Gal-1 Expression Analysis in the GLIOCAT Multicenter Study: Role as a Prognostic Factor and an Immune-Suppressive Biomarker.

Glioblastoma (GBM) is the most frequent primary malignant brain tumor and has a dismal prognosis. Unfortunately, despite the recent revolution of immune checkpoint inhibitors in many solid tumors, these have not shown a benefit in overall survival in GBM patients. Therefore, new potential treatment targets as well as diagnostic, prognostic, and/or predictive biomarkers are needed to improve outcomes in this population. The ß-galactoside binding protein Galectin-1 (Gal-1) is a protein with a wide range of pro-tumor functions such as proliferation, invasion, angiogenesis, and immune suppression. Here, we evaluated Gal-1 expression by immunohistochemistry in a homogenously treated cohort of GBM (the GLIOCAT project) and correlated its expression with clinical and molecular data. We observed that Gal-1 is a negative prognostic factor in GBM. Interestingly, we observed higher levels of Gal-1 expression in the mesenchymal/classical subtypes compared to the less aggressive proneural subtype. We also observed a Gal-1 expression correlation with immune suppressive signatures of CD4 T-cells and macrophages, as well as with several GBM established biomarkers, including SHC1, PD-L1, PAX2, MEOX2, YKL-40, TCIRG1, YWHAG, OLIG2, SOX2, Ki-67, and SOX11. Moreover, Gal-1 levels were significantly lower in grade 4 IDH-1 mutant astrocytomas, which have a better prognosis. Our results confirm the role of Gal-1 as a prognostic factor and also suggest its value as an immune-suppressive biomarker in GBM.

Amyloid Beta-Peptide Increases BACE1 Translation through the Phosphorylation of the Eukaryotic Initiation Factor-2α.

Alzheimer's disease (AD) is tightly linked to oxidative stress since amyloid beta-peptide (Aß) aggregates generate free radicals. Moreover, the aggregation of Aß is increased by oxidative stress, and the neurotoxicity induced by the oligomers and fibrils is in part mediated by free radicals. Interestingly, it has been reported that oxidative stress can also induce BACE1 transcription and expression. BACE1 is the key enzyme in the cleavage of the amyloid precursor protein to produce Aß, and the expression of this enzyme has been previously shown to be enhanced in the brains of Alzheimer's patients. Here, we have found that BACE1 expression is increased in the hippocampi from AD patients at both the early (Braak stage II) and late (Braak stage VI) stages of the disease as studied by immunohistochemistry and western blot. To address the role of Aß and oxidative stress in the regulation of BACE1 expression, we have analyzed the effect of subtoxic concentrations of Aß oligomers (0.25 µM) and H2O2 (10 mM) on a human neuroblastoma cell line. Firstly, our results show that Aß oligomers and H2O2 induce an increase of BACE1 mRNA as we studied by qPCR. Regarding BACE1 translation, it is dependent on the phosphorylation of the eukaryotic initiation factor 2α (eIF2α), since BACE1 mRNA bears a 5'UTR that avoids its translation under basal conditions. BACE1 5'UTR contains four upstream initiating codons (uAUGs), and its translation is activated when eIF2α is phosphorylated. Consistently, we have obtained that Aß oligomers and H2O2 increase the levels of BACE1 and p-eIF2α assayed by western blot and confocal microscopy. Our results suggest that Aß oligomers increase BACE1 translation by phosphorylating eIF2α in a process that involves oxidative stress and conforms a pathophysiological loop, where the Aß once aggregated favors its own production continuously by the increase in BACE1 expression as observed in AD patients.

Defective Cyclin B1 Induction in Trastuzumab-emtansine (T-DM1) Acquired Resistance in HER2-positive Breast Cancer.

