Anthony Kong

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer

IntroductionBreast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice.MethodsMore than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer ‘stem’ cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account.ResultsThe 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working.ConclusionsWith resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

HER2 Phosphorylation Is Maintained by a PKB Negative Feedback Loop in Response to Anti-HER2 Herceptin in Breast Cancer

Herceptin (trastuzumab) is used in patients with breast cancer who have HER2 (ErbB2)-positive tumours. However, its mechanisms of action and how acquired resistance to Herceptin occurs are still poorly understood. It was previously thought that the anti-HER2 monoclonal antibody Herceptin inhibits HER2 signalling, but recent studies have shown that Herceptin does not decrease HER2 phosphorylation. Its failure to abolish HER2 phosphorylation may be a key to why acquired resistance inevitably occurs for all responders if Herceptin is given as monotherapy. To date, no studies have explained why Herceptin does not abolish HER2 phosphorylation. The objective of this study was to investigate why Herceptin did not decrease HER2 phosphorylation despite being an anti-HER2 monoclonal antibody. We also investigated the effects of acute and chronic Herceptin treatment on HER3 and PKB phosphorylation in HER2-positive breast cancer cells. Using both Förster resonance energy transfer (FRET) methodology and conventional Western blot, we have found the molecular mechanisms whereby Herceptin fails to abolish HER2 phosphorylation. HER2 phosphorylation is maintained by ligand-mediated activation of EGFR, HER3, and HER4 receptors, resulting in their dimerisation with HER2. The release of HER ligands was mediated by ADAM17 through a PKB negative feedback loop. The feedback loop was activated because of the inhibition of PKB by Herceptin treatment since up-regulation of HER ligands and ADAM17 also occurred when PKB phosphorylation was inhibited by a PKB inhibitor (Akt inhibitor VIII, Akti-1/2). The combination of Herceptin with ADAM17 inhibitors or the panHER inhibitor JNJ-26483327 was able to abrogate the feedback loop and decrease HER2 phosphorylation. Furthermore, the combination of Herceptin with JNJ-26483327 was synergistic in tumour inhibition in a BT474 xenograft model. We have determined that a PKB negative feedback loop links ADAM17 and HER ligands in maintaining HER2 phosphorylation during Herceptin treatment. The activation of other HER receptors via ADAM17 may mediate acquired resistance to Herceptin in HER2-overexpressing breast cancer. This finding offers treatment opportunities for overcoming resistance in these patients. We propose that Herceptin should be combined with a panHER inhibitor or an ADAM inhibitor to overcome the acquired drug resistance for patients with HER2-positive breast cancer. Our results may also have implications for resistance to other therapies targeting HER receptors.

Adjuvant Radiotherapy for Stage I Endometrial Cancer: An Updated Cochrane Systematic Review and Meta-analysis

BACKGROUND The role of adjuvant radiotherapy in stage I endometrial cancer has changed in recent years. This updated Cochrane systematic review aimed to reexamine the efficacy and toxicity of adjuvant radiotherapy vs no treatment in stage I endometrial cancer. METHODS We searched various databases including The Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, and the Specialised Register of the Cochrane Gynaecological Cancer Review Group (CGCRG) for randomized controlled trials that met the predefined inclusion criteria. The primary outcome was overall survival (OS); secondary outcomes were endometrial cancer-specific survival, locoregional recurrence, distant recurrence, and toxicity. Hazard ratios (HRs) were estimated and pooled if possible; otherwise, dichotomous data were extracted. All statistical tests were two-sided. RESULTS Of the eight included trials, seven trials (3628 women) compared external beam radiotherapy (EBRT) and no EBRT (or vaginal brachytherapy [VBT]), and one trial (645 women) compared VBT and no additional treatment. EBRT statistically significantly reduced locoregional recurrence compared with no EBRT (or VBT alone) (HR = 0.36, 95% confidence Interval [CI] = 0.25 to 0.52; P < .001), but this did not translate into an improvement in OS (HR = 0.99, 95% CI = 0.82 to 1.20; P = .95), endometrial cancer-specific survival (HR = 0.96, 95% CI = 0.72 to 1.28; P = .80), or distant recurrence rates (risk ratio = 1.04, 95% CI = 0.80 to 1.35; P = .77). EBRT was associated with an increased risk of severe acute toxicity, severe late toxicity, and reduced quality of life scores. CONCLUSIONS EBRT reduces the risk of locoregional recurrence but has no statistically significant impact on cancer-related deaths or OS. However, EBRT is associated with clinically and statistically significant morbidity and a reduction in quality of life.

