Present and future breast cancer management--bench to bedside and back: a positioning paper of academia, regulatory authorities and pharmaceutical industry.

Insights into tumour biology of breast cancer have led the path towards the introduction of targeted treatment approaches; still, breast cancer-related mortality remains relatively high. Efforts in the field of basic research revealed new druggable targets which now await validation within the context of clinical trials. Therefore, questions concerning the optimal design of future studies are becoming even more pertinent. Aspects such as the ideal end point, availability of predictive markers to identify the optimal cohort for drug testing, or potential mechanisms of resistance need to be resolved. An expert panel representing the academic community, the pharmaceutical industry, as well as European Regulatory Authorities met in Vienna, Austria, in November 2012, in order to discuss breast cancer biology, identification of novel biological targets and optimal drug development with the aim of treatment individualization. This article summarizes statements and perspectives provided by the meeting participants.

The European Medicines Agency review of sacituzumab govitecan for the treatment of triple-negative breast cancer.

Sacituzumab govitecan (SG) is an antineoplastic agent which combines a humanized monoclonal antibody binding to trophoblast cell surface antigen-2 (Trop-2)-expressing cancer cells, linked with cytotoxic moiety SN-38 (govitecan) with topoisomerase I inhibitor action. On 22 November 2021, a marketing authorization valid through the European Union (EU) was issued under the European Medicines Agency (EMA)'s accelerated assessment program for SG as monotherapy for the treatment of adult patients with unresectable or metastatic triple-negative breast cancer (mTNBC) who have received two or more prior systemic therapies, including at least one of them for advanced disease. The assessment was based on results from an open-label, randomized, phase III trial to evaluate the safety, tolerability, pharmacokinetics and efficacy of SG versus treatment of physician's choice (TPC) in patients with mTNBC who received at least two prior treatments including at least one of them for advanced disease. The efficacy results in the overall population, based on mature data, showed a statistically significant improvement of SG over TPC in progression-free survival (PFS) and overall survival (OS). The median PFS was 4.8 months versus 1.7 months [hazard ratio (HR) = 0.43, n = 529; 95% CI 0.35-0.54; P < 0.0001] and the median OS was 11.8 months versus 6.9 months (HR = 0.51, n = 529; 95% CI 0.41-0.62; P < 0.0001). The most common (>30%) side effects of SG were diarrhea, neutropenia, nausea, fatigue, alopecia, anemia, constipation and vomiting. The aim of this manuscript is to summarize the scientific review of the application leading to regulatory approval in the EU.

Glitazone-like action of glimepiride and glibenclamide in primary human adipocytes.

AIM: Sulphonylureas (SUs) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. In addition, SUs have pleiotropic effects on other tissues. Conflicting findings have been reported regarding the effects of SUs on adipocytes. We have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. METHODS: Primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. Lipid accumulation was assessed by Oil Red O staining and determination of triglyceride content; gene expression was measured by RT PCR and Western blotting. RESULTS: Initially, we characterized the genes regulated during human pre-adipocyte differentiation by performing global microarray analysis. Treatment with glimepiride and glibenclamide caused an increased accumulation of lipid droplets and triglycerides. In addition, genes involved in lipid metabolism were induced and chemokine expression was decreased. Interestingly, the effects of SUs were generally qualitatively and quantitatively similar to those of pioglitazone. In direct comparison, glibenclamide was more potent than glimepiride with respect to the induction of fatty acid binding protein 4 (FABP4) (EC(50) 0.32 vs. 2.8 µM), an important adipocyte marker gene. SU-induced differentiation was virtually completely blocked by the peroxisome proliferator-activated receptor γ (PPARγ)-antagonist T0070907 but not affected by diazoxide, indicating PPARγ activation by SUs. Repaglinide had no effect on adipogenesis, although it causes insulin liberation like SUs. CONCLUSIONS: In primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating PPARγ. Thus, SUs but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation.

The European Medicines Agency review of entrectinib for the treatment of adult or paediatric patients with solid tumours who have a neurotrophic tyrosine receptor kinase gene fusions and adult patients with non-small-cell lung cancer harbouring ROS1 rearrangements.

