Neratinib and Ado-Trastuzumab-Emtansine for Pre-treated and Untreated HER2-positive Breast Cancer Brain Metastases: Translational Breast Cancer Research Consortium Trial 022.

PURPOSE: Treatment options for HER2-positive breast cancer brain metastases (BCBM) remain limited. We previously reported central nervous system (CNS) activity for neratinib and neratinib-capecitabine. Preclinical data suggest that neratinib may overcome resistance to ado-trastuzumab-emtansine (T-DM1) when given in combination. In TBCRC 022's cohort 4, we examined the efficacy of neratinib plus T-DM1 in patients with HER2-positive BCBM. PATIENTS AND METHODS: In this multicenter, phase II study, patients with measurable HER2-positive BCBM received neratinib 160 mg daily plus T-DM1 3.6 mg/kg intravenously every 21 days in three parallel-enrolling cohorts (cohort 4A-previously untreated BCBM, cohorts 4B and 4C- BCBM progressing after local CNS-directed therapy without [4B] and with [4C] prior exposure to T-DM1). Cycle 1 diarrheal prophylaxis was required. The primary endpoint was the Response Assessment in Neuro-Oncology-Brain Metastases (RANO-BM) by cohort. Overall survival (OS) and toxicity were also assessed. RESULTS: Between 2018-2021, 6, 17, and 21 patients enrolled to cohorts 4A, 4B, and 4C. Enrollment was stopped prematurely for slow accrual. The CNS objective response rate in cohorts 4A, 4B, and 4C was 33.3% (95% confidence interval [CI]: 4.3-77.7%), 35.3% (95% CI: 14.2-61.7%), and 28.6% (95% CI: 11.3-52.2%), respectively; 38.1-50% experienced stable disease for ≥6 months or response. Diarrhea was the most common grade 3 toxicity (22.7%). Median OS was 30.2 months (cohort 4A; 95% CI: 21.9, not reached [NR]), 23.3 months (cohort 4B; 95% CI: 17.6, NR), and 20.9 months (cohort 4C; 95% CI: 14.9, NR). CONCLUSION: We observed Intracranial activity for neratinib plus T-DM1, including those with prior T-DM1 exposure, suggesting synergistic effects with neratinib. Our data provide additional evidence for neratinib-based combinations in patients with HER2-positive BCBM, even those who are heavily pre-treated.

Response-guided neoadjuvant sacituzumab govitecan for localized triple-negative breast cancer: results from the NeoSTAR trial.

BACKGROUND: Sacituzumab govitecan (SG), a novel antibody-drug conjugate (ADC) targeting TROP2, is approved for pre-treated metastatic triple-negative breast cancer (mTNBC). We conducted an investigator-initiated clinical trial evaluating neoadjuvant (NA) SG (NCT04230109), and report primary results. PATIENTS AND METHODS: Participants with early-stage TNBC received NA SG for four cycles. The primary objective was to assess pathological complete response (pCR) rate in breast and lymph nodes (ypT0/isN0) to SG. Secondary objectives included overall response rate (ORR), safety, event-free survival (EFS), and predictive biomarkers. A response-guided approach was utilized, and subsequent systemic therapy decisions were at the discretion of the treating physician. RESULTS: From July 2020 to August 2021, 50 participants were enrolled (median age = 48.5 years; 13 clinical stage I disease, 26 stage II, 11 stage III). Forty-nine (98%) completed four cycles of SG. Overall, the pCR rate with SG alone was 30% [n = 15, 95% confidence interval (CI) 18% to 45%]. The ORR per RECIST V1.1 after SG alone was 64% (n = 32/50, 95% CI 77% to 98%). Higher Ki-67 and tumor-infiltrating lymphocytes (TILs) were predictive of pCR to SG (P = 0.007 for Ki-67 and 0.002 for TILs), while baseline TROP2 expression was not (P = 0.440). Common adverse events were nausea (82%), fatigue (76%), alopecia (76%), neutropenia (44%), and rash (48%). With a median follow-up time of 18.9 months (95% CI 16.3-21.9 months), the 2-year EFS for all participants was 95%. Among participants with a pCR with SG (n = 15), the 2-year EFS was 100%. CONCLUSIONS: In the first NA trial with an ADC in localized TNBC, SG demonstrated single-agent efficacy and feasibility of response-guided escalation/de-escalation. Further research on optimal duration of SG as well as NA combination strategies, including immunotherapy, are needed.

The immune profile of small HER2-positive breast cancers: a secondary analysis from the APT trial.

