Event-free survival by residual cancer burden with pembrolizumab in early-stage TNBC: exploratory analysis from KEYNOTE-522.

BACKGROUND: KEYNOTE-522 demonstrated statistically significant improvements in pathological complete response (pCR) with neoadjuvant pembrolizumab plus chemotherapy and event-free survival (EFS) with neoadjuvant pembrolizumab plus chemotherapy followed by adjuvant pembrolizumab in patients with high-risk, early-stage triple-negative breast cancer (TNBC). Prior studies have shown the prognostic value of the residual cancer burden (RCB) index to quantify the extent of residual disease after neoadjuvant chemotherapy. In this preplanned exploratory analysis, we assessed RCB distribution and EFS within RCB categories by treatment group. PATIENTS AND METHODS: A total of 1174 patients with stage T1c/N1-2 or T2-4/N0-2 TNBC were randomized 2 : 1 to pembrolizumab 200 mg or placebo every 3 weeks given with four cycles of paclitaxel + carboplatin, followed by four cycles of doxorubicin or epirubicin + cyclophosphamide. After surgery, patients received pembrolizumab or placebo for nine cycles or until recurrence or unacceptable toxicity. Primary endpoints are pCR and EFS. RCB is a prespecified exploratory endpoint. The association between EFS and RCB was assessed using a Cox regression model. RESULTS: Pembrolizumab shifted patients into lower RCB categories across the entire spectrum compared with placebo. There were more patients in the pembrolizumab group with RCB-0 (pCR), and fewer patients in the pembrolizumab group with RCB-1, RCB-2, and RCB-3. The corresponding hazard ratios (95% confidence intervals) for EFS were 0.70 (0.38-1.31), 0.92 (0.39-2.20), 0.52 (0.32-0.82), and 1.24 (0.69-2.23). The most common first EFS events were distant recurrences, with fewer in the pembrolizumab group across all RCB categories. Among patients with RCB-0/1, more than half [21/38 (55.3%)] of all events were central nervous system recurrences, with 13/22 (59.1%) in the pembrolizumab group and 8/16 (50.0%) in the placebo group. CONCLUSIONS: Addition of pembrolizumab to chemotherapy resulted in fewer EFS events in the RCB-0, RCB-1, and RCB-2 categories, with the greatest benefit in RCB-2. These findings demonstrate that pembrolizumab not only increased pCR rates, but also improved EFS among most patients who do not have a pCR.

Risk-adapted modulation through de-intensification of cancer treatments: an ESMO classification.

BACKGROUND: The landscape of clinical trials testing risk-adapted modulations of cancer treatments is complex. Multiple trial designs, endpoints, and thresholds for non-inferiority have been used; however, no consensus or convention has ever been agreed to categorise biomarkers useful to inform the treatment intensity modulation of cancer treatments. METHODS: An expert subgroup under the European Society for Medical Oncology (ESMO) Precision Medicine Working Group shaped an international collaborative project to develop a classification system for biomarkers used in the cancer treatment de-intensification, based on a tiered approach. A group of disease-oriented clinical, translational, methodology and public health experts, and patients' representatives provided an analysis of the status quo, and scanned the horizon of ongoing clinical trials. The classification was developed through multiple rounds of expert revisions and inputs. RESULTS: The working group agreed on a univocal definition of treatment de-intensification. Evidence of reduction in the dose-density, intensity, or cumulative dose, including intermittent schedules or shorter treatment duration or deletion of segment(s) of the standard regimens, compound(s), or treatment modality must be demonstrated, to define a treatment de-intensification. De-intensified regimens must also portend a positive impact on toxicity, quality of life, health system burden, or financial toxicity. ESMO classification categorises the biomarkers for treatment modulation in three tiers, based on the level of evidence. Tier A includes biomarkers validated in prospective, randomised, non-inferiority clinical trials. The working group agreed that in non-inferiority clinical trials, boundaries are highly dependent upon the disease scenario and endpoint being studied and that the absolute differences in the outcomes are the most relevant measures, rather than relative differences. Biomarkers tested in single-arm studies with a threshold of non-inferiority are classified as Tier B. Tier C is when the validation occurs in prospective-retrospective quality cohort investigations. CONCLUSIONS: ESMO classification for the risk-guided intensity modulation of cancer treatments provides a set of evidence-based criteria to categorise biomarkers deemed to inform de-intensification of cancer treatments, in risk-defined patients. The classification aims at harmonising definitions on this matter, therefore offering a common language for all the relevant stakeholders, including clinicians, patients, decision-makers, and for clinical trials.