Purpose: Trastuzumab-emtansine (T-DM1) is a standard treatment in advanced HER2-positive breast cancer. However, resistance inevitably occurs. We aimed to identify mechanisms of acquired T-DM1 resistance.Experimental Design: HER2-positive breast cancer cells (HCC1954, HCC1419, SKBR3, and BT474) were treated in a pulse-fashion with T-DM1 to induce a resistant phenotype. Cellular and molecular effects of T-DM1 in parental versus resistant cells were compared. CDK1 kinase activity and cyclin B1 expression were assayed under various conditions. Genetic modifications to up- or downregulate cyclin B1 were conducted. Effects of T-DM1 on cyclin B1 levels, proliferation, and apoptosis were assayed in human HER2-positive breast cancer explants.Results: We obtained three cell lines with different levels of acquired T-DM1 resistance (HCC1954/TDR, HCC1419/TDR, and SKBR3/TDR cells). HER2 remained amplified in the resistant cells. Binding to HER2 and intracellular uptake of T-DM1 were maintained in resistant cells. T-DM1 induced cyclin B1 accumulation in sensitive but not resistant cells. Cyclin B1 knockdown by siRNA in parental cells induced T-DM1 resistance, while increased levels of cyclin B1 by silencing cdc20 partially sensitized resistant cells. In a series of 18 HER2-positive breast cancer fresh explants, T-DM1 effects on proliferation and apoptosis paralleled cyclin B1 accumulation.Conclusions: Defective cyclin B1 induction by T-DM1 mediates acquired resistance in HER2-positive breast cancer cells. These results support the testing of cyclin B1 induction upon T-DM1 treatment as a pharmacodynamic predictor in HER2-positive breast cancer. Clin Cancer Res; 23(22); 7006-19. ©2017 AACR.

Dabrafenib in an elderly patient with metastatic melanoma and BRAF V600R mutation: a case report.

BACKGROUND: Approximately 50 % of malignant melanomas harbor activating point mutations in the BRAF gene. Typically, these mutations result in the substitution of the amino acid valine at codon 600 of the gene, and 90-95 % of mutations are either BRAF (V600E) or BRAF (V600K). Specific BRAF inhibitors such as dabrafenib and vemurafenib are the mainstays of treatment in patients with metastatic BRAF-mutant malignant melanomas. The third most common BRAF mutation is V600R, which also leads to increased BRAF signaling. Although evidence exists about the activity of dabrafenib and vemurafenib in patients with the BRAF (V600R) mutation, these patients have been systematically excluded from recent trials with targeted therapies. CASE PRESENTATION: Here, we report the positive results in terms of survival and quality of life obtained with dabrafenib in an 80-year-old Caucasian male patient with a Charlson Comorbidity Index of 8 diagnosed with metastatic malignant melanoma harboring the BRAF (V600R) mutation. Our patient was treated with dabrafenib for 7 months with minimal toxicity. We also report exploratory analyses of circulating tumor DNA during targeted treatment. Interestingly, the mutation was not detected after starting treatment and became detectable before radiological disease progression. CONCLUSIONS: Our report suggests that (1) a relevant benefit can be obtained with a BRAF inhibitor in real-world patients with a malignant melanoma harboring a BRAF (V600R) mutation, and that (2) circulating tumor DNA detection might be of help in assessing tumor burden in everyday clinical practice. The results reported here should encourage the inclusion of patients with BRAF (V600R)-mutated malignant melanomas in future prospective clinical trials with BRAF inhibitors.

Cytoplasmic accumulation of NCoR in malignant melanoma: consequences of altered gene repression and prognostic significance.

Invasive malignant melanoma (MM) is an aggressive tumor with no curative therapy available in advanced stages. Nuclear corepressor (NCoR) is an essential regulator of gene transcription, and its function has been found deregulated in different types of cancer. In colorectal cancer cells, loss of nuclear NCoR is induced by Inhibitor of kappa B kinase (IKK) through the phosphorylation of specific serine residues. We here investigate whether NCoR function impacts in MM, which might have important diagnostic and prognostic significance. By IHC, we here determined the subcellular distribution of NCoR in a cohort of 63 primary invasive MM samples, and analyzed its possible correlation with specific clinical parameters. We therefore used a microarray-based strategy to determine global gene expression differences in samples with similar tumor stage, which differ in the presence of cytoplasmic or nuclear NCoR. We found that loss of nuclear NCoR results in upregulation of a specific cancer-related genetic signature, and is significantly associated with MM progression. Inhibition of IKK activity in melanoma cells reverts NCoR nuclear distribution and specific NCoR-regulated gene transcription. Analysis of public database demonstrated that inactivating NCoR mutations are highly prevalent in MM, showing features of driver oncogene.

