Biomarker testing strategies in non-small cell lung cancer in the real-world setting: analysis of methods in the Prospective Central Lung Cancer Biomarker Registry (LungPath) from the Spanish Society of Pathology (SEAP).

AIMS: The aim of this study is to extend the analysis of the Lung Cancer Biomarker Testing Registry (LungPath), by analysing the techniques used in the determination of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1) and programmed death ligand-1 (PD-L1) for the diagnostic of patients with advanced non-small-cell lung cancer (NSCLC). METHODS: Information of the technique used for the determination of EGFR, ALK, ROS1 and PD-L1 was recorded from March 2018 to January 2019 from 44 centres, but only 34 centres matched with the 38 centres previously analysed, allowing to analyse the techniques used in 8970 matched determinations of EGFR, ALK, ROS1 and PD-L1. Therefore, a by-centre analysis studied the level of implementation of the techniques in the 44 centres, while a by-determination analysis made it possible to assess the overall frequency of the techniques used on the 9134 matched samples. RESULTS: By-centre analysis showed that only 46.5% and 25.6% of the centres used reflex strategies for ALK and ROS1 determination, respectively. By-determination analysis showed that 94.4% of EGFR determinations were performed by PCR, 80.7% of ALK determinations were performed by IHC with clone D5F3, while 55.7% of ROS1 determinations were performed by IHC with clone D4D6. 22C3 were the PD-L1 clone more used (43.5%) followed by SP263 clone (31.1%). CONCLUSIONS: The real-world evidence obtained from LungPath shows the effort of Spanish hospitals in performing biomarker determination in NSCLC with different methodologies despite that next-generation sequencing (NGS) utilisation in the year of the analysis was low. Biomarker determination results could be optimised with the incorporation of sequencing methods such as NGS in pathology departments.

Real-world biomarker testing rate and positivity rate in NSCLC in Spain: Prospective Central Lung Cancer Biomarker Testing Registry (LungPath) from the Spanish Society of Pathology (SEAP).

AIM: The aim of this study was to describe the testing rate and frequency of molecular alterations observed in the Lung Cancer Biomarker Testing Registry (LungPath). METHODS: A descriptive study of NSCLC biomarker determinations collected from March 2018 to January 2019, from 38 Spanish hospitals, was carried out. Only adenocarcinoma and not otherwise specified histologies were included for epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1) and programmed death ligand-1 (PD-L1) expression. The testing rate and the positivity rate were calculated. Multivariate logistic regression was used to explore the joint relationship between independent explanatory factors and both testing and positivity rates. Two models were adjusted: one with sample type and histology as independent factors, and the other adding the testing rate or the positivity rate of the other biomarkers. RESULTS: 3226 patient samples were analysed, where EGFR, ALK, ROS1 and PD-L1 information was collected (a total of 12 904 determinations). Overall, 9118 (71.4%) determinations were finally assessed. EGFR (91.4%) and ALK (80.1%) were the mainly tested biomarkers. Positivity rates for EGFR, ALK, ROS1 and PD-L1 were 13.6%, 3.4%, 2.0% and 49.2%, respectively. Multivariate models showed a lower testing rate for ALK in surgical pieces, fine-needle aspiration or other types of samples versus biopsies. CONCLUSIONS: Despite the high testing rate in EGFR and ALK in NSCLC, the real-world evidence obtained from the LungPath demonstrates that ROS1 and PD-L1 were not determined in a significant portion of patients. LungPath provides crucial information to improve the coverage in molecular testing in lung cancer, to monitor the positivity rate and the introduction of new biomarker testing in clinical practice.

Impact on prognosis of the regional distribution of MGMT methylation with respect to the CpG island methylator phenotype and age in glioma patients.

