. Barlow, Carlos Caldas, Lisa A. Carey, Luc Dirix, Tanja Fehm, Jose A. Garcia-Saenz, Paola Gazzaniga, Daniele Generali, Lorenzo Gerratana, Rafael Gisbert-Criado, William Jacot, Zefei Jiang, Evi Lianidou, Mark J.M. Magbanua, Luis Manso, Dimitrios Mavroudis, Volkmar Müller, Elisabetta Munzone, Klaus Pantel, Jean-Yves Pierga, Brigitte Rack, Sabine Riethdorf, Hope S. Rugo, Kostandinos Sideras, Stefan Sleijfer, Jeffrey Smerage, Justin Stebbing, Leon W.M.M. Terstappen, José Vidal-Martínez, Rita Zamarchi, Karthik Giridhar, Thomas W.P. Friedl, Minetta C. Liu. Clinical utility of repeated circulating tumor cell (CTC) enumeration as early treatment monitoring tool in metastatic breast cancer (MBC) - a global pooled analysis with individual patient data [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS4-08.

Abstract PS13-02: Global myocardial perfusion is reduced in premenopausal breast cancer patients treated with ovarian function suppression and aromatase inhibitor therapy: An adenosine stress T1 map CMR study

Abstract Introduction: Premenopausal women with high-risk hormone receptor-positive (HR+) breast cancer (BC) undergo abrupt menopause induction with aromatase inhibitor (AI) anti-estrogen therapy and ovarian function suppression (OFS). This treatment improves recurrence-free survival but may increase cardiovascular (CV) risk associated with early hypoestrogenemia as observed in women with non-cancerous reasons for early loss of ovarian function. We sought to identify if OFS+AI therapy is associated with myocardial perfusion changes, a subclinical marker of coronary heart disease. Methods: We evaluated women with paired adenosine cardiovascular magnetic resonance imaging scans at 3-to 6-month intervals using cines and T1 maps in standard short-axis planes. As increased blood flow from vasodilation increases adenosine stress parametric T1 values of the myocardial tissue, the myocardial perfusion reactivity was calculated from the percent increase of stress versus native (pre-stress) T1 maps. Double-blinded post-processing of images was performed in CircleCVI software. Statistical analyses were performed in MATLAB (p<0.05 defined as statistically significant) using paired t-tests for within-group comparisons and two-sample t-tests for between-group comparisons. Results: Twenty-one premenopausal women (16 Caucasian, 5 African American; median age 44.7 years) were accrued in 16 months - 14 with HR+ BC within 3 years of initiating OFS+AI (median 8 months) and 7 with triple negative BC within 3 years of chemotherapy (median 10 months) for comparators. Global myocardial perfusion reactivity to adenosine stress declined in HR+ women on OFS+AI therapy during the 3-6 month study interval (-1.3%; 95%CI: -3.4-0.7%) which was significantly different (p=0.02) from changes observed in comparators who had an improvement in perfusion during the interval (3.2%; 95%CI: -1.2-7.6%) (Table). Left ventricular (LV) function remained unchanged during the 3-6 