Vías de señalización intracelular en el carcinoma escamoso infiltrante de cérvix uterino. Identificación de nuevos marcadores pronósticos / Intracellular pathways in infiltrating squamous cell carcinoma of the cervix. Identification of new prognostic markers

Antecedentes y objetivos: Los receptores de tirosincinasa están frecuentemente activados en los tumores humanos malignos. Esta activación lleva a las células a altos niveles de proliferación, migración, desdiferenciación, diseminación y resistencia a la apoptosis. Las vías de se˜nalización dependientes de la activación de los receptores de la familia ErbB/HER en las que se han descrito alteraciones son las vías RAS-RAF-ERK, PIK3-AKT, MAPK y NFKB. El estudio de la actividad de estas vías puede ayudar a identificar aquellos tumores con alta agresividad y puede informar acerca de puntos de interés con potencial terapéutico. Material y métodos: Realizamos estudio inmunohistoquímica de EGFR, p-EGFR, p38, p-ERK ½, JNK, p-AKT, p65, p50, p52, relB, c-Rel y MAPK-1 en 32 carcinomas escamosos infiltrantes de cérvix uterino en una matriz de tejidos. Resultados: Solamente la inmunoexpresión de p-ERK mostró correlación con el estadio de la enfermedad (p < 0,001) y la recidiva (p < 0,001). Además, la inmunoexpresión de MKP1 se correlacionaba inversamente con los niveles de inmunoexpresión de p-JNK (p = 0,036) y p-p38 (p = 0,011), indicando que MKP1 podría tener actividad anti-apoptótica. Esta hipótesis es reforzada por la demostración de la correlación entre la detección de MKP y la mala respuesta al tratamiento (quimioterapia y radioterapia). Conclusiones: Nuestras observaciones indican que los tumores más agresivos muestran inmunoexpresión alta de p-ERK y MKP1 y bajos niveles de p-JNK y p-p38, estos últimos favoreciendo la resistencia a la quimioterapia. La inmunoexpresión de p-ERK y MKP1 podrían usarse como factores pronósticos y dianas terapéuticas en estos tumores (AU)

Background: Tyrosine-kinase receptors are frequently activated in malignant human tumours. This activation results in high levels of proliferation, migration, dedifferentiation, dissemination and resistance to apoptosis of the tumour cells. Alterations in the following signalling pathways related to ErbB/HER: RAS-RAF-ERK, PIK3-AKT, MAPK, and NFKB have been described. Studying these signaling pathways could help identify more aggressive tumours and thus provide information about potential changes in therapy. Material and methods: We performed an immunohistochemical study of EGFR, p-EGFR, p38, p-ERK ½, JNK, p-AKT, p65, p50, p52, relB, c-Rel and MAPK-1 in 32 infiltrating squamous cell carcinomas of the uterine cervix using a tissue array. Results: Only p-ERK immunoexpression correlated with the stage of the disease (P < .001) and the relapse (P < .001). Furthermore, the MKP1 immunoexpression inversely correlated with the immunoexpression of p-JNK (P = .036) and p-p38 (P = .011) levels, thereby indicating that MKP1 could exert anti-apoptotic activity. This hypothesis has been further reinforced by the recently reported correlation between MKP detection and poor response to treatment (chemo- and radiotherapy) (AU)

Targeting epithelial-to-mesenchymal transition with Met inhibitors reverts chemoresistance in small cell lung cancer.