Testosterone therapy in HIV wasting syndrome: systematic review and meta-analysis

Many HIV patients develop weight loss, which increases morbidity and mortality. We aimed to assess the effects of testosterone therapy on lean body mass, total body weight, over-all exercise functional capacity, and perceived quality of life in patients with HIV wasting syndrome and its adverse effects. We systematically reviewed randomised, placebo-controlled trials that compared the effects of testosterone therapy with placebo in HIV patients with wasting. Eight trials met the inclusion criteria and 417 randomised patients were included. Only six trials used lean-body mass, fat-free mass, or body-cell mass as outcome measures. The meta-analysis of the six trials showed a difference in the lean body mass between the testosterone group and placebo group of 1.22 kg (95% CI 0.23-2.22) for the random effect model and 0.51 kg (0.09-0.93) for fixed effect. However, the difference was much greater in the three trials that used the intramuscular route-3.34 kg in the post-hoc analysis. All eight trials included total body weight as an outcome measure, the meta-analysis of which showed a difference of 1.04 kg (-0.01-2.10) between testosterone group and placebo group by random effect and 0.63 kg (-0.01-1.28) for fixed effect models. Over-all, the incidence of adverse effects is similar in both groups. Testosterone therapy has been shown in this review to increase lean body mass more than placebo. The increase is even greater if the therapy is given intramuscularly. There is also a small positive effect in total body weight. The study is, however, limited by the small numbers and heterogeneity of the population, which potentially introduced bias into the methods and results. Testosterone therapy may be considered in patients with HIV wasting syndrome to reverse muscle loss, but there is a concern about the adverse metabolic effects of long-term testosterone administration and long-term follow-up for these patients is needed.

Prognostic Value of an Activation State Marker for Epidermal Growth Factor Receptor in Tissue Microarrays of Head and Neck Cancer

Overexpression and mutation of epidermal growth factor receptors (EGFR) have been shown to be important in the prognosis of several cancers, including head and neck cancers. However, our inability to define the activation status of these and other receptors limits our ability to assess the importance of these pathways and to exploit effectively new molecularly targeted treatments directed at their catalytic activities. Here we describe the use of automated, high-throughput fluorescence lifetime imaging microscopy to measure EGFR autophosphorylation status by fluorescence resonance energy transfer (FRET) in head and neck tumors. We have correlated FRET efficiency with the clinical and survival data. The results from head and neck arrays show that high FRET efficiency is correlated with worsening disease-free survival but not with overall survival. This powerful tool could be exploited as a new independent quantitative prognostic factor in clinical decisions and cancer management.

Temozolomide as second-line chemotherapy for relapsed gliomas.

AbstractBackground: Temozolomide, an imidazotetrazine prodrug has shown activity in phase II studies in patients with high-grade glioma at first recurrence. We assessed the efficacy of Temozolomide as second-line therapy following failure of PCV chemotherapy in patients with recurrent/progressive gliomas. Patients and methods: Between September 1994 and November 2000, 32 patients with high-grade gliomas at second recurrence/progression received Temozolomide as salvage therapy and results were received retrospectively. Results: Of 32 assessable patients 7 had clinical improvement; there were no imaging responses. Median survival of the cohort was 4 months, with 28% alive at 6 months. Age, performance status, histology and previous response to PCV chemotherapy did not predict for clinical response to Temozolomide. Conclusion: In the small cohort of patients with recurrent malignant glioma who failed PCV chemotherapy Temozolomide demonstrated limited activity as second-line treatment although this remains within the confidence intervals of response seen in patients with glioblastoma.

