Newly identified form of phenotypic plasticity of cancer: immunogenic mimicry.

Cancer plasticity is now a recognized new hallmark of cancer which is due to disturbances of cell differentiation programs. It is manifested not only in various forms like the best-known epithelial-mesenchymal transition (EMT) but also in vasculogenic and megakaryocytic mimicries regulated by EMT-specific or less-specific transcription factors such as HIF1a or STAT1/2. Studies in the past decades provided ample data that cancer plasticity can be manifested also in the expression of a vast array of immune cell genes; best-known examples are PDL1/CD274, CD47, or IDO, and we termed it immunogenic mimicry (IGM). However, unlike other types of plasticities which are epigenetically regulated, expression of IGM genes are frequently due to gene amplifications. It is important that the majority of the IGM genes are regulated by interferons (IFNs) suggesting that their protein expressions are regulated by the immune microenvironment. Most of the IGM genes have been shown to be involved in immune escape of cancers broadening the repertoire of these mechanisms and offering novel targets for immunotherapeutics.

Farnesyl-transferase inhibitors show synergistic anticancer effects in combination with novel KRAS-G12C inhibitors.

BACKGROUND: Inhibition of mutant KRAS challenged cancer research for decades. Recently, allele-specific inhibitors were approved for the treatment of KRAS-G12C mutant lung cancer. However, de novo and acquired resistance limit their efficacy and several combinations are in clinical development. Our study shows the potential of combining G12C inhibitors with farnesyl-transferase inhibitors. METHODS: Combinations of clinically approved farnesyl-transferase inhibitors and KRAS G12C inhibitors are tested on human lung, colorectal and pancreatic adenocarcinoma cells in vitro in 2D, 3D and subcutaneous xenograft models of lung adenocarcinoma. Treatment effects on migration, proliferation, apoptosis, farnesylation and RAS signaling were measured by histopathological analyses, videomicroscopy, cell cycle analyses, immunoblot, immunofluorescence and RAS pulldown. RESULTS: Combination of tipifarnib with sotorasib shows synergistic inhibitory effects on lung adenocarcinoma cells in vitro in 2D and 3D. Mechanistically, we present antiproliferative effect of the combination and interference with compensatory HRAS activation and RHEB and lamin farnesylation. Enhanced efficacy of sotorasib in combination with tipifarnib is recapitulated in the subcutaneous xenograft model of lung adenocarcinoma. Finally, combination of additional KRAS G1C and farnesyl-transferase inhibitors also shows synergism in lung, colorectal and pancreatic adenocarcinoma cellular models. DISCUSSION: Our findings warrant the clinical exploration of KRAS-G12C inhibitors in combination with farnesyl-transferase inhibitors.

Comparison of standard mismatch repair deficiency and microsatellite instability tests in a large cancer series.

BACKGROUND: The tumor-agnostic indication of immune checkpoint inhibitors to treat cancers with mismatch repair deficiency (dMMR)/microsatellite instability (MSI) increased the demand for such tests beyond Lynch syndrome. International guideline recommendations accept immunohistochemistry (IHC) for dMMR or molecular techniques (PCR or NGS) for MSI status determinations considering the two tests are equal, although there are scattered reports contradicting to this presumption. MATERIALS AND METHODS: Here we have directly compared four protein MMR immunohistochemistry (IHC) to MSI Pentaplex PCR test in a large cancer patient cohort (n = 1306) of our diagnostic center where the two tests have been run parallel in 703 cases. RESULTS: In this study we have found a high discrepancy rate (19.3%) of the two tests which was independent of the tumor types. The MSI PCR sensitivity for MMR IHC status was found to be very low resulting in a relatively low positive and negative predicting values. As a consequence, the correlation of the two tests was low (kappa < 0.7). During analysis of the possible contributing factors of this poor performance, we have excluded low tumor percentage of the samples, but identified dMMR phenotypes (classic versus non-classic or unusual) as possible contributors. CONCLUSION: Although our cohort did not include samples with identified technical errors, our data strongly support previous reports that unidentified preanalytical factors might have the major influence on the poor performance of the MSI PCR and MMR IHC. Furthermore, the case is open whether the two test types are equally powerful predictive markers of immunotherapies.