Treatment Efficacy Score-continuous residual cancer burden-based metric to compare neoadjuvant chemotherapy efficacy between randomized trial arms in breast cancer trials.

BACKGROUND: Difference in pathologic complete response (pCR) rate after neoadjuvant chemotherapy does not capture the impact of treatment on downstaging of residual cancer in the experimental arm. We developed a method to compare the entire distribution of residual cancer burden (RCB) values between clinical trial arms to better quantify the differences in cytotoxic efficacy of treatments. PATIENTS AND METHODS: The Treatment Efficacy Score (TES) reflects the area between the weighted cumulative distribution functions of RCB values from two trial arms. TES is based on a modified Kolmogorov-Smirnov test with added weight function to capture the importance of high RCB values and uses the area under the difference between two distribution functions as a statistical metric. The higher the TES the greater the shift to lower RCB values in the experimental arm. We developed TES from the durvalumab + olaparib arm (n = 72) and corresponding controls (n = 282) of the I-SPY2 trial. The 11 other experimental arms and control cohorts (n = 947) were used as validation sets to assess the performance of TES. We compared TES to Kolmogorov-Smirnov, Mann-Whitney, and Fisher's exact tests to identify trial arms with higher cytotoxic efficacy and assessed associations with trial arm level survival differences. Significance was assessed with a permutation test. RESULTS: In the validation set, TES identified arms with a higher pCR rate but was more accurate to identify regimens as less effective if treatment did not reduce the frequency of high RCB values, even if the pCR rate improved. The correlation between TES and survival was higher than the correlation between the pCR rate difference and survival. CONCLUSIONS: TES quantifies the difference between the entire distribution of pathologic responses observed in trial arms and could serve as a better early surrogate to predict trial arm level survival differences than pCR rate difference alone.

Predicted sensitivity to endocrine therapy for stage II-III hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer before chemo-endocrine therapy.

BACKGROUND: We proposed that a test for sensitivity to the adjuvant endocrine therapy component of treatment for patients with stage II-III breast cancer (SET2,3) should measure transcription related to estrogen and progesterone receptors (SETER/PR index) adjusted for a baseline prognostic index (BPI) combining clinical tumor and nodal stage with molecular subtype by RNA4 (ESR1, PGR, ERBB2, and AURKA). PATIENTS AND METHODS: Patients with clinically high-risk, hormone receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-negative (HR+/HER2-) breast cancer received neoadjuvant taxane-anthracycline chemotherapy, surgery with measurement of residual cancer burden (RCB), and then adjuvant endocrine therapy. SET2,3 was measured from pre-treatment tumor biopsies, evaluated first in an MD Anderson Cancer Center (MDACC) cohort (n = 307, 11 years' follow-up, U133A microarrays), cut point was determined, and then independent, blinded evaluation was carried out in the I-SPY2 trial (n = 268, high-risk MammaPrint result, 3.8 years' follow-up, Agilent-44K microarrays, NCI Clinical Trials ID: NCT01042379). Primary outcome measure was distant relapse-free survival. Multivariate Cox regression models tested prognostic independence of SET2,3 relative to RCB and other molecular prognostic signatures, and whether other prognostic signatures could substitute for SETER/PR or RNA4 components of SET2,3. RESULTS: SET2,3 added independent prognostic information to RCB in the MDACC cohort: SET2,3 [hazard ratio (HR) 0.23, P = 0.004] and RCB (HR 1.77, P < 0.001); and the I-SPY2 trial: SET2,3 (HR 0.27, P = 0.031) and RCB (HR 1.68, P = 0.008). SET2,3 provided similar prognostic information irrespective of whether RCB-II or RCB-III after chemotherapy, and in both luminal subtypes. Conversely, RCB was most strongly prognostic in cancers with low SET2,3 status (MDACC P < 0.001, I-SPY2 P < 0.001). Other molecular signatures were not independently prognostic; they could effectively substitute for RNA4 subtype within the BPI component of SET2,3, but they could not effectively substitute for SETER/PR index. CONCLUSIONS: SET2,3 added independent prognostic information to chemotherapy response (RCB) and baseline prognostic score or subtype. Approximately 40% of patients with clinically high-risk HR+/HER2- disease had high SET2,3 and could be considered for clinical trials of neoadjuvant endocrine-based treatment.