Potential of PET to Predict the Response to Trastuzumab Treatment in an ErbB2-Positive Human Xenograft Tumor Model

Currently, an alteration in the gross volume of a tumor is used to assess its response to trastuzumab; however, this approach provides only a late indication of response. Tissue-sample ex vivo assays are potentially valuable, but their procurement through biopsies is invasive and might be biased by tumor heterogeneity. We studied the feasibility of using PET to quantify changes in ErbB2 (HER2/neu) expression and to predict the response to trastuzumab in BT474 breast cancer xenografts with N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM)-HER2:342 Affibody. Methods: Mice bearing BT474 tumors were given trastuzumab (50 mg/kg loading dose, 25 mg/kg maintenance dose, administered intraperitoneally twice a week) or saline (control) for a total of 5 doses. Tumor size was monitored twice a week. Animals were scanned before the treatment, at 48 h, and 2 wk after the beginning of therapy. After the final scan, PET results were correlated with tumor response and immunohistochemical assessment of ErbB2 level, as well as with vasculature in the treated tumors. Results: Analysis of PET images indicated that tracer uptake was significantly reduced after 1 dose of trastuzumab, compared with baseline, suggesting applicability as an early indicator of changes in ErbB2 expression. After 5 doses of trastuzumab, the overall decrease in 18F-FBEM-HER2:342 Affibody uptake also correlated with tumor response and downregulation of ErbB2 expression by immunohistochemical assessment. However, individual animals had different responses. There was a correlation between bigger PET changes and a higher vessel count in the tumors, suggesting that an increased number of vessels could lead to better trastuzumab delivery. We confirmed that the difference in average vessel count in the tumors was not related to the size of the tumors and therefore was not due to the selection of more vascular tumors. This finding is consistent with previous findings demonstrating that the number of vessels in a tumor could be a useful prognostic marker for treatment response. Conclusion: Our data suggest that Affibody-based PET can noninvasively provide specific information on changes in receptor expression and could be a valuable strategy for predicting tumor response to trastuzumab.

Profile of neratinib and its potential in the treatment of breast cancer

The HER (ErbB) receptor tyrosine kinase receptors are implicated in many cancers and several anti-HER treatments are now approved. In recent years, a new group of compounds that bind irreversibly to the adenosine triphosphate binding pocket of HER receptors have been developed. One of these compounds, neratinib, has passed preclinical phases and is currently undergoing various clinical trials. This manuscript reviews the preclinical as well as clinical data on neratinib. As a pan-HER inhibitor, this irreversible tyrosine kinase inhibitor binds and inhibits the tyrosine kinase activity of epidermal growth factor receptors, EGFR (or HER1), HER2 and HER4, which leads to reduced phosphorylation and activation of downstream signaling pathways. Neratinib has been shown to be effective against HER2-overexpressing or mutant tumors in vitro and in vivo. Neratinib is currently being investigated in various clinical trials in breast cancers and other solid tumors, including those with HER2 mutation. Earlier studies have already shown promising clinical activity for neratinib. However, more translational research is required to investigate biomarkers that could help to predict response and resistance for selection of appropriate patients for treatment with neratinib, either as monotherapy or in combination with other drug(s).

Upregulation of ADAM proteases and HER ligands through a feedback loop mediates acquired resistance to trastuzumab in HER2-amplified breast cancer

The response rarely sustains long among the responders for Herceptin (trastuzumab) monotherapy treatment. It is still poorly understood how Herceptin exerts its mechanism of action and how the acquired resistance to this drug occurs.

Reply: PET Prediction of Response to Trastuzumab in ErbB2-Positive Human Xenograft Model