Gluing GAP to RAS Mutants: A New Approach to an Old Problem in Cancer Drug Development.

Mutated genes may lead to cancer development in numerous tissues. While more than 600 cancer-causing genes are known today, some of the most widespread mutations are connected to the RAS gene; RAS mutations are found in approximately 25% of all human tumors. Specifically, KRAS mutations are involved in the three most lethal cancers in the U.S., namely pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma. These cancers are among the most difficult to treat, and they are frequently excluded from chemotherapeutic attacks as hopeless cases. The mutated KRAS proteins have specific three-dimensional conformations, which perturb functional interaction with the GAP protein on the GAP-RAS complex surface, leading to a signaling cascade and uncontrolled cell growth. Here, we describe a gluing docking method for finding small molecules that bind to both the GAP and the mutated KRAS molecules. These small molecules glue together the GAP and the mutated KRAS molecules and may serve as new cancer drugs for the most lethal, most difficult-to-treat, carcinomas. As a proof of concept, we identify two new, drug-like small molecules with the new method; these compounds specifically inhibit the growth of the PANC-1 cell line with KRAS mutation G12D in vitro and in vivo. Importantly, the two new compounds show significantly lower IC50 and higher specificity against the G12D KRAS mutant human pancreatic cancer cell line PANC-1, as compared to the recently described selective G12D KRAS inhibitor MRTX-1133.

Molecular Pathology of Skin Melanoma: Epidemiology, Differential Diagnostics, Prognosis and Therapy Prediction.

Similar to other malignancies, TCGA network efforts identified the detailed genomic picture of skin melanoma, laying down the basis of molecular classification. On the other hand, genome-wide association studies discovered the genetic background of the hereditary melanomas and the susceptibility genes. These genetic studies helped to fine-tune the differential diagnostics of malignant melanocytic lesions, using either FISH tests or the myPath gene expression signature. Although the original genomic studies on skin melanoma were mostly based on primary tumors, data started to accumulate on the genetic diversity of the progressing disease. The prognostication of skin melanoma is still based on staging but can be completed with gene expression analysis (DecisionDx). Meanwhile, this genetic knowledge base of skin melanoma did not turn to the expected wide array of target therapies, except the BRAF inhibitors. The major breakthrough of melanoma therapy was the introduction of immune checkpoint inhibitors, which showed outstanding efficacy in skin melanoma, probably due to their high immunogenicity. Unfortunately, beyond BRAF, KIT mutations and tumor mutation burden, no clinically validated predictive markers exist in melanoma, although several promising biomarkers have been described, such as the expression of immune-related genes or mutations in the IFN-signaling pathway. After the initial success of either target or immunotherapies, sooner or later, relapses occur in the majority of patients, due to various induced genetic alterations, the diagnosis of which could be developed to novel predictive genetic markers.

Identification of a Tumor Cell Associated Type I IFN Resistance Gene Expression Signature of Human Melanoma, the Components of Which Have a Predictive Potential for Immunotherapy.

We developed a human melanoma model using the HT168-M1 cell line to induce IFN-α2 resistance in vitro (HT168-M1res), which was proven to be maintained in vivo in SCID mice. Comparing the mRNA profile of in vitro cultured HT168-M1res cells to its sensitive counterpart, we found 79 differentially expressed genes (DEGs). We found that only a 13-gene core of the DEGs was stable in vitro and only a 4-gene core was stable in vivo. Using an in silico cohort of IFN-treated melanoma tissues, we validated a differentially expressed 9-gene core of the DEGs. Furthermore, using an in silico cohort of immune checkpoint inhibitor (ICI)-treated melanoma tissues, we tested the predictive power of the DEGs for the response rate. Analysis of the top four upregulated and top four downregulated genes of the DEGs identified WFDC1, EFNA3, DDX10, and PTBP1 as predictive genes, and analysis of the "stable" genes of DEGs for predictive potential of ICI response revealed another 13 genes, out of which CDCA4, SOX4, DEK, and HSPA1B were identified as IFN-regulated genes. Interestingly, the IFN treatment associated genes and the ICI-therapy predictive genes overlapped by three genes: WFDC1, BCAN, and MT2A, suggesting a connection between the two biological processes.