Clinical and molecular prognostic factors in gliomas include age, IDH mutation, the glioma CpG island methylator phenotype (G-CIMP+) and promoter methylation of the O(6)-methylguanine DNA-methyltransferase (MGMT) gene. Among these markers, a predictive value was reported in glioblastomas (GBM) for MGMT promoter methylation, in particular in elderly GBM patients. In this study, methylation data from 46 glioma samples with the Illumina 450K platform were obtained and extended using external data to include a total of 247 glioma samples. Methylation analysis of the whole MGMT gene with this platform revealed two strongly survival-associated CpG regions within the promoter and the gene body, which were confirmed in a reported dataset of high grade-gliomas. Methylation at the promoter (CpG 25, cg12981137 and the prognostic model MGMT-STP27) and at the gene body CpG 165 (cg07933035), were significantly associated with better overall survival, and strongly correlated with G-CIMP+ status. In this series, the prognostic value of MGMT methylation at the promoter was not observed in G-CIMP- cases, although around 50 % of them were MGMT-methylated. These results were also obtained in an homogeneously-treated series of chemoradiated G-CIMP- GBMs analyzed by MSP and qMSP, and confirmed in a reported pyrosequencing-analyzed series of gliomas. Interestingly, in contrast to the MGMT promoter, gene body methylation was of prognostic value in G-CIMP-patients older than 65 years. Our study highlights the relevance of the prognostic value of the different regions of methylation throughout the MGMT gene that could be affected by specific G-CIMP profiles and age groups.

Molecular classification defines 4 prognostically distinct glioma groups irrespective of diagnosis and grade.

According to World Health Organization criteria, diffuse gliomas are divided into several histological subtypes, including astrocytomas, oligodendrogliomas, and oligoastrocytomas, and 4 malignancy grades (I-IV). Molecular alterations, such as the isocitrate dehydrogenase gene (IDH) mutation or 1p/19q loss, are found in these tumors but are not included in the current classification system. Recently, mutation of α thalassemia/mental retardation syndrome X-linked (ATRX) gene and its loss of expression have been reported in infiltrating gliomas. We evaluated ATRX protein expression in 272 gliomas and its association with molecular and clinical features. Loss of ATRX expression was more common in tumors with an astrocytic component (astrocytomas II/III, 46.4%; oligoastrocytomas, 47.5%) but was uncommon in oligodendrogliomas (7.3%) and glioblastomas (0.9%). In astrocytic tumors, loss of ATRX expression was significantly associated with longer overall survival. Remarkably, on the basis of IDH mutation, 1p/19q codeletion, and ATRX expression, our study defined 4 molecularly and prognostically different groups of gliomas, showing the relevance of ATRX expression as a new marker for refining the molecular classification of gliomas and for distinguishing clinically distinct prognostic subgroups of patients.

Preclinical activity of the oral proteasome inhibitor MLN9708 in Myeloma bone disease.

PURPOSE: MLN9708 (ixazomib citrate), which hydrolyzes to pharmacologically active MLN2238 (ixazomib), is a next-generation proteasome inhibitor with demonstrated preclinical and clinical antimyeloma activity, but yet with an unknown effect on myeloma bone disease. Here, we investigated its bone anabolic and antiresorptive effects in the myeloma setting and in comparison with bortezomib in preclinical models. EXPERIMENTAL DESIGN: The in vitro effect of MLN2238 was tested on osteoclasts and osteoclast precursors from healthy donors and patients with myeloma, and on osteoprogenitors derived from bone marrow mesenchymal stem cells also from both origins. We used an in vivo model of bone marrow-disseminated human myeloma to evaluate MLN2238 antimyeloma and bone activities. RESULTS: Clinically achievable concentrations of MLN2238 markedly inhibited in vitro osteoclastogenesis and osteoclast resorption; these effects involved blockade of RANKL (receptor activator of NF-κB ligand)-induced NF-κB activation, F-actin ring disruption, and diminished expression of αVß3 integrin. A similar range of MLN2238 concentrations promoted in vitro osteoblastogenesis and osteoblast activity (even in osteoprogenitors from patients with myeloma), partly mediated by activation of TCF/ß-catenin signaling and upregulation of the IRE1 component of the unfolded protein response. In a mouse model of bone marrow-disseminated human multiple myeloma, orally administered MLN2238 was equally effective as bortezomib to control tumor burden and also provided a marked benefit in associated bone disease (sustained by both bone anabolic and anticatabolic activities). CONCLUSION: Given favorable data on pharmacologic properties and emerging clinical safety profile of MLN9708, it is conceivable that this proteasome inhibitor may achieve bone beneficial effects in addition to its antimyeloma activity in patients with myeloma.

Neuronal Galectin-4 is required for axon growth and for the organization of axonal membrane L1 delivery and clustering.

