Immunohistochemistry for diagnosis and prognosis of breast cancer: a review.

Breast cancer is the most prevalent malignant tumor and main oncologic cause of mortality in women. Although most diagnosis of breast pathology is accomplished using hematoxylin and eosin stained sections, some cases require immunohistochemistry for proper evaluation. We investigated the latter cases including distinctions between ductal and lobular carcinoma, in situ and invasive carcinoma, typical ductal hyperplasia and atypical ductal hyperplasia/ductal carcinoma in situ, papillary and spindle cell lesion assessment, metastasis evaluation, and assessment of prognostic and therapy markers. E-cadherin is used to differentiate ductal and lobular carcinoma; 34ßE12, CK8, p120 catenin and ß-catenin also produce consistent results. Myoepithelial cell (MEC) stains are used to evaluate in situ and invasive carcinoma; calponin, smooth muscle myosin heavy chain and p63 are sensitive/specific markers. 34ßE12 and CK5/6 are positive in ductal hyperplasia, which enables its differentiation from atypical ductal hyperplasia and ductal carcinoma in situ. CK 5/6, ER and MEC markers are consistent options for evaluating papillary lesions. Spindle cell lesions can be assessed using ß-catenin, SMA, CD34, p63, CKs and hormone receptors. It is important to differentiate primary carcinomas from metastases; the most commonly used markers to identify breast origin include mammaglobin, GCDFP-15, GATA3 and ER, although none of these is completely sensitive or specific. Immunohistochemistry can be used to evaluate central prognostic and predictive factors including molecular subtypes, HER2, hormone receptors, proliferation markers (Ki-67) and lymph-vascular invasion markers including ERG, CD31, CD34, factor VIII and podoplanin. Owing to the complexity of mammary lesions, diagnosis also depends on each particular situation, evaluation of cytological characteristics revealed by immunochemistry and correlation with histological findings.

Early-drug development in the era of immuno-oncology: are we ready to face the challenges?

The classical development of drugs has progressively faded away, and we are currently in an era of seamless drug-development, where first-in-human trials include unusually big expansion cohorts in the search for early signs of activity and rapid regulatory approval. The fierce competition between different pharmaceutical companies and the hype for immune combinations obliges us to question the current way in which we are evaluating these drugs. In this review, we discuss critical issues and caveats in immunotherapy development. A particular emphasis is put on the limitations of pre-clinical toxicology studies, where both murine models and cynomolgus monkeys have underpredicted toxicity in humans. Moreover, relevant issues surrounding dose determination during phase I trials, such as dose-escalation methods or flat versus body-weight dosing, are discussed. A proposal of how to face these different challenges is offered, in order to achieve maximum efficacy with minimum toxicity for our patients.

Clinical research in small genomically stratified patient populations.

The paradigm of early drug development in cancer is shifting from 'histology-oriented' to 'molecularly oriented' clinical trials. This change can be attributed to the vast amount of tumour biology knowledge generated by large international research initiatives such as The Cancer Genome Atlas (TCGA) and the use of next generation sequencing (NGS) techniques developed in recent years. However, targeting infrequent molecular alterations entails a series of special challenges. The optimal molecular profiling method, the lack of standardised biological thresholds, inter- and intra-tumor heterogeneity, availability of enough tumour material, correct clinical trials design, attrition rate, logistics or costs are only some of the issues that need to be taken into consideration in clinical research in small genomically stratified patient populations. This article examines the most relevant challenges inherent to clinical research in these populations. Moreover, perspectives from the Academia point of view are reviewed as well as initiatives to be taken in forthcoming years.

Serum lactate dehydrogenase as an early marker for outcome in patients treated with anti-PD-1 therapy in metastatic melanoma.

BACKGROUND: Treatment with programmed death receptor-1 (PD-1) antibodies is associated with high response rates in patients with advanced melanoma. Reliable markers for early response and outcome are still sparse. METHODS: We evaluated 66 consecutive patients with advanced/metastatic melanoma treated with nivolumab or pembrolizumab between 2013 and 2014. The main objectives of this study were to investigate whether, first, serum lactate dehydrogenase (LDH) at baseline (normal vs above the upper limit of normal) correlates with overall survival (OS), and, second, whether the change of LDH during treatment predicts response before the first scan and OS in patients with an elevated baseline LDH. RESULTS: After a median follow-up of 9 months, patients with an elevated baseline LDH (N=34) had a significantly shorter OS compared with patients with normal LDH (N=32; 6-month OS: 60.8% vs 81.6% and 12-month OS: 44.2% vs 71.5% (log-rank P=0.0292). In those 34 patients with elevated baseline LDH, the relative change during treatment was significantly associated with an objective response on the first scan: the 11 (32%) patients with partial remission had a mean reduction of -27.3% from elevated baseline LDH. In contrast, patients with progressive disease (N=15) had a mean increase of +39%. Patients with a relative increase over 10% from elevated baseline LDH had a significantly shorter OS compared with patients with ⩽ 10% change (4.3 vs 15.7 months, log-rank P<0.00623). CONCLUSIONS: LDH could be a useful marker at baseline and during treatment to predict early response or progression in patients with advanced melanoma who receive anti-PD-1 therapy.