BACKGROUND: Previous data suggest that the immune microenvironment plays a critical role in human epidermal growth factor receptor 2 (HER2) -positive breast cancer; however, there is little known about the immune profiles of small HER2-positive tumors. In this study, we aimed to characterize the immune microenvironment of small HER2-positive breast cancers included in the Adjuvant paclitaxel and trastuzumab for node-negative, HER2-positive breast cancer (APT) trial and to correlate the immune markers with pathological and molecular tumor characteristics. PATIENTS AND METHODS: The APT trial was a multicenter, single-arm, phase II study of paclitaxel and trastuzumab in patients with node-negative HER2-positive breast cancer. The study included 406 patients with HER2-positive, node-negative breast cancer, measuring up to 3 cm. Exploratory analysis of tumor infiltrating lymphocytes (TIL), programmed death-ligand 1 (PD-L1) expression (by immunohistochemistry), and immune gene signatures using data generated by nCounter PanCancer Pathways Panel (NanoString Technologies, Seattle, WA), and their association with pathological and molecular characteristics was carried out. RESULTS: Of the 406 patients, 328 (81%) had at least one immune assay carried out: 284 cases were evaluated for TIL, 266 for PD-L1, and 213 for immune gene signatures. High TIL (≥60%) were seen with greater frequency in hormone-receptor (HR) negative, histological grades 2 and 3, as well in HER2-enriched and basal-like tumors. Lower stromal PD-L1 (≤1%) expression was seen with greater frequency in HR-positive, histological grade 1, and in luminal tumors. Both TIL and stromal PD-L1 were positively correlated with 10 immune cell signatures, including Th1 and B cell signatures. Luminal B tumors were negatively correlated with those signatures. Significant correlation was seen among these immune markers; however, the magnitude of correlation did not indicate a monotonic relationship between them. CONCLUSION: Immune profiles of small HER2-positive breast cancers differ according to HR status, histological grade, and molecular subtype. Further work is needed to explore the implication of these findings on disease outcome. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov identifier: NCT00542451.

Effects of neratinib on health-related quality of life in women with HER2-positive early-stage breast cancer: longitudinal analyses from the randomized phase III ExteNET trial.

BACKGROUND: We report longitudinal health-related quality-of-life (HRQoL) data from the international, randomized, double-blind, placebo-controlled phase III ExteNET study, which demonstrated an invasive disease-free survival benefit of extended adjuvant therapy with neratinib over placebo in human epidermal growth factor receptor-2-positive early-stage breast cancer. PATIENTS AND METHODS: Women (N = 2840) with early-stage HER2-positive breast cancer who had completed trastuzumab-based adjuvant therapy were randomly assigned to neratinib 240 mg/day or placebo for 12 months. HRQoL was an exploratory end point. Patients completed the Functional Assessment of Cancer Therapy-Breast (FACT-B) and EuroQol 5-Dimensions (EQ-5D) questionnaires at baseline and months 1, 3, 6, 9, and 12. Changes from baseline were compared using analysis of covariance with no imputation for missing values. Sensitivity analyses used alternative methods. Changes in HRQoL scores were regarded as clinically meaningful if they exceeded previously reported important differences (IDs). RESULTS: Of the 2840 patients (intention-to-treat population), 2407 patients were evaluable for FACT-B (neratinib, N = 1171; placebo, N = 1236) and 2427 patients for EQ-5D (neratinib, N = 1186; placebo, N = 1241). Questionnaire completion rates exceeded 85%. Neratinib was associated with a decrease in global HRQoL scores at month 1 compared with placebo (adjusted mean differences: FACT-B total, -2.9 points; EQ-5D index, -0.02), after which between-group differences diminished at later time-points. Except for the FACT-B physical well-being (PWB) subscale at month 1; all between-group differences were less than reported IDs. The FACT-B breast cancer-specific subscale showed small improvements with neratinib at months 3-9, but all were less than IDs. Sensitivity analyses exploring missing data did not change the results. CONCLUSIONS: Extended adjuvant neratinib was associated with a transient, reversible decrease in HRQoL during the first month of treatment, possibly linked to treatment-related diarrhea. With the exception of the PWB subscale at month 1, all neratinib-related HRQoL changes did not reach clinically meaningful thresholds. ClinicalTrials.gov: NCT00878709.

Real world patterns of care in HER2-overexpressing breast cancer: Results of a survey of TEACH clinical trial investigators in 2011.