PURPOSE: Met receptor phosphorylation is associated with poor prognosis in human small cell lung cancer (SCLC). The aim of our work was to investigate the effects of hepatocyte growth factor (HGF)/Met-mediated epithelial-to-mesenchymal transition (EMT) in SCLC and to evaluate the role of Met inhibition in mesenchymal/chemorefractory SCLC models. EXPERIMENTAL DESIGN: SCLC models of HGF-induced EMT were evaluated in vitro and in vivo (subcutaneous xenografts in BALB/c nude mice) for chemosensitivity and response to Met inhibition with PF-2341066 (crizotinib). Human SCLC samples at diagnosis (N = 87) and relapse (N = 5) were evaluated by immunohistochemistry and immunofluorescence for EMT markers and Met status and these were correlated with patient outcome. RESULTS: We identified that the activation of the Met receptor through HGF induced expression of mesenchymal markers, an aggressive phenotype, and chemoresistance. Blockade of this process with the Met inhibitor resensitized cells to chemotherapy in vitro and in vivo. Moreover, mesenchymal markers in human SCLC specimens were associated with Met activation, predicted worse survival, and were upregulated in chemorefractory disease. CONCLUSION: These results provide novel evidence on an important role of Met-dependent EMT in the adverse clinical behavior of SCLC and support clinical trials of Met inhibitors and chemotherapy in this fatal disease.

PI3K signaling pathway is activated by PIK3CA mRNA overexpression and copy gain in prostate tumors, but PIK3CA, BRAF, KRAS and AKT1 mutations are infrequent events.

The phosphatidylinositol 3-kinase (PI3K)-AKT and RAS-MAPK pathways are deregulated in a wide range of human cancers by gain or loss of function in several of their components. Our purpose has been to identify genetic alterations in members of these pathways in prostate cancer. A total of 102 prostate tumors, 79 from prostate cancer alone (group G1) and 23 from bladder and prostate cancer patients (G2), are the subject of this study. In 20 of these 23, the bladder tumors were also analyzed. PIK3CA, KRAS, BRAF and AKT1 mutations were analyzed by direct sequencing, and BRAF also by pyrosequencing. PIK3CA quantitative mRNA expression and fluorescence in situ hybridization (FISH) gains were tested in 25 and 32 prostate tumors from both groups (G1 and G2), respectively. Immunohistochemistry for pAKT was performed in 55 prostate tumors. Of 25 prostate tumors, 10 (40%) had PIK3CA mRNA overexpression that was statistically associated with Gleason score ≥ 7 (P=0.018). PIK3CA copy gain was detected in 9 of 32 (28%) prostate tumors. Of 20 bladder tumors, 3 (15%) displayed mutations in PIK3CA, KRAS and AKT1, the corresponding prostate tumors being wt. We also detected a previously not reported PIK3CA polymorphism (IVS9+91) in two prostate tumors. In all, 56% of prostate tumors overexpressed pAKT. There is a statistical association (P<0.0001) of strong pAKT immunostaining with high Gleason score, and with PIK3CA alterations (mRNA overexpression and/or FISH gains). PIK3CA gene is deregulated by mRNA overexpression and DNA gain in ∼ 40 and 28% of prostate tumors, respectively. High-grade prostate tumors are associated with PIK3CA mRNA overexpression, but not with FISH status. PIK3CA, BRAF, KRAS and AKT1 mutations are very infrequent events in prostate tumors. However, PI3K signaling pathway is activated by PIK3CA FISH gain and/or mRNA overexpression, leading to an increased pAKT protein expression.

Dysplastic nevus: the eye of the hurricane.

Dysplastic nevi were generally recognized, thanks to the contributions of Clark et al. in 1978. These lesions were described in a familial context, which was called the 'B-K mole syndrome'. However, it is worth noting that this was not the first time that these nevi had been described in the literature. If we look back in history, we can find that in 1820, Norris had already described some very similarly pigmented lesions, also in a familial context, just as Cawley subsequently did in 1952. Clark coined the term of dysplastic nevi for lesions presenting in patients with personal and family histories of malignant melanoma, having from 10 to 100 nevus lesions of a certain size, irregular shape and variable pigmentation of more than 5 mm. In addition, he pointed out that histologically, such lesions were principally characterized by the presence of atypical melanocytic hyperplasia.

Grading of atypia in nevi: correlation with melanoma risk.