Targeting protein kinase C in sarcoma.

Protein kinase C (PKC) is a family of serine/threonine tyrosine kinases that regulate many cellular processes including division, proliferation, survival, anoikis and polarity. PKC is abundant in many human cancers and aberrant PKC signalling has been demonstrated in cancer models. On this basis, PKC has become an attractive target for small molecule inhibition within oncology drug development programmes. Sarcoma is a heterogeneous group of mesenchymal malignancies. Due to their relative insensitivity to conventional chemotherapies and the increasing recognition of the driving molecular events of sarcomagenesis, sarcoma provides an excellent platform to test novel therapeutics. In this review we provide a structure-function overview of the PKC family, the rationale for targeting these kinases in sarcoma and the state of play with regard to PKC inhibition in the clinic.

Reversion of Hormone Treatment Resistance with the Addition of an mTOR Inhibitor in Endometrial Stromal Sarcoma.

Background. Endometrial stromal sarcomas (ESS) are a subtype of gynaecological sarcomas characterized by the overexpression of hormone receptors. Hormone treatment is widely used in ESS but primary or acquired resistance is common. The mammalian target of rapamycin (mTOR) pathway has been suggested to play a key role in the mechanisms of hormone resistance. Recent studies in breast and prostate cancer demonstrate that this resistance can be reversed with the addition of an mTOR inhibitor. This phenomenon has never been reported in ESS. Methods. We report the outcome of one patient with pretreated, progressing low grade metastatic ESS treated with medroxyprogesterone acetate in combination with the mTOR inhibitor sirolimus. Results. Partial response was achieved following the addition of sirolimus to the hormone treatment. Response has been maintained for more than 2 years with minimal toxicity and treatment is ongoing. Conclusion. This case suggests that the resistance to the hormone manipulation in ESS can be reversed by the addition of an mTOR pathway inhibitor. This observation is highly encouraging and deserves further investigation.

Phase I study and preclinical efficacy evaluation of the mTOR inhibitor sirolimus plus gemcitabine in patients with advanced solid tumours.

BACKGROUND: We conducted a phase I study in patients with advanced solid tumours to identify the recommended dose, assess pharmacokinetics (PK), pharmacodynamic activity and preclinical antitumour efficacy of the combination of sirolimus and gemcitabine. METHODS: Nineteen patients were treated with sirolimus 2 or 5 mg daily and gemcitabine 800 or 1000 mg m(-2) on days 1 and 8. Dose escalation depended on dose-limiting toxicity (DLT) rate during the first 3-week period. Paired skin biopsies were evaluated for phosphorylated S6 (pS6) as marker of mTOR (mammalian target of rapamycin) inhibition. Pharmacokinetics and preclinical evaluation of efficacy using two different sarcoma cell lines and leiomyosarcoma xenografts were also conducted. RESULTS: Three DLTs were observed: grade 3 transaminitis, grade 3 thrombocytopenia and grade 4 thrombocytopenia. Common treatment-related adverse events included anaemia, neutropenia, thrombocytopenia and transaminitis. Pharmacodynamic analyses demonstrated mTOR inhibition with sirolimus 5 mg and PK showed no influence of sirolimus concentrations on gemcitabine clearance. In vitro and in vivo studies suggested mTOR pathway hyperactivation by gemcitabine that was reversed by sirolimus. Tumour growth in leiomyosarcoma xenografts was dramatically inhibited by the treatment. CONCLUSIONS: Recommended dose was sirolimus 5 mg per 24 h plus gemcitabine 800 mg m(-2). Antitumour activity in preclinical sarcoma models and mTOR signalling inhibition were observed. A phase II study is currently ongoing.

Phase I trial of sorafenib in combination with ifosfamide in patients with advanced sarcoma: a Spanish group for research on sarcomas (GEIS) study.