Entrectinib is an inhibitor of the tyrosine kinases TRKA, TRKB, TRKC [all together known as neurotrophic tyrosine receptor kinases (NTRKs)], ROS1 and anaplastic lymphoma kinase (ALK). On 31 July 2020, a conditional marketing authorisation valid through the European Union (EU) was issued for entrectinib for the treatment of adult and paediatric patients 12 years of age and older with NTRK fusion-positive solid tumours that are locally advanced, metastatic or where surgical resection is likely to result in severe morbidity, and who have not received a prior NTRK inhibitor and have no satisfactory therapy; and also for adult patients with ROS1-positive non-small-cell lung cancer (NSCLC) not previously treated with ROS1 inhibitors. The submission was based on three open-label, multicentre, phase I studies (ALKA, STARTRK-1 and STARTRK-NG) and one phase II study (STARTRK-2). In patients with NTRK-positive solid tumours, the objective response rate (ORR) was 63.5% [95% confidence interval (CI) 51.5% to 74.4%] and the median duration of response (DOR) was 12.9 months (95% CI 9.3-not estimable). In patients with ROS1-positive NSCLC, the ORR was 67.1% (95% CI 59.25% to 74.27%) and the median DOR was 15.7 months (95% CI 13.9-28.6 months). The most frequent adverse events were dysgeusia, fatigue, dizziness, constipation, diarrhoea, nausea, increased weight, paraesthesia, increased creatinine, myalgia, peripheral oedema, vomiting, arthralgia, anaemia and increased AST. The aim of this manuscript is to summarise the scientific review of the application leading to regulatory approval of entrectinib in the EU.

The EMA assessment of encorafenib in combination with cetuximab for the treatment of adult patients with metastatic colorectal carcinoma harbouring the BRAFV600E mutation who have received prior therapy.

On 2 June 2020, a marketing authorisation valid through the European Union (EU) was issued for encorafenib in combination with cetuximab in adult patients with metastatic colorectal carcinoma (mCRC) with the BRAFV600E mutation who had received prior systemic therapy. Encorafenib plus cetuximab was evaluated in a randomised phase III trial of encorafenib plus binimetinib plus cetuximab versus encorafenib plus cetuximab versus cetuximab plus irinotecan or FOLFIRI (control arm) to adult patients with BRAFV600E mCRC who had received prior therapy for metastatic disease. The median overall survival was 9.3 months [95% confidence interval (CI): 8.05-11.30] versus 5.88 months (95% CI: 5.09-7.10) for encorafenib plus cetuximab (doublet) versus the control arm, respectively [hazard ratio (HR) 0.61, 95% CI: 0.48-0.77]. Progression-free survival (PFS) was 4.27 months (95% CI: 4.07-5.45) versus 1.54 months (95% CI: 1.48-1.91) (HR 0.44; 95% CI: 0.35-0.55). The most frequent adverse events in patients receiving encorafenib plus cetuximab were fatigue, nausea, diarrhoea, acneiform dermatitis, abdominal pain, arthralgia, decreased appetite, vomiting and rash. The aim of this manuscript is to summarise the scientific review of the application leading to regulatory approval in the EU.

The EMA review of trastuzumab emtansine (T-DM1) for the adjuvant treatment of adult patients with HER2-positive early breast cancer.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate of trastuzumab [a monoclonal antibody against human epidermal growth factor receptor 2 (HER2)] and DM1 (an inhibitor of tubulin polymerisation). It was initially approved in the European Union for the treatment of adult patients with HER2-positive unresectable locally advanced or metastatic breast cancer (BC) who had previously received trastuzumab and taxanes. On 18 December 2019, a variation of the marketing authorisation was approved extending this use to the adjuvant therapy of adult patients with HER2-positive early BC who have residual invasive disease in the breast and/or lymph nodes after neoadjuvant taxane-based and HER2-targeted therapy. A phase III randomised, multicentre, open-label trial compared T-DM1 with trastuzumab as adjuvant therapy in patients with HER2-positive early BC who had received preoperative chemotherapy and HER2-targeted therapy followed by surgery, with a finding of invasive residual disease in the breast and/or axillary lymph nodes. The study met its primary endpoint by showing an increased 3-year invasive disease-free survival rate in the T-DM1 arm (88.3%) compared with the trastuzumab arm (77.0%), with an unstratified hazard ratio of 0.50 (95% confidence interval: 0.39-0.64). There was a higher incidence of hepatotoxicity (37.3% versus 10.6%), thrombocytopenia (28.5% versus 2.4%), peripheral neuropathy (32.3% versus 16.9%), haemorrhage (29.2% versus 9.6%) and pulmonary toxicity (2.8% versus 0.8%) in the T-DM1 arm compared with the control arm. The aim of this manuscript was to summarise the scientific review of the application leading to regulatory approval of this additional indication in the European Union.

The EMA assessment of pembrolizumab as monotherapy for the first-line treatment of adult patients with metastatic microsatellite instability-high or mismatch repair deficient colorectal cancer.

