Myelofibrosis progression grading based on type I and type III collagen and fibrillin 1 expression boosted by whole slide image analysis.

AIMS: The progression of primary myelofibrosis is characterised by ongoing extracellular matrix deposition graded based on 'reticulin' and 'collagen' fibrosis, as revealed by Gomori's silver impregnation. Here we studied the expression of the major extracellular matrix proteins of fibrosis in relation to diagnostic silver grading supported by image analysis. METHODS AND RESULTS: By using automated immunohistochemistry, in this study we demonstrate that the expression of both types I and III collagens and fibrillin 1 by bone marrow stromal cells can reveal the extracellular matrix scaffolding in line with myelofibrosis progression as classified by silver grading. 'Reticulin' fibrosis indicated by type III collagen expression and 'collagen' fibrosis featured by type I collagen expression were parallel, rather than sequential, events. This is line with the proposed role of type III collagen in regulating type I collagen fibrillogenesis. The uniformly strong fibrillin 1 immune signals offered the best inter-rater agreements and the highest statistical correlations with silver grading of the three markers, which was robustly confirmed by automated whole slide digital image analysis using a machine learning-based algorithm. The progressive up-regulation of fibrillin 1 during myelofibrosis may result from a negative feedback loop as fibrillin microfibrils sequester TGF-ß, the major promoter of fibrosis. This can also reduce TGF-ß-induced RANKL levels, which would stimulate osteoclastogenesis and thus can support osteosclerosis in advanced myelofibrosis. CONCLUSIONS: Through the in-situ detection of these extracellular matrix proteins, our results verify the molecular pathobiology of fibrosis during myelofibrosis progression. In particular, fibrillin 1 immunohistochemistry, with or without image analysis, can complement diagnostic silver grading at decent cell morphology.

Exploring Differential Connexin Expression across Melanocytic Tumor Progression Involving the Tumor Microenvironment.

The incidence of malignant melanoma, one of the deadliest cancers, continues to increase. Here we tested connexin (Cx) expression in primary melanocytes, melanoma cell lines and in a common nevus, dysplastic nevus, and thin, thick, and metastatic melanoma tumor progression series involving the tumor microenvironment by utilizing in silico analysis, qRT-PCR, immunocyto-/histochemistry and dye transfer tests. Primary melanocytes expressed GJA1/Cx43, GJA3/Cx46 and low levels of GJB2/Cx26 and GJC3/Cx30.2 transcripts. In silico data revealed downregulation of GJA1/Cx43 and GJB2/Cx26 mRNA, in addition to upregulated GJB1/Cx32, during melanoma progression. In three melanoma cell lines, we also showed the loss of GJA1/Cx43 and the differential expression of GJB1/Cx32, GJB2/Cx26, GJA3/Cx46 and GJC3/Cx30.2. The dominantly paranuclear localization of connexin proteins explained the ~10⁻90 times less melanoma cell coupling compared to melanocytes. In melanocytic tumor tissues, we confirmed the loss of Cx43 protein, fall of cell membrane and elevated paranuclear Cx32 with moderately increased cytoplasmic Cx26 and paranuclear Cx30.2 positivity during tumor progression. Furthermore, we found Cx43, Cx26 and Cx30 proteins upregulated in the melanoma adjacent epidermis, and Cx43 in the tumor flanking vessels. Therefore, differential connexin expression is involved in melanocytic tumor progression where varying connexin isotypes and levels reflect tumor heterogeneity-related bidirectional adaptive interactions with the microenvironment.

Exosomes in colorectal carcinoma formation: ALIX under the magnifying glass.

Exosomes are small membrane vesicles that have important roles in transporting a great variety of bioactive molecules between epithelial compartment and their microenvironment during tumor formation including colorectal adenoma-carcinoma sequence. We tested the mRNA expression of the top 25 exosome-related markers based on ExoCharta database in healthy (n=49), adenoma (n=49) and colorectal carcinoma (n=49) patients using Affymetrix HGU133 Plus2.0 microarrays. Most related genes showed significantly elevated expression including PGK1, PKM, ANXA5, ENO1, HSP90AB1 and MSN during adenoma-carcinoma sequence. Surprisingly, the expression of ALIX (ALG 2-interacting protein X), involved in multivesicular body (MVB) and exosome formation, was significantly reduced in normal vs adenoma (P=5.02 × 10(-13)) and in normal vs colorectal carcinoma comparisons (P=1.51 × 10(-10)). ALIX also showed significant reduction (P<0.05) at the in situ protein level in the epithelial compartment of adenoma (n=35) and colorectal carcinoma (n=37) patients compared with 27 healthy individuals. Furthermore, significantly reduced ALIX protein levels were accompanied by their gradual transition from diffuse cytoplasmic expression to granular signals, which fell into the 0.6-2 µm diameter size range of MVBs. These ALIX-positive particles were seen in the tumor nests, including tumor-stroma border, which suggest their exosome function. MVB-like structures were also detected in tumor microenvironment including α-smooth muscle actin-positive stromal cells, budding off cancer cells in the tumor front as well as in cancer cells entrapped within lymphoid vessels. In conclusion, we determined the top aberrantly expressed exosome-associated markers and revealed the transition of diffuse ALIX protein signals into a MVB-like pattern during adenoma-carcinoma sequence. These tumor-associated particles seen both in the carcinoma and the surrounding microenvironment can potentially mediate epithelial-stromal interactions involved in the regulation of tumor growth, metastatic invasion and therapy response.

Altered crosstalk in the dipeptidyl peptidase-4-incretin-immune system in type 1 diabetes: A hypothesis generating pilot study.