Immunological differences between primary and metastatic breast cancer.

Background: Little is known about how the immune microenvironment of breast cancer evolves during disease progression. Patients and methods: We compared tumor infiltrating lymphocyte (TIL) count, programmed death-ligand 1 (PD-L1) protein expression by immunohistochemistry and mRNA levels of 730 immune-related genes using Nanostring technology in primary and metastatic cancer samples. Results: TIL counts and PD-L1 positivity were significantly lower in metastases. Immune cell metagenes corresponding to CD8, T-helper, T-reg, Cytotoxic T, Dendritic and Mastoid cells, and expression of 13 of 29 immuno-oncology therapeutic targets in clinical development including PD1, PD-L1, and CTLA4 were significantly lower in metastases. There was also coordinated down regulation of chemoattractant ligand/receptor pairs (CCL19/CCR7, CXCL9/CXCR3, IL15/IL15R), interferon regulated genes (STAT1, IRF-1,-4,-7, IFI-27,-35), granzyme/granulysin, MHC class I and immune proteasome (PSMB-8,-9,-10) expression in metastases. Immunotherapy response predictive signatures were also lower. The expression of macrophage markers (CD163, CCL2/CCR2, CSF1/CSFR1, CXCR4/CXCL12), protumorigenic toll-like receptor pathway genes (CD14/TLR-1,-2,-4,-5,-6/MyD88), HLA-E, ecto-nuclease CD73/NT5E and inhibitory complement receptors (CD-59,-55,-46) remained high in metastases and represent potential therapeutic targets. Conclusions: Metastatic breast cancers are immunologically more inert than the corresponding primary tumors but some immune-oncology targets and macrophage and angiogenesis signatures show preserved expression and suggest therapeutic combinations for clinical testing.

A framework to rank genomic alterations as targets for cancer precision medicine: the ESMO Scale for Clinical Actionability of molecular Targets (ESCAT).

Background: In order to facilitate implementation of precision medicine in clinical management of cancer, there is a need to harmonise and standardise the reporting and interpretation of clinically relevant genomics data. Methods: The European Society for Medical Oncology (ESMO) Translational Research and Precision Medicine Working Group (TR and PM WG) launched a collaborative project to propose a classification system for molecular aberrations based on the evidence available supporting their value as clinical targets. A group of experts from several institutions was assembled to review available evidence, reach a consensus on grading criteria and present a classification system. This was then reviewed, amended and finally approved by the ESMO TR and PM WG and the ESMO leadership. Results: This first version of the ESMO Scale of Clinical Actionability for molecular Targets (ESCAT) defines six levels of clinical evidence for molecular targets according to the implications for patient management: tier I, targets ready for implementation in routine clinical decisions; tier II, investigational targets that likely define a patient population that benefits from a targeted drug but additional data are needed; tier III, clinical benefit previously demonstrated in other tumour types or for similar molecular targets; tier IV, preclinical evidence of actionability; tier V, evidence supporting co-targeting approaches; and tier X, lack of evidence for actionability. Conclusions: The ESCAT defines clinical evidence-based criteria to prioritise genomic alterations as markers to select patients for targeted therapies. This classification system aims to offer a common language for all the relevant stakeholders in cancer medicine and drug development.

Revisiting the definition of estrogen receptor positivity in HER2-negative primary breast cancer.