On 21 January 2021, the European Commission amended the marketing authorisation granted for pembrolizumab to include the first-line treatment of microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) metastatic colorectal cancer (mCRC) in adults. The recommended dose of pembrolizumab was either 200 mg every 3 weeks or 400 mg every 6 weeks by intravenous infusion. Pembrolizumab was evaluated in a phase III, open-label, multicentre, randomised trial versus standard of care (SOC: FOLFOX6/FOLFIRI alone or in combination with bevacizumab/cetuximab) as first-line treatment of locally confirmed mismatch repair-deficient or microsatellite instability-high stage IV CRC. Subjects randomised to the SOC arm had the option to crossover and receive pembrolizumab once disease progressed. Both progression-free survival (PFS) and overall survival were primary endpoints. Pembrolizumab showed a statistically significant improvement in PFS compared with SOC, with a hazard ratio of 0.60 [95% confidence interval (CI): 0.45-0.80], P = 0.0002. Median PFS was 16.5 (95% CI: 5.4-32.4) versus 8.2 (95% CI: 6.1-10.2) months for the pembrolizumab versus SOC arms, respectively. The most frequent adverse events in patients receiving pembrolizumab were diarrhoea, fatigue, pruritus, nausea, increased aspartate aminotransferase, rash, arthralgia, and hypothyroidism. Having reviewed the data submitted, the European Medicines Agency's (EMA's) Committee for Medicinal Products for Human Use (CHMP) considered that the benefit-risk balance was positive. This is the first time the CHMP has issued an opinion for a target population defined by DNA repair deficiency biomarkers. The aim of this manuscript is to summarise the scientific review of the application leading to regulatory approval in the European Union.

The European Medicines Agency review of the initial application of atezolizumab and the role of PD-L1 expression as biomarker for checkpoint inhibitors.

Immune checkpoint inhibitors have revolutionised cancer therapeutics. Translational research evaluating the role of biomarkers is essential to identify the ideal target population for these drugs. From a regulatory perspective, the identification of biomarkers and diagnostic assays is strongly encouraged by the European Medicines Agency (EMA). The aim of this article is to analyse the role of programmed death-ligand 1 (PD-L1) expression as a predictive biomarker in relation to the data submitted for the initial assessment of atezolizumab, a monoclonal antibody targeting human PD-L1. On 20 July 2017, atezolizumab was granted a marketing authorisation valid throughout the European Union (EU) for adult patients with (i) locally advanced or metastatic non-small-cell lung cancer (NSCLC) after chemotherapy and (ii) locally advanced or metastatic urothelial carcinoma (UC) after chemotherapy or cisplatin-ineligibility. Initially, these indications were not restricted by the level of PD-L1 expression, but preliminary data from an ongoing phase III trial in patients with UC led to a restriction in the UC indication to cisplatin-ineligible patients whose tumours have ≥5% PD-L1 expression. Still, the role of PD-L1 expression as predictive biomarker for atezolizumab therapy remains inconclusive and further research is needed. Data in this paper came from the scientific review leading to the initial regulatory approval of atezolizumab in the EU and its complementary application for indication (EMEA/H/C/004143/II/0010). The full scientific assessment report and product information are available on the EMA website (www.ema.europa.eu).

The EMA assessment of avapritinib in the treatment of gastrointestinal stromal tumours harbouring the PDGFRA D842V mutation.

Avapritinib is a protein kinase inhibitor designed to selectively inhibit oncogenic KIT and platelet-derived growth factor receptor alpha (PDGFRA) mutants by targeting the active conformation of the kinase. On 24 September 2020, a marketing authorisation valid through the European Union was issued for avapritinib as treatment of adult patients with unresectable or metastatic gastrointestinal stromal tumours (GIST) harbouring the PDGFRA D842V mutation. The drug was evaluated in an open-label, phase I, first-in-human, dose-escalation, open-label study to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy of avapritinib in adults with unresectable or metastatic GIST. The benefit of avapritinib was observed in patients with GIST harbouring the PDGFRA D842V mutation. The overall response rate was 95% (95% confidence interval 82.3%-99.4%), with a median duration of response of 22.1 months (95% confidence interval 14.1-not estimable months). The most common adverse events were nausea, fatigue, anaemia, periorbital and face oedema, hyperbilirubinaemia, diarrhoea, vomiting, increased lacrimation, and decreased appetite. Most of the reported cognitive effects were mild memory impairment. Rarer events were cases of severe encephalopathy and intracranial or gastrointestinal bleeding. The aim of this manuscript is to summarise the scientific review of the application leading to regulatory approval in the European Union.

Market and patient access to new oncology products in Europe: a current, multidisciplinary perspective.

