Triple Negative Breast Cancer Biopsies Research Articles

Homologous Recombination Deficiency Across Subtypes of Primary Breast Cancer.

Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and associated with response to PARP inhibition (PARPi). Here, we studied the prevalence of HRD in non-TNBC to assess the potential for PARPi in a wider group of patients with breast cancer. HRD status was established using targeted gene panel sequencing (360 genes) and BRCA1 methylation analysis of pretreatment biopsies from 201 patients with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials.gov identifier: NCT02624973). HRD was defined as mutations in BRCA1, BRCA2, BRIP1, BARD1, or PALB2 and/or promoter methylation of BRCA1 (strict definition; HRD-S). In secondary analyses, a wider definition (HRD-W) was used, examining mutations in 20 additional genes. Furthermore, tumor BRCAness (multiplex ligation-dependent probe amplification), PAM50 subtyping, RAD51 nuclear foci to test functional HRD, tumor-infiltrating lymphocyte (TIL), and PD-L1 analyses were performed. HRD-S was present in 5% of non-TNBC cases (n = 9 of 169), contrasting 47% of the TNBC tumors (n = 15 of 32). HRD-W was observed in 23% of non-TNBC (n = 39 of 169) and 59% of TNBC cases (n = 19 of 32). Of 58 non-TNBC and 30 TNBC biopsies examined for RAD51 foci, 4 of 4 (100%) non-TNBC and 13 of 14 (93%) TNBC cases classified as HRD-S had RAD51 low scores. In contrast, 4 of 17 (24%) non-TNBC and 15 of 19 (79%) TNBC biopsies classified as HRD-W exhibited RAD51 low scores. Of nine non-TNBC tumors with HRD-S status, only one had a basal-like PAM50 signature. There was a high concordance between HRD-S and either BRCAness, high TIL density, or high PD-L1 expression (each P < .001). The prevalence of HRD in non-TNBC suggests that therapy targeting HRD should be evaluated in a wider breast cancer patient population. Strict HRD criteria should be implemented to increase diagnostic precision with respect to functional HRD.

Deregulated miRNA Expression in Triple-Negative Breast Cancer of Ancestral Genomic-Characterized Latina Patients.

Among patients with triple-negative breast cancer (TNBC), several studies have suggested that deregulated microRNA (miRNA) expression may be associated with a more aggressive phenotype. Although tumor molecular signatures may be race- and/or ethnicity-specific, there is limited information on the molecular profiles in women with TNBC of Hispanic and Latin American ancestry. We simultaneously profiled TNBC biopsies for the genome-wide copy number and miRNA global expression from 28 Latina women and identified a panel of 28 miRNAs associated with copy number alterations (CNAs). Four selected miRNAs (miR-141-3p, miR-150-5p, miR-182-5p, and miR-661) were validated in a subset of tumor and adjacent non-tumor tissue samples, with miR-182-5p being the most discriminatory among tissue groups (AUC value > 0.8). MiR-141-3p up-regulation was associated with increased cancer recurrence; miR-661 down-regulation with larger tumor size; and down-regulation of miR-150-5p with larger tumor size, high p53 expression, increased cancer recurrence, presence of distant metastasis, and deceased status. This study reinforces the importance of integration analysis of CNAs and miRNAs in TNBC, allowing for the identification of interactions among molecular mechanisms. Additionally, this study emphasizes the significance of considering the patients ancestral background when examining TNBC, as it can influence the relationship between intrinsic tumor molecular characteristics and clinical manifestations of the disease.

Comparison of assessment of programmed death-ligand 1 (PD-L1) status in triple-negative breast cancer biopsies and surgical specimens

AimsProgrammed death-ligand 1 (PD-L1) status in triple-negative breast cancer (TNBC) is important for immune checkpoint inhibitor therapies but may vary between different immunohistochemical assays, scorings and the type of specimen...

CXCR2 Small-Molecule Antagonist Combats Chemoresistance and Enhances Immunotherapy in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer as the absence of cell surface receptors renders it more difficult to be therapeutically targeted. Chemokine receptor 2 (CXCR2) has been suggested not only to promote therapy resistance and suppress immunotherapy but it also to possess a positive cross-talk with the multifunctional cytokine transforming growth factor beta (TGF-β). Here, we showed that CXCR2 and TGF-β signaling were both upregulated in human TNBC biopsies. CXCR2 inhibition abrogated doxorubicin-mediated TGF-β upregulation in 3D in vitro TNBC coculture with PBMCs and eliminated drug resistance in TNBC mammospheres, suggesting a vital role for CXCR2 in TNBC doxorubicin-resistance via TGF-β signaling regulation. Moreover, CXCR2 inhibition improved the efficacy of the immunotherapeutic drug “atezolizumab” where the combined inhibition of CXCR2 and PDL1 in TNBC in vitro coculture showed an additive effect in cytotoxicity. Altogether, the current study suggests CXCR2 inhibitors as a promising approach to improve TNBC treatment if used in combination with chemotherapy and/or immunotherapy.