month interval for all groups (p>0.05). The cardiovascular stress tests identified two HR+ women (14%) with abnormal results during stress imaging who were sent for further cardiovascular evaluations.Conclusions: Women with HR+ BC treated with OFS+AI exhibited a decline in global microcirculatory perfusion during an adenosine cardiac stress test with normal LV functional parameters. Alternatively, triple negative BC patients trended to have an improvement in LV function during the interval with non-significant improvement in myocardial perfusion. This study demonstrates a decline in myocardial perfusion in premenopausal women on OFS+AI therapy, suggesting subclinical coronary artery effects due to early hypoestrogenemia. Future work should confirm these results, identify women at higher risk, and test strategies to mitigate cardiotoxicity in premenopausal women with HR+ BC. Table: Cardiovascular magnetic resonance (CMR) study measures for HR+ BC patients on OFS+ AI therapy and triple negative BC patientsCMR MeasureHormone Receptor-Positive Breast Cancer(n=14)Triple Negative Breast Cancer(n=7)p-value for difference in change by groupBaselineFollow-upp-valueBaselineFollow-upp-valueLV Ejection Fraction, %56 (51, 61)56 (51, 62)0.8255 (50, 60)60 (55, 65)0.060.12EDVindex, mL/m270.6 (63.7, 77.5)69.9 (63.5, 76.3)0.7365.2 (58.0, 72.3)66.8 (51.4, 82.1)0.740.60ESVindex, mL/m231.3 (26.2, 36.5)30.4 (26.2, 34.5)0.3729.4 (23.8, 35.0)27.1 (19.4, 34.8)0.360.55SVindex, mL/m239.3 (34.3, 44.2)39.5 (33.9, 45.1)0.9035.8 (32.3, 39.3)39.7 (31.2, 48.2)0.230.29Myocardial mass index, g/m248.1 (42.7, 53.6)44.6 (40.0, 49.3)0.0843.5 (40.2, 46.8)41.0 (33.3, 48.7)0.420.77Global myocardial perfusion reactivity, %2.8 (1.1, 4.4)1.4 (-0.4, 3.3)0.191.4 (-0.9, 3.7)4.6 (0.9, 8.4)0.130.02Basal SAX myocardial perfusion reactivity, %2.2 (0.7, 3.7)1.4 (0.3, 2.5)0.261.8 (0.2, 3.4)2.1 (-1.7, 5.9)0.850.46Mid SAX myocardial perfusion reactivity, %2.4 (-.3, 5.2)0.5 (-1.5, 2.7)0.221.2 (-1.9, 4.2)3.3 (-0.9, 7.6)0.360.12Apical SAX myocardial perfusion reactivity, %3.4 (1.2, 5.7)2.1 (-1.4, 5.7)0.490.8 (-2.6, 4.1)8.8 (3.7, 13.9)<0.05<0.01LV = left ventricular, EDV = end diastolic volume, ESV = end systolic volume, SV = stroke volume, SAX = short-axis slice; volumetric and mass measures indexed to body surface area. Data are presented as mean (95% Confidence Interval). Citation Format: Alexandra Thomas, Ralph B. D'Agostino, Jr., Katherine C. Ansley, Sung Park, Anuj Kotak, Steven M. Sorscher, Susan Melin, Emily H. Douglas, Sujethra Vasu, Paul A. Romitti, William G. Hundley, Jennifer H. Jordan. Global myocardial perfusion is reduced in premenopausal breast cancer patients treated with ovarian function suppression and aromatase inhibitor therapy: An adenosine stress T1 map CMR study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS13-02.

Abstract PS14-19: Characteristics of breast cancer brain metastases presentation by subtype and validation of the modified breast graded prognostic assessment