Immunologic and immunogenomic aspects of tumor progression.

Tumor progression to metastatic disease is characterized by continuous genetic alterations due to instability of the genome. Immune sensitivity was found to be linked to tumor mutational burden (TMB) and the resulting amount of neoantigens. However, APOBEC activity resulting in increase in TMB causes immune evasion. On the other hand, clonal or acquired genetic loss of HLA class I also hampers immune sensitivity of tumors. Rare amplification of the PD-L1 gene in cancers may render them sensitive to immune checkpoint inhibitors but involvement of broader regions of chromosome 9p may ultimately lead again to immune evasion due to inactivation of the IFN-γ signaling pathway. Such genetic changes may occur not only in the primary tumor but at any phase of progression: in lymphatic as well as in visceral metastases. Accordingly, it is rational to monitor these changes continuously during disease progression similar to target therapies. Moreover, beside temporal variability, genomic features of tumors such as mutation profiles, as well as the tumor immune microenvironment also show considerable inter- and intratumoral spatial heterogeneity, suggesting the necessity of multiple sampling in biomarker studies.

Molecular epidemiology and diagnostics of KRAS mutations in human cancer.

RAS mutation is the most frequent oncogenic alteration in human cancers. KRAS is the most frequently mutated followed by NRAS. The emblematic KRAS mutant cancers are pancreatic, colorectal, lung adenocarcinomas and urogenital cancers. KRAS mutation frequencies are relatively stable worldwide in various cancer types with the one exception of lung adenocarcinoma. The frequencies of KRAS variant alleles appears cancer type specific, reflecting the various carcinogenic processes. In addition to point mutation KRAS, allelic imbalances are also frequent in human cancers leading to the predominance of a mutant allele. KRAS mutant cancers are characterized by typical, cancer-type-specific co-occurring mutations and distinct gene expression signatures. The heterogeneity of KRAS mutant primary cancers is significant, affecting the variant allele frequency, which could lead to unpredictable branching development in metastases. Selection of minute mutant subclones in the primary tumors or metastases during target therapies can also occur frequently in lung or colorectal cancers leading to acquired resistance. Ultrahigh sensitivity techniques are now routinely available for diagnostic purposes, but the proper determination of mutant allele frequency of KRAS in the primary or metastatic tissues may have larger clinical significance.

Horizontal Combination of MEK and PI3K/mTOR Inhibition in BRAF Mutant Tumor Cells with or without Concomitant PI3K Pathway Mutations.

The RAS/RAF and PI3K/Akt pathways play a key regulatory role in cancer and are often hit by oncogenic mutations. Despite molecular targeting, the long-term success of monotherapy is often hampered by de novo or acquired resistance. In the case of concurrent mutations in both pathways, horizontal combination could be a reasonable approach. In our study, we investigated the MEK inhibitor selumetinib and PI3K/mTOR dual inhibitor BEZ235 alone and in combination in BRAF-only mutant and BRAF + PI3K/PTEN double mutant cancer cells using short- and long-term 2D viability assays, spheroid assays, and immunoblots. In the 2D assays, selumetinib was more effective on BRAF-only mutant lines when compared to BRAF + PI3K/PTEN double mutants. Furthermore, combination therapy had an additive effect in most of the lines while synergism was observed in two of the double mutants. Importantly, in the SW1417 BRAF + PI3K double mutant cells, synergism was also confirmed in the spheroid and in the in vivo model. Mechanistically, p-Akt level decreased only in the SW1417 cell line after combination treatment. In conclusion, the presence of concurrent mutations alone did not predict a stronger response to combination treatment. Therefore, additional investigations are warranted to identify predictive factors that can select patients who can benefit from the horizontal combinational inhibition of these two pathways.