Interobserver variability in the assessment of stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative invasive breast carcinoma influences the association with pathological complete response: the IVITA study

High stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC) are associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). Histopathological assessment of sTILs in TNBC biopsies is characterized by substantial interobserver variability, but it is unknown whether this affects its association with pCR. Here, we aimed to investigate the degree of interobserver variability in an international study, and its impact on the relationship between sTILs and pCR. Forty pathologists assessed sTILs as a percentage in digitalized biopsy slides, originating from 41 TNBC patients who were treated with NAC followed by surgery. Pathological response was quantified by the MD Anderson Residual Cancer Burden (RCB) score. Intraclass correlation coefficients (ICCs) were calculated per pathologist duo and Bland–Altman plots were constructed. The relation between sTILs and pCR or RCB class was investigated. The ICCs ranged from −0.376 to 0.947 (mean: 0.659), indicating substantial interobserver variability. Nevertheless, high sTILs scores were significantly associated with pCR for 36 participants (90%), and with RCB class for eight participants (20%). Post hoc sTILs cutoffs at 20% and 40% resulted in variable associations with pCR. The sTILs in TNBC with RCB-II and RCB-III were intermediate to those of RCB-0 and RCB-I, with lowest sTILs observed in RCB-I. However, the limited number of RCB-I cases precludes any definite conclusions due to lack of power, and this observation therefore requires further investigation. In conclusion, sTILs are a robust marker for pCR at the group level. However, if sTILs are to be used to guide the NAC scheme for individual patients, the observed interobserver variability might substantially affect the chance of obtaining a pCR. Future studies should determine the ‘ideal' sTILs threshold, and attempt to fine-tune the patient selection for sTILs-based de-escalation of NAC regimens. At present, there is insufficient evidence for robust and reproducible sTILs-guided therapeutic decisions.

Intra-Tumour Heterogeneity Is One of the Main Sources of Inter-Observer Variation in Scoring Stromal Tumour Infiltrating Lymphocytes in Triple Negative Breast Cancer.

Simple SummaryThe stromal tumour infiltrating lymphocytes (sTILs) within a tumour are a strong predictor of outcome for patients with triple negative breast cancer (TNBC). However, the assessment of sTILs is subject to variation and needs to be standardized in order for it to be used more widely as a biomarker. The aim of this study was to determine the level of consistency that can be achieved when an internet-based scoring aid is used to assist with evaluation of sTILs. Twenty-three breast pathologists across Europe scored sTILs in 49 cases of TNBC taken from a routine diagnostic practice using this aid. The consistency of scoring sTILs was good. However, variation in the distribution of sTILs within the tumour resulted in discordance between pathologists scoring cases, particularly as it caused variability in the selection of regions of the tumour to score. More rigorous training of pathologists is needed for standardization of sTILs assessment, which may potentially be improved using automated approaches. Stromal tumour infiltrating lymphocytes (sTILs) are a strong prognostic marker in triple negative breast cancer (TNBC). Consistency scoring sTILs is good and was excellent when an internet-based scoring aid developed by the TIL-WG was used to score cases in a reproducibility study. This study aimed to evaluate the reproducibility of sTILs assessment using this scoring aid in cases from routine practice and to explore the potential of the tool to overcome variability in scoring. Twenty-three breast pathologists scored sTILs in digitized slides of 49 TNBC biopsies using the scoring aid. Subsequently, fields of view (FOV) from each case were selected by one pathologist and scored by the group using the tool. Inter-observer agreement was good for absolute sTILs (ICC 0.634, 95% CI 0.539–0.735, p < 0.001) but was poor to fair using binary cutpoints. sTILs heterogeneity was the main contributor to disagreement. When pathologists scored the same FOV from each case, inter-observer agreement was excellent for absolute sTILs (ICC 0.798, 95% CI 0.727–0.864, p < 0.001) and good for the 20% (ICC 0.657, 95% CI 0.561–0.756, p < 0.001) and 40% (ICC 0.644, 95% CI 0.546–0.745, p < 0.001) cutpoints. However, there was a wide range of scores for many cases. Reproducibility scoring sTILs is good when the scoring aid is used. Heterogeneity is the main contributor to variance and will need to be overcome for analytic validity to be achieved.