Abstract Purpose: Breast cancer brain metastases (BCBM) diagnosis is increasing in frequency due to improved systemic control and imaging techniques. Differences have been noted in rates of central nervous system (CNS) relapse and biologic subtype. The modified breast graded prognostic assessment (breast-GPA) was initially validated in patients treated between 1996-2013 and considers biologic subtype. In this study, we characterize patients diagnosed with BCBM by subtype and validate the breast-GPA in a modern cohort of patients. Methods: All patients with BCBM treated at our institution with radiotherapy between 2016 and 2019 were identified. Characteristics of patients’ initial brain metastasis diagnosis were retrieved from the clinical chart and radiologic examinations. To test differences between cohorts, the Kruskal-Wallis and Pearson’s chi-square tests were used when appropriate. Overall survival (OS) was calculated from the date of brain metastasis diagnosis to the date of death using the Kaplan-Meier (KM) method, with the log-rank test used to examine differences between groups. Results: A total of 122 BCBM patients were identified. Breast cancer subtypes included hormone receptor (HR)+/HER2- (45%), triple negative (TN) (25%), HR-/HER2+ (16%), and HR+/HER2+ (14%). The first treatment for BCBM patients following diagnosis was whole brain radiation (51%), surgery followed by stereotactic radiation (28%), and stereotactic radiation (21%). The interval between breast cancer diagnosis and diagnosis of BCBM was longest for HR+/HER2- 4.5 years, followed by TN 2.8 years, HR+/HER2+ 2.3 years, HR-/HER2+ 1.9 years, p=0.003. The interval from systemic metastases to BCBM diagnosis trended towards the shortest for TN patients 6.6 months, p=0.15. A total of 34 patients (28%) were diagnosed with leptomeningeal disease (LMD) at initial brain metastases presentation. LMD was diagnosed most commonly at presentation in HR+/HER2- (36%) followed by , TN (26%), HR-/HER2+ (26%), and HR+/HER2+ (6%), p=0.06. No differences were noted based on receptor typessubtype and age, symptomatic intracranial disease, number of brain metastases, type of first intracranial treatment or concurrent systemic metastases at initial BCBM presentation, all p > 0.05. Twenty-four month KM OS rates following diagnosis of brain metastasis for breast-GPA 0-1, 1.5-2, 2.5-3, and 3.5-4 groups were 14%, 27%, 33%, and 86% (p=0.0005), respectively. Conclusions: In our institutional analysis, similarities were noted in the initial presentation of BCBM based on receptor typesubtype. Significant differences were noted in OS based on the modified breast-GPA. Further investigation is needed to determine which subtypes of asymptomatic breast cancer patients are at sufficient risk to warrant brain MRI screening. Citation Format: Yuki Kawahara, Matthew Fahey, Thrisha K Potluri, Matthew N Mills, Nicholas B Figura, Iman R Washington, Roberto Diaz, Timothy J Robinson, Hsiang-Hsuan M Yu, Arnold B Etame, James Liu, Michael A Vogelbaum, Brian J Czerniecki, Peter A Forsyth, Hatem H Soliman, Hyo S Han, Kamran A Ahmed. Characteristics of breast cancer brain metastases presentation by subtype and validation of the modified breast graded prognostic assessment [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS14-19.

Distinct profile of CD34+ cells and plasma-derived extracellular vesicles from triple-negative patients with Myelofibrosis reveals potential markers of aggressive disease

BackgroundMyelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10–15% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TN patients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis.MethodsPeripheral blood was collected from MF patients (n = 29; JAK2V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34+ cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34+ cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, IL6) or after co-culture with EVs from MF patients/HD.ResultsThe absolute numbers of circulating CD34+ cells were massively increased in TN patients. We found that TN CD34+ cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TN patients. Compared to JAK2V617F-mutated patients, the GEP of TN CD34+ cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2V617F-mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TN CD34+ cells. Along with a specific microRNA signature, the circulating EVs from TN patients are enriched with miR-361-5p.ConclusionsDistinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF.

Development of new brain metastases in triple negative breast cancer.

Brain metastases are common in patients with breast cancer, and those with triple negative status have an even higher risk. Triple negative status is currently not considered when managing brain metastases. To determine whether triple negative breast cancer (TNBC) patients with brain metastases have a higher burden of intracranial disease and whether WBRT has a survival benefit in this cohort of patients. We conducted a retrospective cohort study with 85 patients meeting the inclusion criteria. 25% of patients had TNBC. 95% of the patients in this study received SRS and 48% received WBRT. The average number of new brain metastases from time of initial brain imaging to radiation therapy was 0.67 ± 1.1 in the non-TNBC status patients and 2.6 ± 3.7 in the triple negative status patients (p = 0.001). A cox proportional hazards model showed that WBRT does not significantly affect overall survival in patients with TNBC (HR 1.48; 95% CI 0.47-4.67; p = 0.50). Our findings highlight the highly aggressive intracranial nature of TNBC. The rate of new brain metastasis formation is higher in TNBC patients compared to non-TNBC patients. Furthermore, there is no survival benefit for WBRT in TNBC patients. These findings are relevant for clinicians planning brain radiation for TNBC patients as they may find more brain metastases at the time of brain radiation than they anticipated based on initial brain imaging.

