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HER2-positive metastatic breast cancer is incurable and new treatments are needed. Addition of atezolizumab to trastuzumab emtansine might potentiate anticancer immunity and enhance the HER2-targeted cytotoxic activity of trastuzumab emtansine. We aimed to test this combination in HER2-positive advanced breast cancer that had progressed after previous treatment with trastuzumab and a taxane. The KATE2 study is a randomised, double-blind, placebo-controlled, phase 2 study at 68 centres from nine countries across Asia, Australia, North America, and western Europe. Eligible patients were adults (aged ≥18 years) with an Eastern Cooperative Oncology Group performance status of 0 or 1 and centrally confirmed, measurable, HER2-positive advanced breast cancer previously treated with trastuzumab and a taxane. Patients were randomly assigned (2:1) either trastuzumab emtansine (3·6 mg/kg of bodyweight) plus atezolizumab (1200 mg) or trastuzumab emtansine plus placebo; all study drugs were administered by intravenous infusion every 3 weeks. Randomisation was done via an interactive voice and web response system using a permuted block scheme (block size of six) and was stratified by PD-L1 status, world region, and liver metastases. Patients, investigators, and study team members were masked to treatment allocation. The primary endpoint was investigator-assessed progression-free survival in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT02924883, and the study has been completed. Between Sept 26, 2016, and Aug 7, 2017, 330 patients were screened for the study, of whom 202 were randomly allocated either atezolizumab (n=133) or placebo (n=69). At the recommendation of the independent data monitoring committee, treatment assignment was unmasked on Dec 11, 2017, due to futility and the numerically higher frequency of adverse events among patients assigned atezolizumab. This date was set as the clinical cutoff for the primary analysis. Median follow-up was 8·5 months (IQR 6·1–11·5) for patients assigned atezolizumab and 8·4 months (5·3–11·1) for those assigned placebo. Median progression-free survival was 8·2 months (95% CI 5·8–10·7) for patients assigned atezolizumab versus 6·8 months (4·0–11·1) for those assigned placebo (stratified hazard ratio 0·82, 95% CI 0·55–1·23; p=0·33). The most common grade 3 or worse adverse events were thrombocytopenia (17 [13%] among 132 patients who received atezolizumab vs three [4%] among 68 who received placebo), increased aspartate aminotransferase (11 [8%] vs two [3%]), anaemia (seven [5%] vs 0), neutropenia (six [5%] vs three [4%]), and increased alanine aminotransferase (six [5%] vs two [3%]). Serious adverse events occurred in 43 (33%) of 132 patients who received atezolizumab and 13 (19%) of 68 patients who received placebo. One patient who received atezolizumab died due to a treatment-related adverse event (haemophagocytic syndrome). Addition of atezolizumab to trastuzumab emtansine did not show a clinically meaningful improvement in progression-free survival and was associated with more adverse events. Further study of trastuzumab emtansine plus atezolizumab is warranted in a subpopulation of patients with PD-L1-positive, HER2-positive advanced breast cancer. F Hoffman-La Roche.

Background KRISTINE is an open-label, phase III study of trastuzumab emtansine + pertuzumab (T-DM1 + P) versus docetaxel + carboplatin + trastuzumab + pertuzumab (TCH + P) in patients with HER2-positive, stage II–III breast cancer. We investigated the association of biomarkers with clinical outcomes in KRISTINE. Methods Patients were randomized to receive neoadjuvant T-DM1 + P or TCH + P and assessed for pathologic complete response (pCR; ypT0/is, ypN0). HER2 status (per central assessment), hormone receptor status, PIK3CA mutation status, HER2/HER3 mRNA levels, tumor-infiltrating lymphocyte levels, PD-L1 status, and NanoString data were analyzed. pCR rates by treatment arm were compared across biomarker subgroups. Analyses were descriptive. Results Biomarker analyses included data from all 444 patients (T-DM1 + P, n  = 223; TCH + P, n  = 221) enrolled in KRISTINE. Biomarker distribution was balanced across treatment arms. All subgroups with higher HER2 amplification/expression and immune marker levels showed numerically higher pCR rates in both arms. Mutated versus non-mutated PIK3CA tumors were associated with numerically lower pCR rates in the T-DM1 + P arm but not in the TCH + P arm. In a multivariate analysis, Prediction Analysis of Microarray with the 50-gene classifier (PAM50) HER2-enriched subtype, HER2 gene ratio ≥ 4, and PD-L1-positive status positively influenced the pCR rate. Biomarkers associated with lower pCR rates (e.g., low HER2 levels, positive hormone receptor status, mutated PIK3CA ) were more likely to co-occur. Dynamic on-treatment biomarker changes were observed. Differences in the treatment effects for T-DM1 + P versus TCH + P were similar to those observed in the intent-to-treat population for the majority of the biomarker subgroups. Conclusions Although our biomarker analysis did not identify a subgroup of patients that benefited from neoadjuvant T-DM1 + P versus TCH + P, the data revealed that patients with higher HER2 amplification/expression and immune marker levels had improved response irrespective of treatment arm. These analyses confirm the role of HER2 tumor biology and the immune microenvironment in influencing pCR in the neoadjuvant setting and reaffirm the molecular diversity of HER2-positive breast cancer. Trial Registration : ClinicalTrials.gov NCT02131064. Registered 06 May 2014.