BACKGROUND: This phase I trial assessed safety, pharmacokinetics (PK), dose limiting toxicity (DLT), maximum tolerated dose and recommended dose (RD) of the combination of sorafenib plus ifosfamide in patients with advanced sarcoma. METHODS: Twelve sarcoma patients (9 soft-tissue, 3 bone sarcoma) were treated with sorafenib plus ifosfamide (starting doses 200 mg bid and 6 g/m(2) respectively). A 3 + 3 dose escalation design with cohorts of 3-6 patients was used. A study to assess the in vitro efficacy of the combination was also conducted. RESULTS: Three DLTs were observed: fatigue grade 4 with sorafenib 400 mg bid plus ifosfamide 6 g/m(2) and encephalopathy and emesis grade 3 with sorafenib 400 mg bid plus ifosfamide 7.5 g/m(2). Other toxicities included diarrhea, hand-foot syndrome, mucositis, neutropenia, skin rash and thrombocytopenia. There were no relevant effects on PK of sorafenib but an increase in ifosfamide active metabolite 4-hydroxy-ifosfamide was observed. Eight patients achieved stable disease lasting more than 12 weeks. An additive effect was observed in vitro. CONCLUSIONS: RD was sorafenib 400 mg bid plus ifosfamide 6 g/m(2), allowing administration of active doses of both agents. Limited preliminary antitumor activity was also observed. A phase II study is currently ongoing.

Elevated glucose concentration changes the content and cellular localization of AMPA receptors in the retina but not in the hippocampus.

Diabetic retinopathy and diabetic encephalopathy are two common complications of diabetes mellitus. The impairment of glutamatergic neurotransmission in the retina and hippocampus has been suggested to be involved in the pathogenesis of these diabetic complications. In this study, we investigated the effect of elevated glucose concentration and diabetes on the protein content and surface expression of AMPA receptor subunits in the rat retina and hippocampus. We have used two models, cultured retinal and hippocampal cells exposed to elevated glucose concentration and an animal model of streptozotocin-induced type 1 diabetes. The immunoreactivity of GluA1, GluA2 and GluA4 was evaluated by Western blot and immunocytochemistry. The levels of these subunits at the plasma membrane were evaluated by biotinylation and purification of plasma membrane-associated proteins. Elevated glucose concentration increased the total levels of GluA2 subunit of AMPA receptors in retinal neural cells, but not of the subunits GluA1 or GluA4. However, at the plasma membrane, elevated glucose concentration induced an increase of all AMPA receptor subunits. In cultured hippocampal neurons, elevated glucose concentration did not induce significant alterations in the levels of AMPA receptor subunits. In the retinas of diabetic rats there were no persistent changes in the levels of AMPA receptor subunits comparing to aged-matched control retinas. Also, no consistent changes were detected in the levels of GluA1, GluA2 or GluA4 in the hippocampus of diabetic rats. We demonstrate that elevated glucose concentration induces early changes in AMPA receptor subunits, mainly in GluA2 subunit, in retinal neural cells. Conversely, hippocampal neurons seem to remain unaffected by elevated glucose concentration, concerning the expression of AMPA receptors, suggesting that AMPA receptors are more susceptible to the stress caused by elevated glucose concentration in retinal cells than in hippocampal neurons.

Long-term exposure to high glucose induces changes in the content and distribution of some exocytotic proteins in cultured hippocampal neurons.

A few studies have reported the existence of depletion of synaptic vesicles, and changes in neurotransmitter release and in the content of exocytotic proteins in the hippocampus of diabetic rats. Recently, we found that diabetes alters the levels of synaptic proteins in hippocampal nerve terminals. Hyperglycemia is considered the main trigger of diabetic complications, although other factors, such as low insulin levels, also contribute to diabetes-induced changes. Thus, the aim of this work was to evaluate whether long-term elevated glucose per se, which mimics prolonged hyperglycemia, induces significant changes in the content and localization of synaptic proteins involved in exocytosis in hippocampal neurons. Hippocampal cell cultures were cultured for 14 days and were exposed to high glucose (50 mM) or mannitol (osmotic control; 25 mM plus 25 mM glucose), for 7 days. Cell viability and nuclear morphology were evaluated by MTT and Hoechst assays, respectively. The protein levels of vesicle-associated membrane protein-2 (VAMP-2), synaptosomal-associated protein-25 (SNAP-25), syntaxin-1, synapsin-1, synaptophysin, synaptotagmin-1, rabphilin 3a, and also of vesicular glutamate and GABA transporters (VGluT-1 and VGAT), were evaluated by immunoblotting, and its localization was analyzed by immunocytochemistry. The majority of the proteins were not affected. However, elevated glucose decreased the content of SNAP-25 and increased the content of synaptotagmin-1 and VGluT-1. Moreover, there was an accumulation of syntaxin-1, synaptotagmin-1 and VGluT-1 in the cell body of some hippocampal neurons exposed to high glucose. No changes were detected in mannitol-treated cells. In conclusion, elevated glucose per se did not induce significant changes in the content of the majority of the synaptic proteins studied in hippocampal cultures, with the exception of SNAP-25, synaptotagmin-1 and VGluT-1. However, there was an accumulation of some proteins in cell bodies of hippocampal neurons exposed to elevated glucose, suggesting that the trafficking of these proteins to the synapse may be compromised. Moreover, these results also suggest that other factors, in addition to hyperglycemia, certainly contribute to alterations detected in synaptic proteins in diabetic animals.