REPLY: The work that we have recently described tested the feasibility of the use of Affibody molecules (Affibody AB) and PET to predict tumor response to ErbB2-targeted therapy. It is clear that additional studies will be needed to dissect the mechanistic events underlying the observed changes. Differences among tumor cell lines could affect responses as well. Clinical studies show that the assessment of ErbB2 level by immunohistochemistry produces variable results among laboratories. This variation may be due to differences in immunohistochemistry staining techniques and scoring criteria (1). For antigen-retrieval processes, the solution used (e.g., citrate buffer or ethylenediaminetetraacetic acid and their pH), the duration of heating, and antigen retrieval may all affect detection of the ErbB2 antigen by immunohistochemistry (2). Different anti-ErbB2 antibodies used for immunohistochemistry staining have also been shown to produce different degrees of ErbB2 staining in tumors, even in the presence of gene amplification (3), although applying calibration may help in minimizing those differences (4). The HercepTest (Dako) using Dako antibody was proposed as the standardized immunohistochemistry method to overcome the problem of interlaboratory variations. The scoring system uses the intensity of ErbB2 staining as its basis, and an ErbB2-positive tumor is defined as a tumor with greater than 10% of cells stained 31 (5). Despite use of the HercepTest, there still was a high discrepancy between local and central ErbB2 testing in the N9831 Intergroup Adjuvant Trial, with a concordance of only 81.6% for a diagnostic test for the presence of the ErbB2 protein (6). The American Society of Clinical Oncology and College of American Pathologists recommended an algorithm defining positive, equivocal, and negative values for both ErbB2 protein expression and gene amplification. A positive ErbB2 result from immunohistochemistry staining is defined by uniform, intense membrane staining of more than 30% of invasive tumor cells instead of the original 10% (5). However, despite this new algorithm and definition, not all laboratories have adopted this new guideline and there still are variable results in ErbB2 testing among laboratories. Furthermore, both the old and the new “ErbB2 counting” definitions have problems in detecting subtle ErbB2 changes induced by the treatment. For example, if trastuzumab decreases ErbB2 staining from 100% of the cells to 40%, both the old and the new ErbB2 definition will score preand post-treatment samples as “positive” and may fail to detect ErbB2 changes after trastuzumab treatment. After trastuzumab treatment, we found in human breast carcinoma BT474 xenografts a significant reduction of tracer uptake related to ErbB2 decrease rather than tumor size reduction (7). The observable reduction in PET signals could be due to partial-volume effect, but this possibility is rather unlikely since the images were acquired with high contrast and in the absence of background activity. When large enough regions are drawn around the tumor, the partial-volume effect does not cause any loss of signal, and the signal that is measured indicates the actual activity distribution. Moreover, for PET quantification, we deliberately chose the value related to maximum counts per pixel within the tumor that is least affected by partial-volume effect. Importantly, we have also shown that tumor ErbB2 membrane staining and PET changes correlated with tumor volume after 5 doses of trastuzumab treatment (7). There was a correlation between PET and immunohistochemistry, and the radionuclide concentrations measured with PET agreed with the radioactivity concentrations obtained by g-counting (data were not presented). Although there was a large overlap in ErbB2 staining between the trastuzumab-treated and control groups, we found a significant reduction of ErbB2 downregulation after 5 doses of trastuzumab. This finding is consistent with several cell line experiments from different groups finding that trastuzumab downregulates ErbB2 receptors (8–10). After a single dose of trastuzumab, we could clearly see the differences in ErbB2 membrane staining between control and trastuzumab-treated samples, but the intensity percentage scoring failed to detect these changes (7). The dose and duration of trastuzumab will clearly affect the amount of ErbB2 downregulation and the detection of ErbB2 changes by immunohistochemistry. In our paper, the dose of trastuzumab was deliberately high (50 mg/kg loading dose followed by 4 more doses of 25 mg/kg each) to ensure that changes in receptor expression ErbB2 would be possible (7). Reddy et al. (11) treated BT474 xenografts with a lower dose of 10 mg/kg for only 6 d. They found a decrease in PET tracer using C6.5 diabody but could not detect any ErbB2 changes by immunohistochemistry. They concluded that “The exact mechanism by which trastuzumab treatment inhibits C6.5db binding is not yet understood.” On the other hand, McLarty et al. (12) reported that trastuzumab reduced ErbB2 membrane staining in SKBR3 cells and in MDAMB361 and MDAMB361 xenograft models. In this case, mice were treated only with 4 mg of trastuzumab per kilogram for 3 d or 3 wk. At 3 d, the authors did not see ErbB2 membrane changes, but 3 wk later immunohistochemistry analysis of tumor tissues indicated significant ErbB2 downregulation, associated with almost complete eradication of viable tumor cells. This finding is consistent with our study as we did not see a difference in intensity percentage scoring after a single dose of trastuzumab (7); we observed differences in ErbB2 membrane staining after 5 doses of the drug (7). We believe that the differences seen in ErbB2 staining between Reddy et al. (11), McLarty et al. (12), and our study (7) may be related to the dose and duration of trastuzumab used. However, the differences may also be related to the ErbB2 testing methods and the scoring criteria, which could not detect subtle ErbB2 changes after trastuzumab treatment. Trastuzumab was used as monotherapy before surgery in patients with primary operable ErbB2-positive breast tumors in a pilot study by Gennari et al. (13). They observed no change in ErbB2-positive staining using monoclonal antibody CB11 in the trastuzumab-treated samples. However, they provided figures from only 1 patient, as shown in their Figures 2A and 2B (13). Although these figures suggest some changes between preand posttreatment samples, the authors found no variations in the ErbB2 status (13). Furthermore, the use of a different anti-ErbB2 antibody and different scoring criteria may also have contributed