Humanin Promotes Tumor Progression in Experimental Triple Negative Breast Cancer

Humanin (HN) is a mitochondrial-derived peptide with cytoprotective effect in many tissues. Administration of HN analogs has been proposed as therapeutic approach for degenerative diseases. Although HN has been shown to protect normal tissues from chemotherapy, its role in tumor pathogenesis is poorly understood. Here, we evaluated the effect of HN on the progression of experimental triple negative breast cancer (TNBC). The meta-analysis of transcriptomic data from The Cancer Genome Atlas indicated that HN and its receptors are expressed in breast cancer specimens. By immunohistochemistry we observed up-regulation of HN in TNBC biopsies when compared to mammary gland sections from healthy donors. Addition of exogenous HN protected TNBC cells from apoptotic stimuli whereas shRNA-mediated HN silencing reduced their viability and enhanced their chemo-sensitivity. Systemic administration of HN in TNBC-bearing mice reduced tumor apoptotic rate, impaired the antitumor and anti-metastatic effect of chemotherapy and stimulated tumor progression, accelerating tumor growth and development of spontaneous lung metastases. These findings suggest that HN may exert pro-tumoral effects and thus, caution should be taken when using exogenous HN to treat degenerative diseases. In addition, our study suggests that HN blockade could constitute a therapeutic strategy to improve the efficacy of chemotherapy in breast cancer.

Small Non-Coding RNA Profiling Identifies miR-181a-5p as a Mediator of Estrogen Receptor Beta-Induced Inhibition of Cholesterol Biosynthesis in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease, representing the most aggressive breast cancer (BC) subtype with limited treatment options due to a lack of estrogen receptor alpha (ERα), progesterone receptor (PR), and Erb-B2 receptor tyrosine kinase 2 (HER2/neu) expression. Estrogen receptor beta (ERβ) is present in a fraction of TNBC patients, where its expression correlates with improved patient outcomes, supported by the fact that it exerts oncosuppressive effects in TNBC cell models in vitro. ERβ is involved in microRNA-mediated regulation of gene expression in hormone-responsive BC cells and could mediate its actions through small noncoding RNAs (sncRNAs) in TNBCs also. To verify this possibility, smallRNA sequencing was performed on three ERβ-expressing cell lines from different TNBC molecular subtypes. Several sncRNAs resulted modulated by ERβ, with a subset being regulated in a tumor subtype-independent manner. Interestingly, sncRNA profiling of 12 ERβ+and 32 ERβ− primary TNBC biopsies identified 7 microRNAs, 1 PIWI-interacting RNA (piRNA), and 1 transfer RNA (tRNA) differentially expressed in ERβ+ compared to ERβ− tumors and cell lines. Among them, miR-181a-5p was found to be overexpressed in ERβ+ tumors and predicted target key components of the cholesterol biosynthesis pathway previously found to be inhibited by ERβ in TNBC cells.

A Review of the Potential Role of Human Cytomegalovirus (HCMV) Infections in Breast Cancer Carcinogenesis and Abnormal Immunity.

Previously recognized classical human onco-viruses can regulate complex neoplastic events, and are estimated to play a role during carcinogenesis in 15–20% of cancer cases. Although the DNA and gene products of several viruses have been found in breast tumors, none of the classical onco-viruses have definitely been linked to the initiation of breast cancer. However, recent evidence shows that human cytomegalovirus (HCMV) gene products are found in >90% of tumors and metastases of breast cancers, and their increased expression can be correlated to a more aggressive breast cancer phenotype. Supporting the active role of HCMV in breast cancer, a specific HCMV strain, HCMV-DB, was recently shown to exert oncogenic transformational activity in breast epithelial cells in vitro, and to give rise to fast-growing, triple-negative breast tumors when injected into immune deficient mice. The same observation holds true for clinical studies implying increased HCMV protein expression in triple negative breast cancer biopsies. In addition to functionally being able to hijack tumor-promoting cellular events, HCMV is known to exhibit a wide range of immunosuppressive effects, which can have radical impact on the tumor microenvironment. HCMV infected cells can avoid recognition and elimination by the immune system by orchestrating polarization of immunosuppressive type II macrophages, preventing antigen presentation, by expressing T cell inhibitory molecules, and possibly, by the induction of regulatory T (Treg) cell responses. These actions would be especially deleterious for the antigenic activation and proliferation of tumor specific CD8+ cytotoxic T lymphocytes (CTLs), whose effector functions have recently been targeted by successful, experimental immunotherapy protocols. The recognition of alternative causes and drivers of breast cancer is a pivotal research topic for the development of diagnostics and novel, effective preventive and therapeutic strategies targeting both tumor cells and their microenvironments.