BACKGROUND: HER2-overexpressing breast cancer (BC) is common among young patients and poses a public health burden. Adjuvant anti-HER2/neu therapy with trastuzumab reduces the risk of recurrence and improves survival. METHODS: A web-based survey was sent to 386 physicians of the "TEACH" trial in 2011 to determine access to HER2/neu testing and treatment patterns for HER2-overexpressing BC. RESULTS: There were 151 responders (39%) from 28 countries. Ninety-seven percent reported HER2/neu expression is routinely measured in their institutions by immunohistochemistry (85%), FISH (80%) and other methods (16%). Twenty percent of responders from Asia reported that the test was not routinely available. Forty-eight percent of participants reported instances when adjuvant HER2-directed therapy was recommended to a patient who eventually did not receive it. Reasons for not receiving trastuzumab was cost (73%, p < 0.0001) in low- and middle-income countries and co-morbidities in high-income countries (43%, p = 0.003). CONCLUSIONS: This survey reflects the availability of HER2/neu testing and anti-HER2/neu therapy among physicians who participated in TEACH. A high proportion of women with HER2-overexpressing BC may not receive standard adjuvant therapy due to unavailability of the test and cost of therapy. Despite having some limitations, such as a possible selection bias of participating physicians, variable definitions of access to healthcare among respondents, and changes in trastuzumab availability since 2011, our results demonstrate that access to care and region of practice impact the implementation of cancer treatments.

Clinical outcomes of ethnic minority women in MA.17: a trial of letrozole after 5 years of tamoxifen in postmenopausal women with early stage breast cancer.

BACKGROUND: Aromatase inhibitors are widely employed in the adjuvant treatment of early stage breast cancer. The impact of aromatase inhibitors has not been established in ethnic minority women. PATIENTS AND METHODS: The purpose of this study was to evaluate the impact of letrozole on minority women in MA.17, a placebo-controlled trial of letrozole following 5 years of tamoxifen in postmenopausal women with early stage breast cancer. Retrospective comparison of disease-free survival (DFS), side effects, and mean changes in quality of life (QOL) scores from baseline between Caucasian and minority women was performed. RESULTS: Minority (n = 352) and Caucasian (n = 4708) women were analyzed. There was no difference between these groups in DFS (91.6% versus 92.4% respectively for 4 year DFS). Letrozole, compared with placebo, significantly improved DFS for Caucasians (HR = 0.55; P < 0.0001) but not for minorities (HR = 1.39; P = 0.53). Among women who received letrozole, minorities had a significantly lower incidence of hot flashes (49% versus 58%; P = 0.02), fatigue (29% versus 39%; P = 0.005), and arthritis (2% versus 7%; P = 0.006) compared with Caucasians. Mean change in QOL scores for minority women who received letrozole demonstrated improved mental health at the 6-month assessment (P = 0.02) and less bodily pain at the 12-month assessment (P = 0.046). CONCLUSION: Letrozole improved DFS in Caucasians but a definite benefit in minority women has not yet been demonstrated. Minority women tolerated letrozole better than Caucasians in terms of toxicity. These results need confirmation in other trials of aromatase inhibitors.

P388 leukaemia cells resistant to the anthracycline menogaril lack multidrug resistant phenotype.

Menogaril is an anthracycline presently in Phase II clinical trials. Menogaril-resistant mouse leukaemia P388 cells were developed in vitro by 4 months of exposure to step-wise increasing concentrations of menogaril after which resistant cells (P388/MEN) were cloned in 320 ng ml-1 menogaril. P388/MEN cells were 40-fold more resistant to menogaril in vitro compared to P388/O and were also resistant in vivo. Resistance to menogaril was stable for at least 2 months in the absence of the drug. The results indicate that P388/MEN, although resistant to an anthracycline, did not display the typical multidrug resistant phenotype. It was not cross-resistant to several structurally unrelated drugs such as actinomycin D, cisplatin, or vinblastine, but it was cross-resistant to the anthracycline, adriamycin. Uptake and efflux of menogaril was similar in sensitive and resistant cell lines. Also, resistance was not reversed by verapamil. No major karyotypic difference was noted between P388/O and P388/MEN. There was no significant amplification or overexpression of the mdr gene in P388/MEN compared to P388/O. In contrast to P388/MEN, P388 cells resistant to adriamycin displayed the typical multidrug resistant phenotype. Glutathione content of P388/MEN cells was similar to that of P388/O and depletion of glutathione did not potentiate menogaril cytotoxicity. Therefore, we conclude that glutathione is not likely to be involved in menogaril resistance to P388/MEN cells.

Characterization of B16 melanoma cells resistant to the CC-1065 analogue U-71,184.