Characterization of cellular and humoral immune responses to alkaline phosphatase from fertile hydatid cysts in the human peripheral blood.

Hydatid cyst, the larval stage of the tapeworm Echinococcus granulosus and a causative agent of cystic echinococcosis, possesses a vast number of antigenic peptides that are constantly presented in the host immune system during infection. Here, we sought to provide more information about the cellular/humoral components engaged in the peripheral immune reactions to the fertile-cyst-derived Echinococcus alkaline phosphatase (E.ALP) in human hosts. Lymphoproliferative and cytokine responses after recall of E.ALP suggested the presence of specific immune reactions against the antigen. Interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin (IL)-10 had the highest fold increase over the spontaneous levels in response to hydatid crude antigen (HCA). Recall of E.ALP, as well as its encounter, boosted IFN-γ, TNF-α, IL-2, and IL-6 responses in peripheral blood mononuclear cells cultures (PBMCs). The HCA-driven levels of all the cytokines in the culture supernatants of normal PBMCs were higher than those measured after E.ALP encounter. Immunoglobulin G (IgG)-profile in response to HCA showed the dominance of specific IgG1, IgG2, and IgG4 antibodies, but relatively lower affinity of IgG3 to this antigen. IgG1 and IgG3 were the only isotypes detected in serum responses to E.ALP. Our findings suggested that E.ALP contributes to the early phase of immune responses to the parasite, likely by induction of proinflammatory profiles and clonal expansion of high-affinity IgG1- and IgG3-secreting plasma cells, suggesting the value of E.ALP as a candidate to develop novel therapeutic and immunization strategies.

The role of lipid signaling in the progression of malignant melanoma.

In the past decades, a vast amount of data accumulated on the role of lipid signaling pathways in the progression of malignant melanoma, the most metastatic/aggressive human cancer type. Genomic studies identified that PTEN loss is the leading factor behind the activation of the PI3K-signaling pathway in melanoma, mutations of which are one of the main resistance mechanisms behind target therapy failures. On the other hand, illegitimate expressions of megakaryocytic genes p12-lipoxyganse, cyclooxygenase-2, and phosphodiestherase-2/autotaxin (ATX) are mostly involved in the regulation of motility signaling in melanoma through various G-protein-coupled bioactive lipid receptors. Furthermore, endocannabinoid signaling can also be a novel paracrine survival factor in melanoma. Last but not least, prenylation inhibitors acting even on mutated small GTP-ases, such as NRAS of melanoma may offer novel therapeutic opportunities. As regards melanoma, the most effective therapy nowadays is immunotherapy, with the resistance mechanisms also possibly involving the lipid signaling activities of melanoma cells, which further supports the idea of their being therapeutic targets.

Clinical protein science in translational medicine targeting malignant melanoma.

Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry-based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression. In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry-based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution. These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma. A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer. The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level. Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.

Tumor necrosis correlates with PD-L1 and PD-1 expression in lung adenocarcinoma.

Background: Predictive biomarkers for immunotherapy in lung cancer are intensively investigated; however, correlations between PD-L1/PD-1 expressions and clinical features or histopathological tumor characteristics determined on hematoxylin and eosin stained sections have not extensively been studied. Material and methods: We determined PD-L1 expression of tumor cells (TC) and immune cells (IC), and PD-1 expression of IC by immunohistochemistry in 268 lung adenocarcinoma (LADC) patients, and correlated the data with smoking, COPD, tumor grade, necrosis, lepidic growth pattern, vascular invasion, density of stromal IC, and EGFR/KRAS status of the tumors. Results: There was a positive correlation between PD-L1 expression of TC and IC, as well as PD-L1 and PD-1 expression of IC. Tumor necrosis was associated with higher PD-L1 expression of TC and PD-1 expression of IC. A negative correlation was observed between lepidic growth pattern and PD-L1 expression of TC and PD-L1/PD-1 expression of IC. EGFR mutation seemed to negatively correlate with PD-1 expression of IC, but this tendency could not be verified when applying corrections for multiple comparisons. No significant effect of the KRAS mutation on any of the studied variables could be established. Conclusion: Here we first demonstrate that the presence of necrosis correlates with higher PD-L1 expression of TC and PD-1 expression of IC in LADC. Further studies are required to determine the predictive value of this observation in LADC patients receiving immunotherapy.