Abstract 2901: Clonal dynamics and phenotypic evolution during chemoresistance and metastasis revealed by patient-derived xenograft models of triple negative breast cancer

Abstract Half of all triple negative breast cancer (TNBC) patients harbor significant residual cancer burden following standard neoadjuvant chemotherapy treatment, resulting in distant metastasis and death for most of these patients. Intra-tumor heterogeneity (ITH) is pervasive in TNBC as is a barrier to development of effective therapeutic strategies, but the relative contributions of heterogeneous tumor cell populations to chemoresistance and metastasis are not well understood. To investigate the clonal dynamics that accompany chemotherapy treatment and metastasis, we employed orthotopic patient-derived xenograft (PDX) models of treatment-naïve TNBC, thus enabling experimentation with heterogeneous populations of human tumor cells that have undergone minimal manipulation. To monitor the fates of PDX tumor cell lineages as they metastasized, we introduced a pooled lentiviral barcode library (Cellecta) into freshly dissociated PDX tumor cells which were orthotopically engrafted into recipient mice. Genomic analyses, including barcode enumeration, whole-exome sequencing, custom targeted DNA sequencing, and transcriptome sequencing, were conducted to characterize the clonal dynamics that accompanied metastasis. Of the thousands of diverse primary tumor clones, only ~2% harbored metastatic capacity. Of those, a rare population of the exact same clones predominated metastases in lung, liver, and brain, the three most common sites of human TNBC metastasis. These studies provide a quantitative map of the clonal architecture of multi-organ metastasis in TNBC and reveal that identical subclones can thrive in diverse secondary organ microenvironments. NACT resistance leads to metastasis and death for most patients, yet the origins of chemoresistance in TNBC are unclear. We modeled NACT resistance in an array of PDX models derived from treatment-naïve TNBC biopsies in alignment with an ongoing neoadjuvant clinical trial (NCT02276443). Upon partial response to NACT, tumors entered a transient drug-tolerant state characterized by distinct histologic, proteomic, and transcriptomic features that were reverted as tumors regrew after cessation of treatment. Barcode-mediated lineage tracking and whole-exome sequencing revealed that the drug-tolerant state was not mediated by clonal selection. Based on transcriptomic and metabolic features of the drug-tolerant state, we conducted preclinical trials with an inhibitor of mitochondrial oxidative phosphorylation (IACS-010759), which significantly delayed the regrowth of residual tumors in PDX models. Together, these studies revealed that TNBCs can resist NACT through non-selective adaptation of a reversible phenotypic state, and that inhibition of oxidative phosphorylation may be a promising therapy in the neoadjuvant setting for TNBC. Citation Format: Gloria V. Echeverria, Zhongqi Ge, Sahil Seth, Emily Powell, Xiaomei Zhang, Sabrina Jeter-Jones, Xinhui Zhou, Yan Jiang, Aaron McCoy, Shirong Cai, Yizheng Tu, Michael Peoples, Yuting Sun, Huan Qiu, Christopher Bristow, Alessandro Carugo, Jiansu Shao, Stacy L. Moulder, William F. Symmans, Timothy P. Heffernan, Jeffrey T. Chang, Helen M. Piwnica-Worms. Clonal dynamics and phenotypic evolution during chemoresistance and metastasis revealed by patient-derived xenograft models of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2901.

The role of stromal tumor infiltrating lymphocytes (sTILs) as a predictive biomarker for carboplatin-based neoadjuvant chemotherapy (NACT) in triple-negative breast cancer (TNBC).