Immune Infiltrates in Breast Cancer: Recent Updates and Clinical Implications.

In recent decades, the increasing interest in the field of immunotherapy has fostered an intense investigation of the breast cancer (BC) immune microenvironment. In this context, tumor-infiltrating lymphocytes (TILs) have emerged as a clinically relevant and highly reproducible biomarker capable of affecting BC prognosis and response to treatment. Indeed, the evaluation of TILs on primary tumors proved to be strongly prognostic in triple-negative (TN) BC patients treated with either adjuvant or neoadjuvant chemotherapy, as well as in early TNBC patients not receiving any systemic treatment, thus gaining level-1b evidence in this setting. In addition, a strong relationship between TILs and pathologic complete response after neoadjuvant chemotherapy has been reported in all BC subtypes and the prognostic role of higher TILs in early HER2-positive breast cancer patients has also been demonstrated. The interest in BC immune infiltrates has been further fueled by the introduction of the first immune checkpoint inhibitors in the treatment armamentarium of advanced TNBC in patients with PD-L1-positive status by FDA-approved assays. However, despite these advances, a biomarker capable of reliably and exhaustively predicting immunotherapy benefit in BC is still lacking, highlighting the imperative need to further deepen this issue. Finally, more comprehensive evaluation of immune infiltrates integrating both the quantity and quality of tumor-infiltrating immune cells and incorporation of TILs in composite scores encompassing other clinically or biologically relevant biomarkers, as well as the adoption of software-based and/or machine learning platforms for a more comprehensive characterization of BC immune infiltrates, are emerging as promising strategies potentially capable of optimizing patient selection and stratification in the research field. In the present review, we summarize available evidence and recent updates on immune infiltrates in BC, focusing on current clinical applications, potential clinical implications and major unresolved issues.

BARD1 Pathogenic Variants are Associated with Triple-Negative Breast Cancer in a Spanish Hereditary Breast and Ovarian Cancer Cohort.

Only a small fraction of hereditary breast and/or ovarian cancer (HBOC) cases are caused by germline variants in the high-penetrance breast cancer 1 and 2 genes (BRCA1 and BRCA2). BRCA1-associated ring domain 1 (BARD1), nuclear partner of BRCA1, has been suggested as a potential HBOC risk gene, although its prevalence and penetrance are variable according to populations and type of tumor. We aimed to investigate the prevalence of BARD1 truncating variants in a cohort of patients with clinical suspicion of HBOC. A comprehensive BARD1 screening by multigene panel analysis was performed in 4015 unrelated patients according to our regional guidelines for genetic testing in hereditary cancer. In addition, 51,202 Genome Aggregation Database (gnomAD) non-Finnish, non-cancer European individuals were used as a control population. In our patient cohort, we identified 19 patients with heterozygous BARD1 truncating variants (0.47%), whereas the frequency observed in the gnomAD controls was 0.12%. We found a statistically significant association of truncating BARD1 variants with overall risk (odds ratio (OR) = 3.78; CI = 2.10–6.48; p = 1.16 × 10−5). This association remained significant in the hereditary breast cancer (HBC) group (OR = 4.18; CI = 2.10–7.70; p = 5.45 × 10−5). Furthermore, deleterious BARD1 variants were enriched among triple-negative BC patients (OR = 5.40; CI = 1.77–18.15; p = 0.001) compared to other BC subtypes. Our results support the role of BARD1 as a moderate penetrance BC predisposing gene and highlight a stronger association with triple-negative tumors.

Eribulin Mesylate for the Treatment of Metastatic Hormone-refractory and Triple-negative Breast Cancer: A Multi-institutional Real-world Report on Efficacy and Safety.