Long-Term Vemurafenib Exposure Induced Alterations of Cell Phenotypes in Melanoma: Increased Cell Migration and Its Association with EGFR Expression.

Acquired resistance during BRAF inhibitor therapy remains a major challenge for melanoma treatment. Accordingly, we evaluated the phenotypical and molecular changes of isogeneic human V600E BRAF-mutant melanoma cell line pairs pre- and post-treatment with vemurafenib. Three treatment naïve lines were subjected to in vitro long-term vemurafenib treatment while three pairs were pre- and post-treatment patient-derived lines. Molecular and phenotypical changes were assessed by Sulforhodamine-B (SRB) assay, quantitative RT-PCR (q-RT-PCR), immunoblot, and time-lapse microscopy. We found that five out of six post-treatment cells had higher migration activity than pretreatment cells. However, no unequivocal correlation between increased migration and classic epithelial-mesenchymal transition (EMT) markers could be identified. In fast migrating cells, the microphthalmia-associated transcription factor (MITF) and epidermal growth factor receptor (EGFR) mRNA levels were considerably lower and significantly higher, respectively. Interestingly, high EGFR expression was associated with elevated migration but not with proliferation. Cells with high EGFR expression showed significantly decreased sensitivity to vemurafenib treatment, and had higher Erk activation and FRA-1 expression. Importantly, melanoma cells with higher EGFR expression were more resistant to the EGFR inhibitor erlotinib treatment than cells with lower expression, with respect to both proliferation and migration inhibition. Finally, EGFR-high melanoma cells were characterized by higher PD-L1 expression, which might in turn indicate that immunotherapy may be an effective approach in these cases.

The Antitumor Effect of Lipophilic Bisphosphonate BPH1222 in Melanoma Models: The Role of the PI3K/Akt Pathway and the Small G Protein Rheb.

Malignant melanoma is one of the most metastatic cancer types, and despite recent success with novel treatment strategies, there is still a group of patients who do not respond to any therapies. Earlier, the prenylation inhibitor hydrophilic bisphosphonate zoledronic acid (ZA) was found to inhibit melanoma growth in vitro, but only a weaker effect was observed in vivo due to its hydrophilic properties. Recently, lipophilic bisphosphonates (such as BPH1222) were developed. Accordingly, for the first time, we compared the effect of BPH1222 to ZA in eight melanoma lines using viability, cell-cycle, clonogenic and spheroid assays, videomicroscopy, immunoblot, and xenograft experiments. Based on 2D and spheroid assays, the majority of cell lines were more sensitive to BPH. The activation of Akt and S6 proteins, but not Erk, was inhibited by BPH. Additionally, BPH had a stronger apoptotic effect than ZA, and the changes of Rheb showed a correlation with apoptosis. In vitro, only M24met cells were more sensitive to ZA than to BPH; however, in vivo growth of M24met was inhibited more strongly by BPH. Here, we present that lipophilic BPH is more effective on melanoma cells than ZA and identify the PI3K pathway, particularly Rheb as an important mediator of growth inhibition.

Pathogenic and targetable genetic alterations in 70 urachal adenocarcinomas.