e12085 Background: High numbers of sTILs are predictive of pCR in TNBCs. This study examines the role of sTILs as a predictive biomarker for carboplatin (Cb) based NACT. Methods: sTILs were scored on 88 pre NACT TNBC biopsies as per the International TILs Working Group and were scored as continuous and categorical variables: 0-10%; 11-25%; 26-49%; ≥50%. OR was calculated using non-pCR as baseline parameter. Results: Patients with sTILs &gt; 10% had increased pCR rates compared to those with sTILs ≤10%: pCR breast (B) (61% vs 31% p = 0.004); pCR breast/axilla (BA) (52% vs 29% p = 0.021). In those treated with Cb, there were no differences in pCR B between sTILs &gt; 10% or ≤10% (67% vs 53%; p = 0.355) or pCR BA (60% vs 53%; p = 0.500). In those who did not receive Cb, sTILs &gt; 10% had increased pCR B (57% vs 15%; p = 0.002) and pCR BA (46% vs 12%; p = 0.005) compared to sTILs ≤10%. Similar trends were seen with sTILs &gt; 25% and ≥50%: differences were seen in patients who did not receive Cb, but not in those treated with Cb. In LPBCs, the addition of Cb did not significantly increase pCR rates: 67% vs 83% for Cb and non-Cb regimens (p = 0.583). In non-LPBC, pCR rates were increased with Cb: pCR B 59% vs 31% (p = 0.017), and pCR BA 56% vs 23% (p = 0.005) for Cb and non-Cb regimens. The association between sTILs and pCR was significant in multivariable models adjusting for grade and Cb. In patients who did not receive Cb (n = 54), increasing sTILs were associated with increased pCR B (OR 0.15 p = 0.004; OR 0.27 p = 0.039; OR 0.10 p = 0.05 for &gt; 10%, &gt; 25%, ≥50%) and pCR BA (OR 0.18 p = 0.019; OR 0.08 p = 0.029 for &gt; 10%, ≥50%). In patients treated with Cb (n = 30), increasing sTILs were not associated with pCR B (OR 1.97 p = 0.581; OR 0.27 p = 0.334; OR 0.50 p = 0.719) or pCR BA (OR 2.93 p = 0.372; OR 0.79 p = 0.831; OR 0.40 p = 0.622 for &gt; 10%, &gt; 25%, ≥50%). Conclusions: sTILs are predictive of pCR: as sTILs increase, pCR rates increase. Tumors with high sTILs had high pCR rates to anthracycline-taxane (AT) NACT. The effect of increasing sTILs on pCR was most notable in patients who did not receive Cb based NACT, suggesting that tumors with high sTILs are inherently sensitive to AT based chemotherapy. Intensifying NACT with Cb could be used to increase pCR rates in patients with low sTILs. sTILs should be explored as a biomarker to intensify chemotherapy in those with low sTILs, and to de-escalate chemotherapy in tumors with high sTILs.

Heart myxoma develops oncogenic and metastatic phenotype

Heart myxomas have been frequently considered as benign lesions associated with Carney's complex. However, after surgical removal, myxomas re-emerge causing dysfunctional heart. To identify whether cardiac myxomas may develop a metastatic phenotype as occurs in malignant cancers, a profile of several proteins involved in malignancy such as oncogenes (c-MYC, K-RAS and H-RAS), cancer-associated metabolic transcriptional factors (HIF-1α, p53 and PPAR-γ) and epithelial-mesenchymal transition proteins (fibronectin, vimentin, β-catenin, SNAIL and MMP-9) were evaluated in seven samples from a cohort of patients with atrial and ventricular myxomas. The analysis was also performed in: (1) cardiac tissue surrounding the area where myxoma was removed; (2) non-cancer heart tissue (NCHT); and (3) malignant triple negative breast cancer biopsies for comparative purposes. Statistical analysis applying univariate (Kruskal-Wallis and Dunn's tests) and multivariate analyses (PCA, principal component analysis) revealed that heart myxomas (7-15 times) and myxoma surrounding tissue (22-99 times) vs. NCHT showed high content of c-MYC, p53, vimentin, and HIF-1α, indicating that both myxoma and its surrounding area express oncogenes and malignancy-related proteins as occurs in triple negative breast cancer. Based on ROC (receiver operating characteristics) statistical analysis, c-MYC, HIF-1α, p53, and vimentin may be considered potential biomarkers for malignancy detection in myxoma.

Genetic alterations of triple negative breast cancer (TNBC) in women from Northeastern Mexico.

Triple negative breast cancer (TNBC) is a subtype of breast cancer of heterogeneous nature that is negative for estrogen receptor (ER), progesterone receptor (PR) and growth factor human epidermal 2 (HER2) following immunohistochemical analysis. TNBC is frequently characterized by relapse and reduced survival. To date, there is no targeted therapy for this type of cancer. Chemotherapy, radiotherapy, and surgery remain as the standard treatments options. The lack of a target therapy and the heterogeneity of TNBC highlight the need to seek new therapeutic options. In this study, fresh tissue samples of TNBC were analyzed with a panel of 48 driver genes (212 amplicons) that are likely to be therapeutic targets. We found intron variants, missense, stop gained and splicing variants in TP53, PIK3CA and FLT3 genes. Interestingly, all the analyzed samples had at least two variants in the TP53 gene, one being a drug response variant, rs1042522, found in 94% of our samples. We also found seven additional variants not previously reported in the TP53 gene, to the best of our knowledge, with probable deleterious characteristics of the tumor suppressor gene. We found four genetic variants in the PIK3CA gene, including two missense variants. The rs2491231 variant in the FLT3 gene was identified in 84% (16/19) of the samples, which not yet reported for TNBC, to the best of our knowledge. In conclusion, genetic variants in TP53 were found in all TNBC tumors, with rs1042522 being the most frequent (94% of TNBC biopsies), which had not been previously reported in TNBC. Also, we found two missense variants in the PIK3CA gene. These results justify the validation of these genetic variants in a large cohort, as well as the extensive study of their impact on the prognosis and therapy management of TBNC.