U-71,184 is a CC-1065 analogue which is highly cytotoxic in vitro and has a broad spectrum of antitumor activity in vivo. Against B16 cells, U-71,184 was 8-fold and 253-fold more potent than Actinomycin D and Adriamycin, respectively. U-71,184 killed 90% of B16 cells at 0.01 ng/ml levels of drug in the medium, which was equivalent to an intracellular concentration of about 8 pg/10(6) cell (= 2 x 10(-8) pmol/cell). A B16 cell line resistant to U-71,184 developed after 3 months of in vitro exposure to gradually increasing concentrations of the drug. The sensitive and resistant cell lines were cloned and a B16/R clone was selected which was 60 to 100 times more resistant to U-71,184 than the cloned sensitive parent (B16/S). Cells grown in the absence of U-71,184 for 2 months retained resistance to the drug. B16/R was slightly cross-resistant only to Adriamycin but not to Actinomycin D, vinblastine, or colchicine. Among alkylating agents, it was slightly cross-resistant to Melphalan but not to 1,3-bis(2-chloroethyl)-1-nitrosourea or cisplatin. B16/R did not overexpress mdr mRNA. Therefore, this cell line does not exhibit the multidrug-resistant phenotype. Most karyotypes of B16/R had a marker chromosome which carried an aberrantly staining region apparently containing repetitive replication of the same segment. Resistance can be partly accounted for by the approximately 10-fold lesser uptake of [3H]-U-71,184 in B16/R, as compared to B16/S. B16/R was cross-resistant in varying degrees to several other CC-1065 analogues. The ratio of the 50% lethal dose of U-71,184 for B16/R, as compared to B16/S, was about 60 (i.e., R/S = 60). In comparison, the following compounds had an R/S ratio of less than 20 (i.e., modest level of cross-resistance to U-71,184): U-68,819, U-73,975, U-75,500, U-75,559, and CC-1065. In contrast, the following compounds had an R/S ratio greater than 20 (i.e., highly cross-resistant to U-71,184): U-71,184 analogues U-71,185, U-73,903, and U-75,012; U-73,975 analogues U-75,613, U-75,032, and U-73,896; and CC-1065 enantiomer U-76,915. We cannot yet explain the difference in the level of cross-resistance between these compounds in vitro. B16/S and B16/R cells were tumorigenic in mice and B16/R was resistant to U-71,184 in vivo. There was no clear indication of cross-resistance of B16/R in vivo to Adriamycin, Actinomycin D, cisplatin, or Melphalan. However, U-73,975, a compound with modest cross-resistance in vitro, was significantly cross-resistant in vivo.

Interaction of CC-1065 and its analogues with mouse DNA and chromatin.

CC-1065 is a potent antitumor antibiotic which is cytotoxic to P388 and L1210 leukemia cells in vitro and in vivo. CC-1065 covalently binds to calf thymus DNA preferentially to adenine-thymine regions at N3 of adenine. Here, we compare CC-1065 interaction with P388-derived chromatin, DNA, and histones as measured by electronic absorption and circular dichroism. Two CC-1065 analogues (U-71,184 and its enantiomer, U-71,185) which show different biological activities from CC-1065 were also studied. The shape and temporal behavior of the induced circular dichroism curves generated by CC-1065 or its analogues bound to chromatin were similar to CC-1065 plus DNA. This suggested that CC-1065 and its analogues bind to the minor groove of chromatin DNA in a manner similar to calf thymus DNA. However, the binding of CC-1065 and its analogues to DNA induced a more intense circular dichroism band than binding to chromatin. The order of interaction for both chromatin and DNA was CC-1065 greater than U-71,184 greater than U-71,185. In contrast to the essentially irreversible binding to DNA after 24-h incubation, binding to chromatin was primarily a reversible interaction, the degree of reversibility being U-71,185 greater than U-71,184 = CC-1065. CC-1065 binds weakly and nonspecifically to histones.

Differences in the nuclear matrix phosphoproteins of a wild-type and nitrogen mustard-resistant rat breast carcinoma cell line.