Urachal cancer (UrC) is a rare but aggressive malignancy often diagnosed in advanced stages requiring systemic treatment. Although cytotoxic chemotherapy is of limited effectiveness, prospective clinical studies can hardly be conducted. Targeted therapeutic treatment approaches and potentially immunotherapy based on a biological rationale may provide an alternative strategy. We therefore subjected 70 urachal adenocarcinomas to targeted next-generation sequencing, conducted in situ and immunohistochemical analyses (including PD-L1 and DNA mismatch repair proteins [MMR]) and evaluated the microsatellite instability (MSI) status. The analytical findings were correlated with clinicopathological and outcome data and Kaplan-Meier and univariable/multivariable Cox regression analyses were performed. The patients had a mean age of 50 years, 66% were male and a 5-year overall survival (OS) of 58% and recurrence-free survival (RFS) of 45% was detected. Sequence variations were observed in TP53 (66%), KRAS (21%), BRAF (4%), PIK3CA (4%), FGFR1 (1%), MET (1%), NRAS (1%), and PDGFRA (1%). Gene amplifications were found in EGFR (5%), ERBB2 (2%), and MET (2%). We detected no evidence of MMR-deficiency (MMR-d)/MSI-high (MSI-h), whereas 10 of 63 cases (16%) expressed PD-L1. Therefore, anti-PD-1/PD-L1 immunotherapy approaches might be tested in UrC. Importantly, we found aberrations in intracellular signal transduction pathways (RAS/RAF/PI3K) in 31% of UrCs with potential implications for anti-EGFR therapy. Less frequent potentially actionable genetic alterations were additionally detected in ERBB2 (HER2), MET, FGFR1, and PDGFRA. The molecular profile strengthens the notion that UrC is a distinct entity on the genomic level with closer resemblance to colorectal than to bladder cancer.

Expression of calcium pumps is differentially regulated by histone deacetylase inhibitors and estrogen receptor alpha in breast cancer cells.

BACKGROUND: Remodeling of Ca2+ signaling is an important step in cancer progression, and altered expression of members of the Ca2+ signaling toolkit including the plasma membrane Ca2+ ATPases (PMCA proteins encoded by ATP2B genes) is common in tumors. METHODS: In this study PMCAs were examined in breast cancer datasets and in a variety of breast cancer cell lines representing different subtypes. We investigated how estrogen receptor alpha (ER-α) and histone deacetylase (HDAC) inhibitors regulate the expression of these pumps. RESULTS: Three distinct datasets displayed significantly lower ATP2B4 mRNA expression in invasive breast cancer tissue samples compared to normal breast tissue, whereas the expression of ATP2B1 and ATP2B2 was not altered. Studying the protein expression profiles of Ca2+ pumps in a variety of breast cancer cell lines revealed low PMCA4b expression in the ER-α positive cells, and its marked upregulation upon HDAC inhibitor treatments. PMCA4b expression was also positively regulated by the ER-α pathway in MCF-7 cells that led to enhanced Ca2+ extrusion capacity in response to 17ß-estradiol (E2) treatment. E2-induced PMCA4b expression was further augmented by HDAC inhibitors. Surprisingly, E2 did not affect the expression of PMCA4b in other ER-α positive cells ZR-75-1, T-47D and BT-474. These findings were in good accordance with ChIP-seq data analysis that revealed an ER-α binding site in the ATP2B4 gene in MCF-7 cells but not in other ER-α positive tumor cells. In the triple negative cells PMCA4b expression was relatively high, and the effect of HDAC inhibitor treatment was less pronounced as compared to that of the ER-α positive cells. Although, the expression of PMCA4b was relatively high in the triple negative cells, a fraction of the protein was found in intracellular compartments that could interfere with the cellular function of the protein. CONCLUSIONS: Our results suggest that the expression of Ca2+ pumps is highly regulated in breast cancer cells in a subtype specific manner. Our results suggest that hormonal imbalances, epigenetic modifications and impaired protein trafficking could interfere with the expression and cellular function of PMCA4b in the course of breast cancer progression.

Pan-RAF and MEK vertical inhibition enhances therapeutic response in non-V600 BRAF mutant cells.