Eribulin mesylate (EM) is a fully synthetic macrocyclic ketone analogue of the marine natural product halichondrin. EM has been reported to be active in metastatic breast cancer. In this paper, we report efficacy and safety of data of EM in a retrospective, real-world series of patients with poor prognosis, hormone-refractory, or triple-negative metastatic breast cancer. The analysis was carried out at 4 interrelated oncology centers. EM was delivered at the dose of 1.4 mg/m2 in 100 mL of normal saline over 2 to 5 minutes on days 1 and 8 every 21 days. EM was continued until disease progression or unacceptable toxicity. Side effects were reported every cycle as per standard clinical practice and graded according to NCI-CTCAE, version 4.0. Time-to-progression and overall survival were reported. In this series of 90 patients the overall response rate was 22%, and 21% and 23% in the hormonal-resistant group and the triple-negative one, respectively. Stable disease was recorded in 24%, 21%, and 27%, respectively, in the whole series, the hormonal-resistant group, and the triple-negative one, respectively. Time-to-progression was 3.5 months (range, 1 to 22 mo) in the whole series and 3.0 months (range, 1 to 14.7 mo) and 3.4 months (range, 2.2 to 16.2 mo) in the hormonal-resistant group and the triple-negative one, respectively. Overall survival reached a median of 11.4 months. This multicenter study, albeit retrospective, demonstrates the activity of this combination as third-line chemotherapy option in a challenging clinical setting such as triple-negative or hormone-resistant patients with breast cancer progressing after several lines of hormonal manipulations.

Superiority of fecal carcinoembryonic antigen as diagnosis marker for adenomatous polyposis coli and asymptomatic colorectal cancer.

Background:Non-invasive diagnostic tools of adenomatous polyposis coli (APC) and asymptomatic colorectal cancer (CRC) are urgently needed. Although fecal carcinoembryonic antigen (FCEA) has been documented in some studies, the diagnostic potential for the detection of APC and asymptomatic CRC has not been described yet.Methods:This is a retrospective study. The pre-diagnostic serum carcinoembryonic antigen (SCEA) and fecal occult blood test (FOBT) levels were retrospectively analyzed in 212 patients with intestinal diseases group (IDG) and 224 controls. The levels of FCEA across all the studied groups were measured using electronic chemiluminescence immunoassay (ECLIA), and their sensitivity and specificity were used to evaluate their diagnostic potential. The individual diagnostic accuracy of the three indices, as well as their combined diagnostic potential, was compared using the receiver operating characteristic (ROC) curve and chi-square test.Results:The FCEA had low sensitivity (50%) and high specificity (93.91%) for the diagnosis of IDG, with the area under the curve (AUC) value of 0.781. The AUC of FCEA was higher than that of SCEA for the diagnosis of APC and CRC in the APC, asymptomatic CRC, and APC + CRC-stage I patients. The AUCs of FCEA were 0.708 and 0.691 for the ‘double-negative patients’ and ‘triple-negative patients’, respectively. In addition, FCEA could diagnose 45.5% of the ‘double-negative’ patients, 43.3% of the asymptomatic patients, and 42.9% of the ‘triple-negative’ patients. The combination of FCEA and FOBT improved the diagnostic value (AUC = 0.916).Conclusion:FCEA has been demonstrated to be a favorable diagnostic marker in intestinal diseases, especially in the APC, asymptomatic CRC, and ‘double-negative’ or ‘triple-negative’ CRC patients.