Axon membrane glycoproteins are essential for neuronal differentiation, although the mechanisms underlying their polarized sorting and organization are poorly understood. We describe here that galectin-4 (Gal-4), a lectin highly expressed in gastrointestinal tissues and involved in epithelial glycoprotein transport, is expressed by hippocampal and cortical neurons where it is sorted to discrete segments of the axonal membrane in a microtubule- and sulfatide-dependent manner. Gal-4 knockdown retards axon growth, an effect that can be rescued by recombinant Gal-4 addition. This Gal-4 reduction, as inhibition of sulfatide synthesis does, lowers the presence and clustered organization of axon growth-promoting molecule NCAM L1 at the axon membrane. Furthermore, we find that Gal-4 interacts with L1 by specifically binding to LacNAc branch ends of L1 N-glycans. Impairing the maturation of these N-glycans precludes Gal-4/L1 association resulting in a failure of L1 membrane cluster organization. In all, Gal-4 sorts to axon plasma membrane segments by binding to sulfatide-containing microtubule-associated carriers and being bivalent, it organizes the transport of L1, and likely other axonal glycoproteins, by attaching them to the carriers through their LacNAc termini. This mechanism would underlie L1 functional organization on the plasma membrane, required for proper axon growth.

Dasatinib as a bone-modifying agent: anabolic and anti-resorptive effects.

BACKGROUND: Bone loss, in malignant or non-malignant diseases, is caused by increased osteoclast resorption and/or reduced osteoblast bone formation, and is commonly associated with skeletal complications. Thus, there is a need to identify new agents capable of influencing bone remodeling. We aimed to further pre-clinically evaluate the effects of dasatinib (BMS-354825), a multitargeted tyrosine kinase inhibitor, on osteoblast and osteoclast differentiation and function. METHODS: For studies on osteoblasts, primary human bone marrow mensenchymal stem cells (hMSCs) together with the hMSC-TERT and the MG-63 cell lines were employed. Osteoclasts were generated from peripheral blood mononuclear cells (PBMC) of healthy volunteers. Skeletally-immature CD1 mice were used in the in vivo model. RESULTS: Dasatinib inhibited the platelet derived growth factor receptor-ß (PDGFR-ß), c-Src and c-Kit phosphorylation in hMSC-TERT and MG-63 cell lines, which was associated with decreased cell proliferation and activation of canonical Wnt signaling. Treatment of MSCs from healthy donors, but also from multiple myeloma patients with low doses of dasatinib (2-5 nM), promoted its osteogenic differentiation and matrix mineralization. The bone anabolic effect of dasatinib was also observed in vivo by targeting endogenous osteoprogenitors, as assessed by elevated serum levels of bone formation markers, and increased trabecular microarchitecture and number of osteoblast-like cells. By in vitro exposure of hemopoietic progenitors to a similar range of dasatinib concentrations (1-2 nM), novel biological sequelae relative to inhibition of osteoclast formation and resorptive function were identified, including F-actin ring disruption, reduced levels of c-Fos and of nuclear factor of activated T cells 1 (NFATc1) in the nucleus, together with lowered cathepsin K, αVß3 integrin and CCR1 expression. CONCLUSIONS: Low dasatinib concentrations show convergent bone anabolic and reduced bone resorption effects, which suggests its potential use for the treatment of bone diseases such as osteoporosis, osteolytic bone metastasis and myeloma bone disease.

In vitro and in vivo rationale for the triple combination of panobinostat (LBH589) and dexamethasone with either bortezomib or lenalidomide in multiple myeloma.