Expression of Androgen Receptor in Triple Negative Breast Cancer Biopsies and Its Correlation with Prognostic Markers

Expression of Androgen Receptor in Triple Negative Breast Cancer Biopsies and Its Correlation with Prognostic Markers

Abstract 180: PhAc-ALGP-Dox is a new tumor selective peptide prodrug of doxorubicin that shows improved efficacy and systemic tolerance in triple negative breast cancer models

Abstract Use of traditional chemotherapeutic drugs is restricted by severe side effects and lack of tumor specificity of these cytotoxic agents. Less toxic prodrug that can be selectively activated in tumor tissue have been explored in attempts to improve the therapeutic index. Some approaches to the development of tumor activating prodrugs take advantage of inherent properties of the tumor, for example, selective enzyme expression, hypoxia, or low extracellular pH in the vicinity of the tumor. CoBioRes makes use of capped, tetrapeptidic prodrug of existing chemotherapeutics. Doxorubicin (Dox) was the first chemotherapeutic that was linked to the tetrapeptide sequence and tested experimentally (PhAc-ALGP-Dox). This approach makes the prodrug impermeable to cell membranes of both normal and tumor cells. Furthermore, it remains stable in blood by the use of the capping group on the amino-terminal group of the peptide that prevents aspecific activation by circulating esopeptidases. The prodrug as such is thus not active. The peptide sequences have been developed to be sensitive to the hydrolytic action of a selected group of peptidases that are released within the tumor microenvironment. These peptidases, which accumulate in the tumors, also play an important role in cancer cell invasion and metastasis. What makes this approach unique is that it is not targeting a single enzyme, but requires, for its selectivity, a two-step activation based on enzymes with an increasing selectivity. In vivo activation starts when the tetrapeptide prodrug is exposed to endonucleases (CD10 and THOP1) leading to the formation of intermediates that are still inactive and poorly permeable through cell membranes. This leads to a tissue distribution that is determined by the expression of the endonucleases. Since these are known to be overexpressed in several tumors, and only present at lower level in some normal tissues, the prodrug will be preferentially located and activated in the vicinity of tumors. The second step of the activation is the cleavage at the prolyl by dipeptidases FAP and DPPIV, releasing the free doxorubicin. The simultaneous expression of all the enzymes involved in the prodrug activation was confirmed both in human triple negative breast cancer biopsies and in experimental triple negative breast cancer models. In vitro analysis of PhAc-ALGP-Dox reveals higher tumor cells tropism and higher cytotoxicity in cancer cells than in normal cells. In vivo PhAc-ALGP-Dox gives promising preliminary results in terms of systemic tolerance even at high concentration, and in vivo efficacy in xenograft mouse models compared to free doxorubicin. Taken together, these results provide a strong rationale for further investigation aimed at unleashing the potential clinical value of this compound. Citation Format: Andrea Casazza, Massimiliano Mazzone, Peter Pokreisz. PhAc-ALGP-Dox is a new tumor selective peptide prodrug of doxorubicin that shows improved efficacy and systemic tolerance in triple negative breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 180. doi:10.1158/1538-7445.AM2017-180

Abstract P1-08-07: A novel pharmacodynamic assay to measure glutaminase inhibition following oral administration of CB-839 in triple negative breast cancer biopsies