MiRNA-195 expression in the tumor tissues of female Brazilian breast cancer patients with operable disease

OBJECTIVE:This study aimed to assess miRNA-195 expression in the tumor tissues from a cohort of Brazilian female breast cancer patients undergoing neoadjuvant chemotherapy (NAC) and evaluate its correlation with various clinicopathological markers.METHODS:Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to evaluate the miRNA-195 expression in tumor tissues from a cohort of female breast cancer patients undergoing NAC. This expression was then correlated with the occurrence of several distinct breast cancer molecular subtypes and other clinicopathological variables.RESULTS:A total of 55 patients were included in this study, 28 (50.9%) of whom were treated using NAC. Tumor miRNA-195 expression was suppressed in breast cancer patients, regardless of their exposure to systemic treatments, histological grade, size, nodal status, and tumor-node-metastasis (TNM) staging. This was more pronounced in luminal and triple-negative patients, and patient’s response to NAC was correlated with an increase in miRNA-195 expression.CONCLUSION:miRNA-195 is downregulated in the tumor tissues of Brazilian breast cancer patients regardless of NAC exposure; this reinforces its role as a tumor suppressor and a potential biomarker for chemotherapy response.

Salmonella typhimurium A1-R Exquisitely Targets and Arrests a Matrix-producing Triple-negative Breast Carcinoma in a PDOX Model.

Triple-negative matrix-producing breast carcinoma (MPBC) is rare, recalcitrant, and highly aggressive. The present study aimed to determine the efficacy of tumor-targeting leucine-arginine auxotroph Salmonella typhimurium (S. typhimurium) A1-R on a triple-negative MPBC in a patient-derived orthotopic xenograft (PDOX) model. The PDOX MPBC model was established in the left second mammary gland of nude mice by surgical orthotopic implantation (SOI). PDOX models were randomized into two groups when the tumor volume reached over 70 mm3: a control group (n=6); and a tumor-targeting S. typhimurium A1-R group (n=7), [intravenous (i.v.) injection of S. typhimurium A1-R via the tail vein, weekly, for two weeks]. All mice were sacrificed on day 14. Tumor volume and body weight were measured once per week. S. typhimurium A1-R exquisitely targeted and arrested the growth of the MPBC PDOX compared to the control group (p=0.017). S. typhimurium A1-R has future clinical potential for triple-negative MPBC patients.

Sentinel Lymph Node Biopsy May Prevent Unnecessary Axillary Dissection in Patients with Inflammatory Breast Cancer Who Respond to Systemic Treatment

Background and Objectives: Inflammatory breast cancer (IBC) is a rare and aggressive breast cancer treated up-front with systemic treatment. Both breast-conserving surgery and sentinel lymph node biopsy (SLNB) are controversial issues in the management of IBC. In this study, we aimed to assess the feasibility of SLNB in pathologically proven node-positive IBC patients. Methods: All patients with a histopathological diagnosis of IBC and biopsy-proven metastatic axillary lymph nodes underwent systemic treatment. Patients with a complete clinical response in the axilla who underwent SLNB followed by standard axillary dissection were analyzed. Results: The study consisted of 25 female patients. The identification rate (IR) and the false negativity rate (FNR) were 17/25 and 2/10, respectively. Overall, 9/25 and 7/25 of patients had a complete pathological response (pCR) in the breast and axilla after systemic treatment, respectively. Although the pCR in the axilla was 2/4 in nonluminal HER2-positive patients, the highest IR 4/4 and the lowest FNR 0/2 were determined in these patients. In triple-negative patients, however, the IR was 2/4 and the FNR was found to be 0/2. Conclusions: SLNB may be considered in selected axilla-downstaged IBC patients including patients with a pCR with HER2-positive and triple-negative tumors. Axillary dissection may be, therefore, omitted in those with negative SLNs.