To air challenging issues related to patient and market access to new anticancer agents, the Biotherapy Development Association--an international group focused on developing targeted cancer therapies using biological agents--convened a meeting on 29 November 2007 in Brussels, Belgium. The meeting provided a forum for representatives of pharmaceutical companies and academia to interact with European regulatory and postregulatory agencies. The goal was to increase all parties' understanding of their counterparts' roles in the development, licensure, and appraisal of new agents for cancer treatment, events guided by an understanding that cancer patients should have rapid and equitable access to life-prolonging treatments. Among the outcomes of the meeting were a greater understanding of the barriers facing drug developers in an evolving postregulatory world, clarity about what regulatory and postregulatory bodies expect to see in dossiers of new anticancer agents as they contemplate licensure and reimbursement, and several sets of recommendations to optimize patients' access to innovative, safe, effective, and fairly priced cancer treatments.

The in ovo carcinogenicity assay (IOCA): a review of an experimental approach for research on carcinogenesis and carcinogenicity testing.

Up to now, the carcinogenicity of a substance, i.e., its cancer-causing effect, has been usually determined with the help of extensive animal testing. The in ovo carcinogenicity assay (IOCA) has been suggested as a rapid and inexpensive, non-animal, method for carcinogenicity testing and for experimental studies on mechanisms of carcinogenesis. The substance to be tested is injected into a fertilized ovum. No later than four days before the hatching date, the embryos are released, and the liver is removed for identifying the effect of hepatocarcinogens. Histological, cytological and molecular biological alterations of the embryonic liver have been induced with a variety of chemical carcinogens. After sufficiently high doses of hepatocarcinogens, tubular structures predominate in the liver and replace the normal trabecular pattern. The cell and nuclear size of the hepatocytes in embryonic liver is severely increased after exposure to chemical carcinogens over a wide dose range including doses that fail to elicit cytotoxicity in the embryonic liver.

The significance of mouse liver tumor formation for carcinogenic risk assessment: results and conclusions from a survey of ten years of testing by the agrochemical industry.

A survey was performed on the results of 138 carcinogenicity studies conducted in various mouse strains by the agrochemical industry over the period 1983-1993. Data for liver tumor incidence, liver weight, and histopathology were collected along with data on genotoxicity. Studies were judged positive or negative for liver tumor formation on the basis of apparent dose response, malignancy, and difference from historical control values using a weight of evidence approach. Thirty-seven studies were judged to be positive for liver tumorigenicity in one or both sexes. There was no evidence showing an influence of the mouse strain and the duration of the study on the proportion of positive studies. Although 8 of the chemicals tested in the 138 studies were positive in the Ames test, only one of these was judged positive for carcinogenicity. Only 6 of the 37 positive chemicals had any other reported positive genotoxicity findings. A clear relationship between hepatomegaly at 1 year after exposure and a positive tumorigenic outcome at 18 months or 2 years after exposure was demonstrated. Whereas the average relative liver weight of top dose animals was 110% of control in negative studies, it was 150% in positive studies. Likewise, very few negative studies demonstrated significant pathological findings after 1 year, whereas the majority of positive studies had significant liver pathology. The implications of these findings for extrapolation to humans are discussed.

Non-persisting early foci of altered hepatocytes induced in rats by N-nitrosomorpholine.

Male Sprague-Dawley rats were treated for 7 weeks with 120 mg/l N-nitrosomorpholine in their drinking water. At the end of the treatment period there were large numbers of enzyme-altered foci in the liver. During the following 10 weeks, the number of foci decreased significantly. This decrease in the number of enzyme-altered foci was due to the disappearance of a special type of focus. The typical features of these non-persisting foci were distinct enzyme histochemical and striking morphological alterations as well as the localization in or close to the third zone, as defined by Rappaport. In contrast to the simultaneously appearing persisting foci, the non-persisting foci were always glycogen-poor or totally glycogen-free. Signs of cell death were frequently found in or near this type of focus. After these non-persisting foci had disappeared, the total number of pre-neoplastic lesions obviously remained constant. We conclude that this disappearance of early appearing, severely altered foci is due to cell loss caused by the non-specific toxic effect of the carcinogen.

N-Nitrosomorpholine induced alterations of unscheduled DNA synthesis, mitochondrial DNA synthesis and cell proliferation in different cell types of liver, kidney, and urogenital organs in the rat.