Abstract Triple negative breast cancer (TNBC) cell lines are highly dependent on glutamine (Gln) for growth and survival. A critical step in Gln utilization is its conversation to glutamate (Glu) by the mitochondrial enzyme glutaminase (GLS). CB-839 is a potent inhibitor of GLS that has anti-proliferative activity in TNBC cell lines and antitumor activity in TNBC xenograft models (Gross et al., Mol. Cancer Ther. 13:890). Across a panel of breast cancer cell lines derived from both receptor positive and TNBC tumors, sensitivity to CB-839 was associated with (i) elevated GLS expression, (ii) elevated GLS activity, and (iii) the TNBC subtype. Importantly, many of the determinants of CB-839 sensitivity in cell lines are also present in primary tumor samples, including high mRNA and protein expression of GLS and a high Glu to Gln ratio in TNBC tumors as compared to receptor positive tumors. These observations motivate the Phase 1 clinical study of CB-839 in TNBC patients. To aid in the selection of a recommended Phase 2 dose, we sought to develop a pharmacodynamic (PD) assay to directly measure the GLS activity in breast tumor lysates in order to determine the extent of GLS inhibition in tumor biopsies from CB-839 treated patients. To develop a robust PD assay, we first identified conditions that maintain the GLS:CB-839 inhibitory complex during preparation of lysates from CB-839 treated samples. High concentrations of KCl (150 mM) and low concentrations of K-phosphate (15 mM) in the lysis buffer, as well as maintaining the lysate at a low temperature stabilized the inhibited complex. Following gel filtration of the lysate to remove unbound CB-839 and exchange the buffer, GLS activity was immediately measured with a coupled enzyme assay. The GLS activity measured at this step reflects the residual activity present in a sample that was exposed to CB-839. To quantify the amount of total GLS present in the sample, we incubated the same lysate for 3 hours at room temperature under conditions of low KCl and high phosphate to allow the the GLS:CB-839 complex to fully dissociate prior to measuring activity. This assay format allows quantitation of the % GLS inhibition from a single tumor lysate sample and eliminates the requirement for multiple biopsies as well as any assay variability due to tumor heterogeneity. We utilized this tumor PD assay to determine the plasma drug levels required for maximal tumor GLS inhibition in a preclinical TNBC model. Mice bearing HCC1806 TNBC tumors were first treated with a range of CB-839 doses. Four hours after oral administration, a 10 mg/kg dose of CB-839 resulted in &amp;gt;90% inhibition of tumor GLS. CB-839 plasma concentrations of 100 nM corresponded to 50% inhibition of tumor GLS, while maximal inhibition occurred at plasma concentrations ≥300 nM. In xenograft studies, maximal anti-tumor efficacy was achieved with BID dosing at 200 mg/kg, a dose and schedule that resulted in trough plasma levels of CB-839 of ≥300 nM and sustained GLS inhibition in tumors. As part of an ongoing Phase 1 trial, this assay will be utilized to monitor tumor PD responses in TNBC patients undergoing treatment. Citation Format: Andy MacKinnon, Mark Bennett, Ethan Emberley, Mathew Gross, Julie Janes, Evan Lewis, Alison Pan, Mirna Rodriguez, Peter Shwonek, Taotao Wang, Jinfu Yang, Frances Zhao, Francesco Parlati. A novel pharmacodynamic assay to measure glutaminase inhibition following oral administration of CB-839 in triple negative breast cancer biopsies [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-08-07.

TTK/hMPS1 is an attractive therapeutic target for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents a subgroup of breast cancers (BC) associated with the most aggressive clinical behavior. No targeted therapy is currently available for the treatment of patients with TNBC. In order to discover potential therapeutic targets, we searched for protein kinases that are overexpressed in human TNBC biopsies and whose silencing in TNBC cell lines causes cell death. A cohort including human BC biopsies obtained at Institut Curie as well as normal tissues has been analyzed at a gene-expression level. The data revealed that the human protein kinase monopolar spindle 1 (hMPS1), also known as TTK and involved in mitotic checkpoint, is specifically overexpressed in TNBC, compared to the other BC subgroups and healthy tissues. We confirmed by immunohistochemistry and reverse phase protein array that TNBC expressed higher levels of TTK protein compared to the other BC subgroups. We then determined the biological effects of TTK depletion by RNA interference, through analyses of tumorigenic capacity and cell viability in different human TNBC cell lines. We found that RNAi-mediated depletion of TTK in various TNBC cell lines severely compromised their viability and their ability to form colonies in an anchorage-independent manner. Moreover, we observed that TTK silencing led to an increase in H2AX phosphorylation, activation of caspases 3/7, sub-G1 cell population accumulation and high annexin V staining, as well as to a decrease in G1 phase cell population and an increased aneuploidy. Altogether, these data indicate that TTK depletion in TNBC cells induces apoptosis. These results point out TTK as a protein kinase overexpressed in TNBC that may represent an attractive therapeutic target specifically for this poor prognosis associated subgroup of breast cancer.

Abstract 4373: LRP5: a potential therapeutic target in triple-negative breast cancer.