Formalin fixation and paraffin embedding present the standard procedures for conserving clinical tissues for histological analysis. However, molecular analysis is impaired by the cross linking properties of formalin. The PAXgene tissue system (PreAnalytix, Switzerland) is a new formalin-free tissue collection device. Aims In this study we aimed to evaluate this new tissue preservation technique in comparison with formalin fixation and fresh frozen tissue samples. Methods 12 melanoma biopsy samples were divided and fixed simultaneously with formalin, PAXgene or fresh frozen in liquid nitrogen and analysed with regard to morphology, immunohistochemistry,  DNA and RNA content and quality. Markers of melanocytic differentiation and tumour cell proliferation were used. Results Morphology was well preserved in PAXPE samples. However, 5 out of 11 immunohistochemical markers showed significantly lower overall staining and staining intensity with PAXPE tissues in comparison with formalin-fixed, paraffin-embedded (FFPE). Increasing membrane permeability through adding a detergent did proportionally increase staining intensity in PAXPE samples. Amplification of different mRNA amplicons showed a direct relationship with the size of the amplicon with greater template integrity observed in PAXPE samples. Sequencing and mutational analysis of DNA samples were comparable for all the different fixation methods, while the level of DNA fragmentation seemed to be lower in PAXPE compared with FFPE tissues. Conclusions The switch from formalin to PAXgene fixation would require a re-evaluation of immunohistochemical markers and staining procedures originally developed for FFPE tissues. Our data demonstrate that PAXPE fixation offers some advantages concerning molecular analysis. However, these advantages would not justify substituting formalin fixation in any routine pathology laboratory.

Following chemotherapy and human epidermal growth factor 2 (HER2)-targeted neoadjuvant therapy for HER2-positive early breast cancer, residual invasive breast cancer at surgery may be HER2-negative on retesting in some patients. We evaluated outcomes with T-DM1 and trastuzumab in patients randomized in the phase III KATHERINE trial based on HER2-positive central testing of the pre-treatment core biopsy with HER2-negative central testing on their corresponding surgical specimen after neoadjuvant treatment. In the 70/845 (8.3%) patients with HER2-negative residual disease on retesting at surgery, there were 11 IDFS events in the 42 trastuzumab-treated patients (26.2%) and none in the 28 T-DM1-treated patients, suggesting that T-DM1 should not be withheld in this patient population.

In specific cell types like keratinocytes, Notch signaling plays an important pro-differentiation and tumor suppressing function, with down-modulation of the Notch1 gene being associated with cancer development. Besides being controlled by p53, little else is known on regulation of Notch1 gene expression in this context. We report here that transcription of this gene is driven by a TATA-less \"sharp peak\" promoter and that the minimal functional region of this promoter, which extends from the -342 bp position to the initiation codon, is differentially active in normal versus cancer cells. This GC rich region lacks p53 binding sites, but binds Klf4 and Sp3. This finding is likely to be of biological significance, as Klf4 and, to a lesser extent, Sp3 are up-regulated in a number of cancer cells where Notch1 expression is down-modulated, and Klf4 over-expression in normal cells is sufficient to down-modulate Notch1 gene transcription. The combined knock-down of Klf4 and Sp3 was necessary for the reverse effect of increasing Notch1 transcription, consistent with the two factors exerting an overlapping repressor function through their binding to the Notch1 promoter.

Background: Activation of somatostatin receptors (sstr1–5) by somatostatin and its analogues exerts an inhibitory effect on hormone secretion and provides the basis for the treatment of a range of endocrine diseases such as acromegaly, Cushing’s disease and neuroendocrine tumors (NET). The lack of well-characterized commercially available sstr subtype-specific antibodies prevents routine identification of the sstr expression profile in patients. Methods: We generated and characterized new mouse monoclonal antibodies (mAbs) targeting the five human sstr subtypes using ELISA and immunohistochemistry, and tested their suitability in formalin-fixed and paraffin-embedded (FFPE) human tissues and archival samples of normal pancreatic tissue and NET. Results: All mAbs were highly specific with no cross-reactivity. The sstr1–5 immunoreactivity in gastrointestinal NET (n = 67) was correlated with clinicopathologic data. With the exception of sstr3, NET were highly positive for all receptor subtypes (42, 63, 6, 32 and 65% of tumors were positive for sstr1, sstr2a, sstr3, sstr4 and sstr5, respectively). sstr1, sstr2a and sstr5 were present at the plasma membrane and in the cytoplasm of tumor cells, whereas sstr3 and sstr4 were almost exclusively cytoplasmic. Immunoreactivity of sstr1, sstr2a and sstr4 tended to decrease as tumor aggressiveness increased. sstr5 showed an opposite pattern, with higher staining in well-differentiated carcinomas compared with well-differentiated tumors. sstr5 immunoreactivity was correlated with the presence of metastases and angioinvasion, suggesting a possible association with more aggressive behavior. Conclusion: Determination of the sstr1–5 by immunohistochemistry using subtype-specific mAbs is feasible in FFPE tissue and may provide a tool for routine clinical practice.

Among persons with residual invasive disease after treatment of HER2-positive early breast cancer, trastuzumab emtansine resulted in 7-year invasive disease–free survival of 80.8%, as compared with 67.1% with trastuzumab.

The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.

Nat. Cell Biol. 10, 902–911 (2008); published online 6th July 2008 The version of this Article initially published lacked sufficient detail on how samples for western blotting were processed. Many experiments involved analysis of multiple proteins of similar molecular weight, making stripping and re-probing of blots often problematic.