Nevi with architectural disorder and cytologic atypia of melanocytes (NAD), aka "dysplastic nevi," have varying degrees of histologic abnormalities, which can be considered on a spectrum of grades of atypia. Somewhat controversial and subjective criteria have been developed for grading of NAD into three categories "mild," "moderate," and "severe." Grading involves architectural and cytological features, which often correlate with each other. Architectural criteria were intraepidermal junctional extension beyond any dermal component, complex distortion of rete ridges, and dermal fibrosis. Cytological criteria were based on nuclear size, dispersion of chromatin, prominence of nucleoli, hyperchromasia and variation in nuclear staining. Few tests have been made of the relationship between specific grades of atypia and patient risk for melanoma. Retrospective review of pathology reports was performed on 20,275 nevi examined between 1989 and 1996. From the total, 6,275 were diagnosed as NAD, which were in 4,481 patients. These patients were divided into those whose worst NAD was mild (2,504), moderate (1,657), or severe (320). Review of accession data revealed that a personal history of melanoma was present in 5.7% of patients with mild, 8.1% with moderate, and 19.7% with severe atypia. The male/female ratios were similar in each group. In the three groups, the mean ages of men were similar and of women were similar, but the mean age of men tended to be 6-11 yrs. older than women in each group. Family histories of melanoma were not considered. The odds ratio as a measure of association between NAD and personal history of melanoma, shows an odds ratio of 4.08 (2.91-5.7) for NAD-severe versus NAD mild, odds ratio 2.81 (2-3.95) for NAD-severe versus NAD-moderate and odds ratio 1.45 (1.13-1.87) for NAD moderate versus NAD-mild. These data show that the probability of having personal history of melanoma, for any given NAD patient, correlates with the NAD grade. Likewise, the risk of melanoma is greater for persons who tend to make nevi with high grade histological atypia.

Expression of the proteoglycans versican and mel-CSPG in dysplastic nevi.

Nevi with architectural disorder and cytologic atypia of melanocytes (NAD) (also called dysplastic nevi) have been controversial with regard to their relationship with melanoma risk and to their gradation in 3 degrees of atypia. Versican and the melanoma-associated proteoglycan (mel-CSPG) are 2 major proteoglycans expressed by malignant melanoma, and they have a role in the regulation of cell adhesion, migration, and differentiation. We evaluated the differences in versican and mel-CSPG expression in nevi, NAD with several degrees of atypia, and primary malignant melanoma. Immunoreactivity for versican was negative in benign melanocytic nevi, positive in NAD (ranging from weakly to intensely positive), and intensely positive in malignant melanoma. Immunostaining for mel-CSPG was negative in benign melanocytic nevi and mild to moderately positive in NAD and melanoma. Our results suggest that versican expression may be of value for distinguishing NAD from benign melanocytic nevi and for distinguishing severe NAD from mild and moderate NAD.

Versican is differentially expressed in human melanoma and may play a role in tumor development.

Undifferentiated human melanoma cell lines produce a large chondroitin sulfate proteoglycan, different from the well-known melanoma-specific proteoglycan mel-PG (Heredia and colleagues, Arch Biochem Biophys, 333: 198-206, 1996). We have identified this proteoglycan as versican and analyzed the expression of versican in several human melanoma cell lines. Versican isoforms are expressed in undifferentiated cell lines but not in differentiated cells, and the isoform expression pattern depends on the degree of cell differentiation. The V0 and V1 isoforms are found on cells with an early degree of differentiation, whereas the V1 isoform is present in cells with an intermediate degree of differentiation. We have also characterized some functional properties of versican on human melanoma cells: the purified proteoglycan stimulates cell growth and inhibits cell adhesion when cells are grown on fibronectin or collagen type I as substrates, and thus may facilitate tumor cell detachment and proliferation. Furthermore, we have analyzed the expression of versican in human melanocytic nevi and melanoma: 10 benign melanocytic nevi, 10 dysplastic nevi, 11 primary malignant melanomas, and 8 metastatic melanomas were tested. Immunoreactivity for versican was negative in benign melanocytic nevi, weakly to strongly positive in dysplastic nevi, and intensely positive in primary malignant melanomas and metastatic melanomas. Our results indicate that versican is involved in the progression of melanomas and may be a reliable marker for clinical diagnosis.