BACKGROUND: Although 1% has been used as cut-off for estrogen receptor (ER) positivity, several studies have reported that tumors with ER < 1% have characteristics similar to those with 1% ≤ ER < 10%. We hypothesized that in patients with human epidermal growth factor 2 (HER2)-negative breast cancer, a cut-off of 10% is more useful than one of 1% in discriminating for both a better pathological complete response (pCR) rate to neoadjuvant chemotherapy and a better long-term outcome with adjuvant hormonal therapy. Our objectives were to identify a percentage of ER expression below which pCR was likely and to determine whether this cut-off value can identify patients who would benefit from adjuvant hormonal therapy. PATIENTS AND METHODS: Patients with stage II or III HER2-negative primary breast cancer who received neoadjuvant chemotherapy followed by definitive surgery between June 1982 and June 2013 were included. Logistic regression models were used to assess the association between each variable and pCR. Cox models were used to analyze time to recurrence and overall survival. The recursive partitioning and regression trees method was used to calculate the cut-off value of ER expression. RESULTS: A total of 3055 patients were analyzed. Low percentage of ER was significantly associated with high pCR rate (OR = 0.99, 95% CI = 0.986-0.994, P < 0.001). The recommended cut-off of ER expression below which pCR was likely was 9.5%. Among patients with ER ≥ 10% tumors, but not those with 1%≤ER < 10% tumors, adjuvant hormonal therapy was significantly associated with long time to recurrence (HR = 0.24, 95% CI = 0.16-0.36, P < 0.001) and overall survival (HR = 0.32, 95% CI = 0.2-0.5, P < 0.001). CONCLUSION: Stage II or III HER2-negative primary breast cancer with ER < 10% behaves clinically like triple-negative breast cancer in terms of pCR and survival outcomes and patients with such tumors may have a limited benefit from adjuvant hormonal therapy. It may be more clinically relevant to define triple-negative breast cancer as HER2-negative breast cancer with <10%, rather than <1%, of ER and/or progesterone receptor expression.

Hybrid capture-based genomic profiling of circulating tumor DNA from patients with estrogen receptor-positive metastatic breast cancer.

BACKGROUND: Genomic changes that occur in breast cancer during the course of disease have been informed by sequencing of primary and metastatic tumor tissue. For patients with relapsed and metastatic disease, evolution of the breast cancer genome highlights the importance of using a recent sample for genomic profiling to guide clinical decision-making. Obtaining a metastatic tissue biopsy can be challenging, and analysis of circulating tumor DNA (ctDNA) from blood may provide a minimally invasive alternative. PATIENTS AND METHODS: Hybrid capture-based genomic profiling was carried out on ctDNA from 254 female patients with estrogen receptor-positive breast cancer. Peripheral blood samples were submitted by clinicians in the course of routine clinical care between May 2016 and March 2017. Sequencing of 62 genes was carried out to a median unique coverage depth of 7503×. Genomic alterations (GAs) in ctDNA were evaluated and compared with matched tissue samples and genomic datasets of tissue from breast cancer. RESULTS: At least 1 GA was reported in 78% of samples. Frequently altered genes were TP53 (38%), ESR1 (31%) and PIK3CA (31%). Temporally matched ctDNA and tissue samples were available for 14 patients; 89% of mutations detected in tissue were also detected in ctDNA. Diverse ESR1 GAs including mutation, rearrangement and amplification, were observed. Multiple concurrent ESR1 GAs were observed in 40% of ESR1-altered cases, suggesting polyclonal origin; ESR1 compound mutations were also observed in two cases. ESR1-altered cases harbored co-occurring GAs in PIK3CA (35%), FGFR1 (16%), ERBB2 (8%), BRCA1/2 (5%), and AKT1 (4%). CONCLUSIONS: GAs relevant to relapsed/metastatic breast cancer management were identified, including diverse ESR1 GAs. Genomic profiling of ctDNA demonstrated sensitive detection of mutations found in tissue. Detection of amplifications was associated with ctDNA fraction. Genomic profiling of ctDNA may provide a complementary and possibly alternative approach to tissue-based genomic testing for patients with estrogen receptor-positive metastatic breast cancer.