BACKGROUND: Currently, there are no available targeted therapy options for non-V600 BRAF mutated tumors. The aim of this study was to investigate the effects of RAF and MEK concurrent inhibition on tumor growth, migration, signaling and apoptosis induction in preclinical models of non-V600 BRAF mutant tumor cell lines. METHODS: Six BRAF mutated human tumor cell lines CRL5885 (G466 V), WM3629 (D594G), WM3670 (G469E), MDAMB231 (G464 V), CRL5922 (L597 V) and A375 (V600E as control) were investigated. Pan-RAF inhibitor (sorafenib or AZ628) and MEK inhibitor (selumetinib) or their combination were used in in vitro viability, video microscopy, immunoblot, cell cycle and TUNEL assays. The in vivo effects of the drugs were assessed in an orthotopic NSG mouse breast cancer model. RESULTS: All cell lines showed a significant growth inhibition with synergism in the sorafenib/AZ628 and selumetinib combination. Combination treatment resulted in higher Erk1/2 inhibition and in increased induction of apoptosis when compared to single agent treatments. However, single selumetinib treatment could cause adverse therapeutic effects, like increased cell migration in certain cells, selumetinib and sorafenib combination treatment lowered migratory capacity in all the cell lines. Importantly, combination resulted in significantly increased tumor growth inhibition in orthotropic xenografts of MDAMB231 cells when compared to sorafenib - but not to selumetinib - treatment. CONCLUSIONS: Our data suggests that combined blocking of RAF and MEK may achieve increased therapeutic response in non-V600 BRAF mutant tumors.

NSCLC molecular testing in Central and Eastern European countries.

BACKGROUND: The introduction of targeted treatments for subsets of non-small cell lung cancer (NSCLC) has highlighted the importance of accurate molecular diagnosis to determine if an actionable genetic alteration is present. Few data are available for Central and Eastern Europe (CEE) on mutation rates, testing rates, and compliance with testing guidelines. METHODS: A questionnaire about molecular testing and NSCLC management was distributed to relevant specialists in nine CEE countries, and pathologists were asked to provide the results of EGFR and ALK testing over a 1-year period. RESULTS: A very high proportion of lung cancer cases are confirmed histologically/cytologically (75-100%), and molecular testing of NSCLC samples has been established in all evaluated CEE countries in 2014. Most countries follow national or international guidelines on which patients to test for EGFR mutations and ALK rearrangements. In most centers at that time, testing was undertaken on request of the clinician rather than on the preferred reflex basis. Immunohistochemistry, followed by fluorescent in situ hybridization confirmation of positive cases, has been widely adopted for ALK testing in the region. Limited reimbursement is a significant barrier to molecular testing in the region and a disincentive to reflex testing. Multidisciplinary tumor boards are established in most of the countries and centers, with 75-100% of cases being discussed at a multidisciplinary tumor board at specialized centers. CONCLUSIONS: Molecular testing is established throughout the CEE region, but improved and unbiased reimbursement remains a major challenge for the future. Increasing the number of patients reviewed by multidisciplinary boards outside of major centers and access to targeted therapy based on the result of molecular testing are other major challenges.

The evidence for and against different modes of tumour cell extravasation in the lung: diapedesis, capillary destruction, necroptosis, and endothelialization.

The development of lung metastasis is a significant negative prognostic factor for cancer patients. The extravasation phase of lung metastasis involves interactions of tumour cells with the pulmonary endothelium. These interactions may have broad biological and medical significance, with potential clinical implications ranging from the discovery of lung metastasis biomarkers to the identification of targets for intervention in preventing lung metastases. Because of the potential significance, the mechanisms of tumour cell extravasation require cautious, systematic studies. Here, we discuss the literature pertaining to the proposed mechanisms of extravasation and critically compare a recently proposed mechanism (tumour cell-induced endothelial necroptosis) with the already described extravasation mechanisms in the lung. We also provide novel data that may help to explain the underlying physiological basis for endothelialization as a mechanism of tumour cell extravasation in the lung. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.