Abstract Background: Triple-negative breast cancer (TNBC) is associated with poor prognosis, only partial response to chemotherapy and lack of clinically established targeted therapies [1]. A deregulation of the Wnt signaling pathway has been described in breast cancers, particularly in TNBC [2–6]. Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical beta-catenin pathway. An overexpression of LRP6 has been reported to enhance Wnt signaling favoring in vitro cell proliferation and in vivo mammary gland hyperplasia and tumor growth [5,7,8]. LRP6 has been claimed to be a potential TNBC therapeutic target [5]. Material and Methods: RNA microarray analysis and reverse phase protein array were performed on 154 samples including biopsies of the various subclasses of breast cancer. MDA-MB-468 and HCC38 cell lines were defined as the most representative in vitro models of the Wnt pathway status found in TNBC biopsies. In order to study the functions of LRP5 or LRP6 in TNBC, we examined in these cell lines the effects of their depletions using RNAi technology on tumorigenesis and on the Wnt3a-induced signaling pathway. Results: Our transcriptomic and proteomic data revealed that both LRP5 and LRP6 are overexpressed in TNBC compared to the other breast cancer subtypes i.e. HER2+/ER-, luminal A and luminal B. Our in vitro studies indicated that the transcriptional activity of beta-catenin/Tcf was strongly reduced when LRP6 was silenced and to a lesser extend when LRP5 was depleted. In accordance with these results, the expression of AXIN2 and other newly identified Wnt target genes, was mainly down-regulated in cells silenced for LRP6. LRP5 and LRP6 knockdown impaired colony formation in soft agar and weakly decreased the number of mammospheres. The inhibition of cell viability observed after LRP5 depletion was the consequence of a programmed cell death as revealed by the increase of annexin V-positive cells, the activation of initiator and effector caspases (8,9,3/7) and the cleavage of poly(ADP-ribose) polymerase. On the contrary, LRP6 depletion inhibited cell viability without promoting apoptosis as reported by others [5]. Conclusions: Altogether our data demonstrate that in TNBC cell lines, LRP5 or LRP6 silencing has an impact on Wnt signaling, cancer stem cell-like activity, tumorigenic properties and cell viability. Most importantly, LRP5 silencing promotes apoptosis, suggesting that LRP5 could represent a promising therapeutic candidate to target in TNBC. Citation Format: Sylvie Maubant, Virginie Maire, Bruno Tesson, David Gentien, Bérengère Marty-Prouvost, Francisco Cruzalegui, Stéphane Depil, Gordon C. Tucker, Sergio Roman-Roman, Thierry Dubois. LRP5: a potential therapeutic target in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4373. doi:10.1158/1538-7445.AM2013-4373

Abstract P6-11-12: Nanoparticle-enhanced chemotherapeutics delivery in drug-resistant triple-negative breast cancer

Abstract Chemotherapeutics delivery is generally poor in tumors characterized by rapid perfusion and low blood volume fraction. Systemically administered nanoparticles can be engineered to overcome abnormal flow conditions to act as intravascular drug depots for the localized delivery of high concentrations of chemotherapeutics. The feasibility of this approach was first demonstrated using melanoma, and is now being further investigated using well-characterized human triple-negative breast cancer biopsies implanted into mice. Intravital microscopy studies of human cancer-in-mice selected for differing vascular morphologies have yielded intriguing preliminary data regarding the role of tumor vascularity in drug and particle delivery. The first-pass perfusion of a 40kDa dextran tracer revealed that BCM-2665 tumors are perfused ∼6x more rapidly than BCM-4195 tumors (11.3 ± 2.3s vs. 67.6 ± 11.0) and contain ∼30% lower volume fraction of blood (0.046 ± 0.011 vs. 0.062 ± 0.015). Interestingly, flow parameters that adversely impact drug accumulation appear to favor plateloid particle accumulation. BCM-2665 xenografts receiving an i.v. injection of 1000×400 nm particles show ∼10x more particle accumulation than BCM-4195 tumors (24.8 ± 3.2 × 103/mm3 vs. 3.5 ± 0.4 × 103/mm3). The ability of these therapeutic particles to reach tumors appears to be primarily driven by flow-related parameters, which we characterize using a combination of intravital microscopy, computed tomography, and mathematical modeling. The total number of particles accumulated within a given tumor appears to be largely driven by the number of particles entering the tumor, since ∼65–70% of entering plateloid particles are retained by the tumor vasculature. This suggests that cytotoxic intravascular drug depots may be a promising strategy for increasing the efficiency of chemotherapeutics delivery to drug-resistant tumors and is the premise of ongoing therapeutic response studies. Clearly if more drugs can be delivered to the tumors, better outcomes can be expected for the patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-11-12.