Pathway level alterations rather than mutations in single genes predict response to HER2-targeted therapies in the neo-ALTTO trial.

Background: We performed whole-exome sequencing of pretreatment biopsies and examined whether genome-wide metrics of overall mutational load, clonal heterogeneity or alterations at variant, gene, and pathway levels are associated with treatment response and survival. Patients and Methods: Two hundred and three biopsies from the NeoALTTO trial were analyzed. Mutations were called with MuTect, and Strelka, using pooled normal DNA. Associations between DNA alterations and outcome were evaluated by logistic and Cox-proportional hazards regression. Results: There were no recurrent single gene mutations significantly associated with pathologic complete response (pCR), except PIK3CA [odds ratio (OR) = 0.42, P = 0.0185]. Mutations in 33 of 714 pathways were significantly associated with response, but different genes were affected in different individuals. PIK3CA was present in 23 of these pathways defining a 'trastuzumab resistance-network' of 459 genes. Cases with mutations in this network had low pCR rates to trastuzumab (2/50, 4%) compared with cases with no mutations (9/16, 56%), OR = 0.035; P < 0.001. Mutations in the 'Regulation of RhoA activity' pathway were associated with higher pCR rate to lapatinib (OR = 14.8, adjusted P = 0.001), lapatinib + trastuzumab (OR = 3.0, adjusted P = 0.09), and all arms combined (OR = 3.77, adjusted P = 0.02). Patients (n = 124) with mutations in the trastuzumab resistance network but intact RhoA pathway had 2% (1/41) pCR rate with trastuzumab alone (OR = 0.026, P = 0.001) but adding lapatinib increased pCR rate to 45% (17/38, OR = 1.68, P = 0.3). Patients (n = 46) who had no mutations in either gene set had 6% pCR rate (1/15) with lapatinib, but had the highest pCR rate, 52% (8/15) with trastuzumab alone. Conclusions: Mutations in the RhoA pathway are associated with pCR to lapatinib and mutations in a PIK3CA-related network are associated with resistance to trastuzumab. The combined mutation status of these two pathways could define patients with very low response rate to trastuzumab alone that can be augmented by adding lapatinib or substituting trastuzumab with lapatinib.

SWOG S0800 (NCI CDR0000636131): addition of bevacizumab to neoadjuvant nab-paclitaxel with dose-dense doxorubicin and cyclophosphamide improves pathologic complete response (pCR) rates in inflammatory or locally advanced breast cancer.

SWOG S0800, a randomized open-label Phase II clinical trial, compared the combination of weekly nab-paclitaxel and bevacizumab followed by dose-dense doxorubicin and cyclophosphamide (AC) with nab-paclitaxel followed or preceded by AC as neoadjuvant treatment for HER2-negative locally advanced breast cancer (LABC) or inflammatory breast cancer (IBC). Patients were randomly allocated (2:1:1) to three neoadjuvant chemotherapy arms: (1) nab-paclitaxel with concurrent bevacizumab followed by AC; (2) nab-paclitaxel followed by AC; or (3) AC followed by nab-paclitaxel. The primary endpoint was pathologic complete response (pCR) with stratification by disease type (non-IBC LABC vs. IBC) and hormone receptor status (positive vs. negative). Overall survival (OS), event-free survival (EFS), and toxicity were secondary endpoints. Analyses were intent-to-treat comparing bevacizumab to the combined control arms. A total of 215 patients were accrued including 11 % with IBC and 32 % with triple-negative breast cancer (TNBC). The addition of bevacizumab significantly increased the pCR rate overall (36 vs. 21 %; p = 0.019) and in TNBC (59 vs. 29 %; p = 0.014), but not in hormone receptor-positive disease (24 vs. 18 %; p = 0.41). Sequence of administration of nab-paclitaxel and AC did not affect the pCR rate. While no significant differences in OS or EFS were seen, a trend favored the addition of bevacizumab for EFS (p = 0.06) in TNBC. Overall, Grade 3-4 adverse events did not differ substantially by treatment arm. The addition of bevacizumab to nab-paclitaxel prior to dose-dense AC neoadjuvant chemotherapy significantly improved the pCR rate compared to chemotherapy alone in patients with triple-negative LABC/IBC and was accompanied by a trend for improved EFS. This suggests reconsideration of the role of bevacizumab in high-risk triple-negative locally advanced breast cancer.