Diagnostic and Therapeutic MicroRNAs in Primary Myelofibrosis

Primary myelofibrosis (PMF) is a pluripotent hematopoietic stem cell-derived malignancy, included in the heterogeneous group of myeloproliferative neoplasms (MPNs). PMF diagnosis is based on a composite assessment of clinical and laboratory data. The three major diagnostic criteria are: screening for driver mutations, exclusion of other conditions that can cause myelofibrosis, and bone marrow biopsy displaying megakaryocyte changes and fibrosis. PMF treatment options are only partially disease-modifying and consist mainly of symptom control. Recently, a new targeted therapy was introduced for PMF patients, JAK-STAT inhibitors (i.e. ruxolitinib). However, specific subgroups of patients do not benefit from the JAK-STAT inhibitors: (1) those who are carrying JAK2 mutations, but ruxolitinib does not reduce the spleen size; (2) triple negative patients (no JAK2, CALR, or MPL mutations); and (3) those who discontinue JAK-STAT therapy because of side effects. These subgroups are in need of new therapeutic approaches. Mature microRNAs (miRNAs) range from 16 to 28 nucleotides (nt) in length and regulate specific messenger RNAs at the post-transcriptional level. Numerous in vitro and in vivo studies have reported specific miRNAs, as well as complex miRNA networks, to be dysregulated in PMF. Several of these miRNAs were shown to be implicated in essential events of PMF pathophysiology: increase of bone marrow fibrosis, progression to acute myeloid leukemia, resistance to JAK-STAT inhibitors, and activation of differentiation of hematopoietic stem/progenitor cells into megakaryocytes. Hence, we propose miRNAs as a potential minimally invasive diagnostic tool for PMF and as therapeutic targets that could address the unmet medical needs of these patients.

Clinical characteristics of essential thrombocythemia patients depend on the mutation status

AbstractThe impact of the mutation status on the clinical course and the outcome of essential thrombocythemia (ET) patients has not yet been completely established. A total of 171 patients with diagnosed ET were tested and subsequently grouped, according to their mutation status – Janus Kinase 2 (JAK2) – 112 patients, calreticulin (CALR) – 36 patients, and thrombopoietin receptor (MPL) – 5 patients. Moreover, 18 individuals were triple-negative (with non-mutated JAK2, CALR, and MPL). CALR-mutated patients preferentially were male, with higher platelets (PLT) counts (mean PLT = 1 002.3) and lower hemoglobin and hematocrit levels at the diagnosis, compared to the JAK2 (mean PLT = 933.6), MPL (mean PLT = 940.8) and triple-negative patients (mean PLT = 822.6) (p = 0.0035). The patients with CALR mutated, and the triple-negative ones had a lower risk of arterial and venous thrombosis (3% and 5.6% cases at the time of diagnosis, respectively) than the patients with JAK2 mutation (7.2%) (p = 0.9210). The overall survival rate did not differ statistically between the groups.

Residual Axillary Burden After Neoadjuvant Chemotherapy (NACT) in Early Breast Cancer in Patients with a priori Clinically Occult Nodal Metastases - a transSENTINA Analysis.

Background Among patients with breast cancer undergoing neoadjuvant chemotherapy (NACT), the association between pathological complete remission (pCR) in the breast and clinical/pathological parameters is well established, whereas the association between these parameters and residual axillary involvement after NACT remains unclear. Methods Patients with clinically occult nodal metastases (i.e. negative by clinical assessment but positive by SLNB prior to NACT, i.e. Arm B of the SENTINA trial) were included in the presented analysis. All patients received a second sentinel lymph node biopsy (SLNB) and axillary dissection after NACT. Univariate and multivariate analyses were carried out to evaluate the association between clinical/pathological parameters and axillary involvement after NACT. Results Arm B of the SENTINA study contained 360 patients, 318 of which were evaluable for this analysis. After NACT, 71/318 (22.3%) patients had involved SLNs or non-SLNs after NACT. Overall, 71/318 (22.3%) patients achieved a pCR in the breast. Associations of extranodal spread, lack of multifocality and pCR in the breast with residual axillary burden were statistically significant. In a descriptive analysis including all patients with clinically negative axilla before NACT in the SENTINA trial 1.2% of triple negative (TN) patients and 0.5% of HER/2 positive patients had residual axillary disease in case of a breast pCR. Conclusions Patients in the SENTINA trial with clinically negative axilla and involved SLNs still carried a significant risk of nodal metastases after NACT. However, the risk of residual axillary burden was particularly low in TN and HER/2 positive tumors in case of a breast pCR.