In order to measure rates of unscheduled DNA synthesis (UDS), mitochondrial DNA synthesis, and cell proliferation, i.e. factors relevant in the early phase of carcinogenesis, young rats received by gavage 200 mg/kg N-nitrosomorpholine (NNM) or vehicle (distilled water), and were injected with 3H-thymidine 24 h later. Autoradiographs from liver, kidney, urethra, prostate, seminal vesicle, and ductus deferens were prepared from deparaffinized sections, using a 250-day exposure time. In the liver, UDS was at least doubled in 2n and 4n hepatocytes. Approximately 3% of these hepatocytes exhibited a fourfold increase in UDS. Such strongly labeled cells were only observed in the liver following NNM exposure. With the exception of renal epithelial cells of the proximal tubule, UDS in epithelial cells of bladder, urethra, ductus deferens, seminal vesicle and prostate was decreased in NNM-exposed rats. Mitochondrial DNA synthesis and cell proliferation were significantly increased only in hepatocytes, and were decreased in all other monitored organs in NNM-exposed rats. The strongly increased UDS and more moderately increased mitochondrial DNA synthesis in a subgroup of hepatocytes suggest that possibly some unrepaired damage persists in the DNA of these cells. The latter cells may be the precursors of so-called foci of hepatocellular alteration, which appear later during the process of carcinogenesis. The increased UDS but decreased rate of proliferation in the renal proximal tubule cells might be related to renal carcinogenesis which is observed in NNM-exposed rats after a long latency period.

Damage to mitochondrial DNA induced by the hepatocarcinogen diethylnitrosamine in ovo.

Different doses of the hepatocarcinogen diethylnitrosamine (DEN) were injected into fertilized turkey eggs 8 days before hatching. The embryos were removed from the eggs after 4 days and liver samples were shock frozen. Mitochondrial DNA (mtDNA) was purified from the samples. Electrophoresis on agarose gels with native mitochondrial DNA and with ribonuclease-treated mitochondrial DNA revealed a DEN-induced effect on the molecular size of the mtDNA. The content of mtDNA of the regular size of 16 kb dose-dependently decreased, whereas the amount of mtDNA fragments of various size increased. Fluorescent staining of the electrophoresis gels allowed the densitometric quantification of the mitochondrial DNA of the regular band at 16 kb and the amount of fragments of irregular size (smear). The diethylnitrosamine-induced effect was dose-dependent over the whole dose range from 1.24 to 6.2 mmol/kg. Even the lowest dose (10 mg DEN per egg) showed clear-cut effects. Mitochondrial damage and malfunction may be mechanistically involved in the neoplastic transformation and in aging phenomena. The in ovo model is a simple and rapid approach for investigations on chemically induced alterations of mtDNA.

Damage to mitochondrial DNA induced by the quinolone Bay y 3118 in embryonic turkey liver.

Quinolones are a class of antibiotics that induce damage to and loss of DNA from bacteria. The structural organization of bacterial DNA is more similar to eukaryotic mitochondrial DNA (mtDNA) than to eukaryotic chromosomal or nuclear DNA (nDNA). Antibiotics affecting the bacterial genome may therefore preferentially damage mtDNA rather than nDNA. We investigated the effect of a quinolone on mtDNA in avian embryonic hepatocytes in ovo. The quinolone Bay y 3118 (1-cyclopropyl-7-(2,8-diazabicyclo[4.3.0]non-8-yl) 6-fluoro-8-chloro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride, chemical structure see Bremm et al. [K.D. Bremm, U. Petersen, K.G. Metzger, R. Endermann, In vitro evaluation of Bay-y 3118, a new full-spectrum fluoroquinolone, Chemotherapy 38 (1992) 376-387] was injected into fertilized turkey eggs 8 days before hatching at doses of 1, 3, 10 and 30 mg per egg. The embryos were removed from the eggs after 4 days and liver samples were shock frozen. Mitochondrial DNA was purified from samples of the embryonic liver. The integrity of mtDNA was investigated by electrophoresis on agarose gels with native mtDNA and with ribonuclease-treated mtDNA. Fluorescent staining of the electrophoresis gels allows the densitometric quantification of the mtDNA of the regular band at 16 kilobases (kb) and the amount of DNA fragments of irregular size (smear). The genotoxic nitrosamine nitrosodiethylamine (NDEA) has previously been shown to reduce the content of mtDNA of the regular size of 16 kb and to induce the occurrence of smaller fragments of mtDNA [H. Enzmann, C. Kühlem, E. Löser, P. Bannasch, Damage to mitochondrial DNA induced by the hepatocarcinogen, diethylnitrosamine in ovo, Mutation Res. 329 (1995) 113-120]. After exposure to 10 and 30 mg Bay y 3118, a dose-dependent induction of damage to the mtDNA was found, whereas exposure to 3 and 1 mg showed no effect. NDEA (25 mg) was used as positive control. Testing chemical compounds in the in ovo model is a simple and rapid approach for investigations on chemically induced alterations of mtDNA.