Immune modulation of pathologic complete response after neoadjuvant HER2-directed therapies in the NeoSphere trial.

BACKGROUND: To investigate in the NeoSphere trial the contribution of the immune system to pathologic complete response in the breast (pCRB) after neoadjuvant docetaxel with trastuzumab (TH), pertuzumab (TP), or both (THP), or monoclonal antibodies alone (HP). PATIENTS AND METHODS: Immune gene mRNA expression (n = 350, 83.8%), lymphocyte infiltration (TIL, n = 243, 58.3%), and PDL1 by immunohistochemistry (n = 305, 73.1%) were correlated with pCRB. We studied five selected genes (IFNG, PD1, PDL1, PDL2, CTLA4) and six immune metagenes corresponding to plasma cells (IGG), T cells (CD8A), antigen-presenting cells (MHC2), and to MHC1 genes (MHC1), STAT1 co-expressed genes (STAT1), and interferon-inducible genes (IF-I). Gene expression data from the NOAH trial were used for validation. RESULTS: TIL as continuous variable and PDL1 protein expression were not significantly associated with pCRB. Expression of immune genes/metagenes had different association with pCRB after THP than after other therapies. With THP, higher expression of PD1 and STAT1, or any among PDL1, CTLA4, MHC1, and IF-I were linked with lower pCRB. In the combined TH/TP/HP treatment group, in multivariate analysis, higher expression of PD1, MHC2, and STAT1 were linked with pCRB, and higher PDL1, MHC1, or IF-I to lower pCRB. In the NOAH, a similar association of higher STAT1 with higher pCRB, and higher MHC1 and IF-I with lower pCRB was found for trastuzumab/chemotherapy but not for chemotherapy treatment only. CONCLUSIONS: The immune system modulates response to therapies containing trastuzumab and pertuzumab. Greatest benefit from THP is observed for low expression of some immune markers (i.e. MHC1, CTLA4). The involvement of PDL1 in resistance supports testing combinations of HER2-directed antibodies and immune-checkpoint inhibitors.

Metabolic isoenzyme shifts in cancer as potential novel therapeutic targets.

The functional redundancy of metabolic enzyme expression may present a new strategy for developing targeted therapies in cancer. To satisfy the increased metabolic demand required during neoplastic transformations and proliferation, cancer cells may rely on additional isoforms of a metabolic enzyme to satisfy the increased demand for metabolic precursors, which could subsequently render cancer cells more vulnerable to isoform-specific inhibitors. In this review, we provide a survey of common isoenzyme shifts that have been reported to be important in cancer metabolism and link those to metabolic pathways that currently have drugs in various stages of development. This phenomenon suggests a potentially new therapeutic strategy for the treatment of cancer by identifying shifts in the expression of metabolic isoenzymes between cancer and normal cells. We also delineate other putative metabolic isoenzymes that could be targets for novel targeted therapies for cancer. Changes in isoenzyme expression that occur during neoplastic transformations or in response to environmental pressure in cancer cells may result in isoenzyme diversity that may subsequently render cancer cells more vulnerable to isoform-specific inhibitors due to reliance on a single isoform to perform a vital enzymatic function.

Open-label randomized clinical trial of standard neoadjuvant chemotherapy with paclitaxel followed by FEC versus the combination of paclitaxel and everolimus followed by FEC in women with triple receptor-negative breast cancer†.