Using polyclonal antibodies raised against a rat liver nuclear envelope protein, lamin protein A, the nuclear matrix proteins of a Walker 256 rat mammary carcinoma wild-type (WS) and a selected cell line with acquired resistance to nitrogen mustards (WR) were found to possess antigenic determinants which were recognized by the antibodies. In one-dimensional immunoblotting analysis, the nuclear matrix protein fractions of both cell lines revealed a common band at Mr 75,000; however only the WS nuclear matrix protein fraction contained a broad band at approximately Mr 70,000. Two-dimensional gel blotting studies of these proteins showed that this Mr 70,000 WS protein had a pI of approximately 7.5. Immunoprecipitation analysis revealed that the altered mobility of this protein could be a function of phosphorylation. The nuclear matrix proteins from both WS and WR cells were shown to bind 3':5'-cyclic adenylic acid (cAMP), as judged by photoaffinity labeling and gel electrophoresis studies. The WS nuclear matrix proteins showed a quantitatively greater level of cAMP binding compared to WR, with predominant binding to proteins with molecular weights of 45,000, 55,000, and 70,000. In WR cells, there was no cAMP binding in the Mr 70,000 region. These data indicate that the Mr 70,000 nuclear matrix lamin proteins are antigenically similar in WS and WR but differ in that the WR protein is hypophosphorylated and does not bind cAMP.

Poly(adenosine diphosphate-ribosylation) of nuclear matrix proteins in alkylating agent resistant and sensitive cell lines.

Using Walker 256 breast carcinoma cell lines either with or without acquired resistance to alkylating agents, the structural framework proteins of the nucleus, the nuclear matrix proteins, were found to be effective acceptors for poly(ADP-ribose). Incubation of isolated nuclei with nicotinamide adenine [32P] dinucleotide ([32P] NAD), followed by the isolation of the nuclear matrix, demonstrated that two polypeptides of approximate molecular weight (Mr) 105 000 and 116 000 were extensively poly(ADP-ribosylated). By an in vitro [32P] NAD assay, the nuclear matrix fraction was found to maintain approx. 15% of the total nuclear matrix activity of poly(ADP-ribose) polymerase. Confirmation that the trichloroacetic acid (TCA) precipitable material represented ADP-ribose units was achieved by enzymatic digestion of the nuclear matrix preparation with snake venom phosphodiesterase (SVP). Within 15 min, greater than 85% of the 32P label was digested by SVP and the final digestion products were found to be phosphoribosyl-AMP (PR-AMP) and adenosine 5'-monophosphate (5'-AMP) by thin layer chromatographic analysis. The average polymer chain length was estimated to be 6-7 ADP-ribose units. Because poly(ADP-ribose) polymerase has a putative role in DNA repair, a comparison of the nuclear matrix fractions from Walker resistant and sensitive tumor cell lines was made. In both cell lines, the quantitative and qualitative patterns of the nuclear matrix associated poly(ADP-ribosylation) were similar.

Enhanced nitrosourea cytotoxicity in cell culture by sodium butyrate.

In HeLa S3 cells, sodium butyrate was found to potentiate the cytotoxicity of chloroethylnitrosoureas and alkylating agents in vitro. Using a soft-agar colony-forming assay, 2.5 and 5.0 mM sodium butyrate pretreatment for 22 h increased the cell killing efficacy of both methyl- and chloroethylnitrosoureas by between 30 and 70%. The potentiation of cytotoxicity of bifunctional nitrogen mustards by butyrate was less than that of nitrosoureas, with a 15-30% increased cell kill at 5 mM butyrate. Sodium butyrate per se reduced plating efficiency and caused growth delay if residual levels (calculated at 100 microM for starting concentrations of 5 mM) were not removed by washing prior to plating.

Acquired drug resistance is accompanied by modification in the karyotype and nuclear matrix of a rat carcinoma cell line.

A Walker 256 breast carcinoma cell line (WR) exhibiting a greater than 20-fold resistance to alkylating agents has been selected from a parent cell line (WS). Karyotypic heterogeneity was apparent, with a number of differences evident between WR and WS cells. The modal chromosome number for WS is 62; for WR, 54; double minutes were found only in WR, whereas spontaneous chromosomal aberrations were present in approx. 40% of the WS cells. No similar aberrations were observed in WR. Using SDS-gel electrophoresis and subsequent silver staining, differences in the profile of nuclear matrix proteins in WR and WS were observed. A diffuse band at approx. 70 kD in the WS was absent in WR cells. This protein was phosphorylated, together with a number of the other major matrix polypeptides. Levels of phosphorylated matrix proteins were approximately equivalent in both WR and WS cell lines, but matrix protein phosphorylation levels were approx. 2-fold higher than corresponding values for bulk nuclear proteins. Selective pressure of drug exposure has resulted in enhanced genetic stability in WR cells and observed karyotype differences are accompanied by modifications in the structural proteins of the nuclear matrix. Whether the observed differences are the cause or result of drug resistance remains to be established.