BACKGROUND: Combinations of drug treatments based on bortezomib or lenalidomide plus steroids have resulted in very high response rates in multiple myeloma. However, most patients still relapse, indicating the need for novel combination partners to increase duration of response or to treat relapsed disease. We explored the antimyeloma activity of triple combinations of these well-established schemes with panobinostat, a novel deacetylase inhibitor with a multi-targeted profile. DESIGN AND METHODS: The activity of these combinations was explored in vitro in cell lines by using MTT and annex-in V, ex vivo by flow cytometry, and in vivo using two different murine models of human myeloma: one bearing a subcutaneous plasmacytoma and another with a disseminated myeloma. Moreover, gene expression profiling and immunohistochemical studies were performed. RESULTS: The addition of panobinostat (LBH589) to dexamethasone and either bortezomib or lenalidomide resulted in clear potentiation in multiple myeloma cell lines, freshly isolated plasma cells, and murine models of multiple myeloma. The quantification of the potency of these combinations by using the Chou-Talalay method showed synergistic combination indices for all of them. This effect derived from the deregulation of a cluster of genes that was completely different from the sum of genes affected by the single agents (895 and 1323 genes exclusively deregulated by panobinostat and dexamethasone plus bortezomib or lenalidomide, respectively). Functional experiments, such as annexin V staining, cell cycle analysis, and immunohistochemical studies also supported this potentiation. Anti-myeloma efficacy was confirmed in an extramedullary plasmacytoma model and a disseminated luciferized model, in which panobinostat also provided a marked benefit in bone disease. CONCLUSIONS: The potent activity, together with the exclusive mechanistic profile, provides the rationale for the clinical evaluation of these drug combinations in multiple myeloma.

Zalypsis: a novel marine-derived compound with potent antimyeloma activity that reveals high sensitivity of malignant plasma cells to DNA double-strand breaks.

Multiple myeloma (MM) remains incurable, and new drugs with novel mechanisms of action are still needed. In this report, we have analyzed the action of Zalypsis, an alkaloid analogous to certain natural marine compounds, in MM. Zalypsis turned out to be the most potent antimyeloma agent we have tested so far, with IC(50) values from picomolar to low nanomolar ranges. It also showed remarkable ex vivo potency in plasma cells from patients and in MM cells in vivo xenografted in mice. Besides the induction of apoptosis and cell cycle arrest, Zalypsis provoked DNA double-strand breaks (DSBs), evidenced by an increase in phospho-histone-H2AX and phospho-CHK2, followed by a striking overexpression of p53 in p53 wild-type cell lines. In addition, in those cell lines in which p53 was mutated, Zalypsis also provoked DSBs and induced cell death, although higher concentrations were required. Immunohistochemical studies in tumors also demonstrated histone-H2AX phosphorylation and p53 overexpression. Gene expression profile studies were concordant with these results, revealing an important deregulation of genes involved in DNA damage response. The potent in vitro and in vivo antimyeloma activity of Zalypsis uncovers the high sensitivity of tumor plasma cells to DSBs and strongly supports the use of this compound in MM patients.

Sarcoma de Ewing/PNET simulador de neoplasia primaria de glándula tiroides / Ewing sarcoma/PNET simulating primary thyroid gland neoplasm

Caso clínico: Los autores presentan un sarcoma de partesblandas cervicales que por su forma de presentación clínica,localización yuxtatiroidea y aspecto histológico simulabaun tumor maligno de glándula tiroides. Resultados:Aunque la histología podía ser compatible con varias formasde carcinoma tiroideo, especialmente con un carcinomamedular, los resultados del estudio inmunohistoquímico ylas alteraciones moleculares del gen EWS mediante FISHen parafina correspondieron a un tumor maligno de célulasredondas de estirpe mesenquimal, concordante con sarcomade Ewing/ PNET. Conclusión: Este caso, de gran dificultaddiagnóstica, sirve para recordar que en el diagnóstico diferencialde tumores tiroideos pobremente diferenciadoshemos de considerar la posibilidad de afectación secundariadel mismo por tumores de estructuras vecinas, ya que lossarcomas primarios de la glándula tiroides son muy infrecuentesy en su mayoría corresponden a formas sarcomatoidesde carcinomas tiroideos (AU)

Case report:We report a case of cervical soft tissue sarcomasimulating primary malignant thyroid neoplasm dueto its location, histopathology and clinical presentation.Results: Although histopathology was consistent with severalforms of thyroid carcinoma, most of all, medullary carcinoma,immunohistochemical analysis and moleculargenetic studies on EWS gene, using FISH on paraffinembeddedtissue, yielded the diagnosis of mesenquimalmalignant round cell tumor consistent with Ewing’s sarcoma/PNET. Conclusion: The message of this difficult case isthat differential diagnosis of poorly differenciated thyroidtumors must include the possibility of secondary involvementfrom neighbouring cervical structures because primarythyroid sarcomas are much less frequent than sarcomatoidvariants of primary thyroid carcinomas (AU)