BACKGROUND: Everolimus synergistically enhances taxane-induced cytotoxicity in breast cancer cells in vitro and in vivo in addition to demonstrating a direct antiproliferative activity. We aim to determine pharmacodynamics changes and response of adding everolimus to standard neoadjuvant chemotherapy in triple-negative breast cancer (TNBC). PATIENTS AND METHODS: Phase II study in patients with primary TNBC randomized to T-FEC (paclitaxel 80 mg/m(2) i.v. weekly for 12 weeks, followed by 5-fluorouracil 500 mg/m(2), epirubicin 100 mg/m(2), and cyclophosphamide 500 mg/m(2) every 3 weeks for four cycles) versus TR-FEC (paclitaxel 80 mg/m(2) i.v. and everolimus 30 mg PO weekly for 12 weeks, followed by FEC). Tumor samples were collected to assess molecular changes in the PI3K/AKT/mTOR pathway, at baseline, 48 h, 12 weeks, and at surgery by reverse phase protein arrays (RPPA). Clinical end points included 12-week clinical response rate (12-week RR), pathological complete response (pCR), and toxicity. RESULTS: Sixty-two patients were registered, and 50 were randomized, 27 received T-FEC, and 23 received TR-FEC. Median age was 48 (range 31-75). There was downregulation of the mTOR pathway at 48 h in the TR-FEC arm. Twelve-week RR by ultrasound were 29.6% versus 47.8%, (P = 0.075), and pCR were 25.9% versus 30.4% (P = 0.76) for T-FEC and TR-FEC, respectively. mTOR downregulation at 48 h did not correlate with 12-week RR in the TR-FEC group (P = 0.58). Main NCI grade 3/4 toxicities included anemia, neutropenia, rash/desquamation, and vomiting in both arms. There was one case of grade 3 pneumonitis in the TR-FEC arm. No grade 3/4 stomatitis occurred. CONCLUSION: The addition of everolimus to paclitaxel was well tolerated. Everolimus downregulated mTOR signaling but downregulation of mTOR at 48 h did not correlate with 12-week RR in the TR-FEC group. CLINICAL TRIAL NUMBER: NCT00499603.

Ink4a/Arf(-/-) and HRAS(G12V) transform mouse mammary cells into triple-negative breast cancer containing tumorigenic CD49f(-) quiescent cells.

Intratumoral heterogeneity within individual breast tumors is a well-known phenomenon that may contribute to drug resistance. This heterogeneity is dependent on several factors, such as types of oncogenic drivers and tumor precursor cells. The purpose of our study was to engineer a mouse mammary tumor model with intratumoral heterogeneity by using defined genetic perturbations. To achieve this, we used mice with knockout (-/-) of Ink4a/Arf, a tumor suppressor locus; these mice are known to be susceptible to non-mammary tumors such as fibrosarcoma. To induce mammary tumors, we retrovirally introduced an oncogene, HRAS(G12V), into Ink4a/Arf(-/-) mammary cells in vitro, and those cells were inoculated into syngeneic mice mammary fat pads. We observed 100% tumorigenesis. The tumors formed were negative for estrogen receptor, progesterone receptor and HER2. Further, they had pathological features similar to those of human triple-negative breast cancer (TNBC) (for example, pushing borders, central necrosis). The tumors were found to be heterogeneous and included two subpopulations: CD49f(-) quiescent cells and CD49f(+)cells. Contrary to our expectation, CD49f(-) quiescent cells had high tumor-initiating potential and CD49f(+)cells had relatively low tumor-initiating potential. Gene expression analysis revealed that CD49f(-) quiescent cells overexpressed epithelial-to-mesenchymal transition-driving genes, reminiscent of tumor-initiating cells and claudin-low breast cancer. Our animal model with intratumoral heterogeneity, derived from defined genetic perturbations, allows us to test novel molecular targeted drugs in a setting that mimics the intratumoral heterogeneity of human TNBC.

Acidic properties of aqueous phosphoric acid solutions: a microscopic view.

We report on new neutron and x-ray diffraction data on D2O:D3PO4 solutions at two concentrations, 1:1 and 3:1. The experimental datasets were modelled simultaneously by the reverse Monte Carlo (RMC) method. From the resulting models, partial radial distribution functions (prdf) and coordination numbers were obtained. The acidity was found to decrease with increasing D3PO4 concentration. The ratio of dissociated acidic protons was estimated by dedicated simulation runs using average coordination number constraints. It was found that in the saturated solution the ratio of dissociated protons cannot exceed 20%.