Both GLP1(7)(-)(36) (via GLP1 receptor) and the dipeptidyl peptidase-4 (DPP4) cleaved form of GLP1 (GLP1(9)(-)(36), independently of GLP1R) may modulate the response of lymphocytes to cytokine stimuli. The incretin axis, CXCR3 (receptor of DPP4 ligand cytokines CXCL9-11) expression on T(reg)s and hematologic parameters were assessed in 34 patients with long standing type 1 diabetes (T1DM) and in 35 healthy controls. Serum DPP4 (sDPP4) activity, plasma total GLP1 and GLP1(7)(-)(36) concentrations were determined. GLP1(9)(-)(36) concentrations were calculated. CXCR3 expression (flow cytometry) was higher on the CD25(-/)(low)Foxp3(+) than on the CD25(+)Foxp3(+) T(reg)s independently from T1DM, suggesting that CD25(-/)(low)Foxp3(+) T(reg)s are possibly waiting for orientational chemotactic stimuli in a "standby mode". The higher sDPP4 activities in T1DM were inversely correlated with GLP1(7)(-)(36) levels and GLP1(9)(-)(36) levels directly with lymphocyte counts in controls. Our results might indicate an altered DPP4-incretin system and altered immunoregulation including a potentially dysfunctional GLP1(9)(-)(36) signaling in T1DM.

Promoter hypermethylation-related reduced somatostatin production promotes uncontrolled cell proliferation in colorectal cancer.

BACKGROUND: Somatostatin (SST) has anti-proliferative and pro-apoptotic effects. Our aims were to analyze and compare the SST expression during normal aging and colorectal carcinogenesis at mRNA and protein levels. Furthermore, we tested the methylation status of SST in biopsy samples, and the cell growth inhibitory effect of the SST analogue octreotide in human colorectal adenocarcinoma cell line. METHODS: Colonic samples were collected from healthy children (n1 = 6), healthy adults (n2 = 41) and colorectal cancer patients (CRCs) (n3 = 34) for SST mRNA expression analysis, using HGU133 Plus2.0 microarrays. Results were validated both on original (n1 = 6; n2 = 6; n3 = 6) and independent samples ((n1 = 6; n2 = 6; n3 = 6) by real-time PCR. SST expressing cells were detected by immunohistochemistry on colonic biopsy samples (n1 = 14; n2 = 20; n3 = 23). The effect of octreotide on cell growth was tested on Caco-2 cell line. SST methylation percentage in biopsy samples (n1 = 5; n2 = 5; n3 = 9) was defined using methylation-sensitive restriction enzyme digestion. RESULTS: In case of normal aging SST mRNA expression did not alter, but decreased in cancer (p < 0.05). The ratio of SST immunoreactive cells was significantly higher in children (0.70% ± 0.79%) compared to CRC (0% ± 0%) (p < 0.05). Octreotide significantly increased the proportion of apoptotic Caco-2 cells. SST showed significantly higher methylation level in tumor samples (30.2% ± 11.6%) compared to healthy young individuals (3.5% ± 1.9%) (p < 0.05). CONCLUSIONS: In cancerous colonic mucosa the reduced SST production may contribute to the uncontrolled cell proliferation. Our observation that in colon cancer cells octreotide significantly enhanced cell death and attenuated cell proliferation suggests that SST may act as a regulator of epithelial cell kinetics. The inhibition of SST expression in CRC can be epigenetically regulated by promoter hypermethylation.

The potential prognostic value of connexin 26 and 46 expression in neoadjuvant-treated breast cancer.

BACKGROUND: Several classification systems are available to assess pathological response to neoadjuvant chemotherapy in breast cancer, but reliable biomarkers to predict the efficiency of primary systemic therapy (PST) are still missing. Deregulation of gap junction channel forming connexins (Cx) has been implicated in carcinogenesis and tumour progression through loss of cell cycle control. In this study we correlated Cx expression and cell proliferation with disease survival and pathological response to neoadjuvant chemotherapy in breast cancers using existing classification systems. METHODS: The expression of Cx26, Cx32, Cx43, Cx46 and Ki67 was evaluated in 96 breast cancer patients prior to and after neoadjuvant chemotherapy using duplicate cores in tissue microarrays (TMA). Cx plaques of <1µm were detected with multilayer, multichannel fluorescence digital microscopy. Current classifications to assess residual tumour burden after primary systemic therapy included the EWGBSP, CPS-EG, Miller-Payne, Sataloff and NSABP systems. RESULTS: In our cohort dominated by hormone receptor (ER/PR) positive and HER2 negative cases, only the CPS-EG classification showed prognostic relevance: cases with scores 1-2 had significantly better overall survival (p=0.015) than cases with scores 3-5. Pre-chemotherapy Cx43 expression correlated positively with hormone receptor status both before and after chemotherapy and had a negative correlation with HER2 expression pre-chemotherapy. There was a positive correlation between Cx32 and HER2 expression pre-chemotherapy and between Cx32 and Ki67 expression post-chemotherapy. A negative correlation was found between post-chemotherapy Cx46 and Ki67 expression. Decreased post-chemotherapy Cx26 expression (<5%) statistically correlated with better overall survival (p=0.011). Moderate or higher Cx46 expression (>20%) pre- and post-chemotherapy correlated with significantly better survival in the intermediate prognostic subgroups of EWGBSP TR2b (p(pre-chemo)=0.006; Sataloff TB (p(pre-chemo)=0.005; p(post-chemo)=0.029) and in Miller-Payne G3 (p(pre-chemo)=0.002; p(post-chemo)=0.012) classifications. Pre-chemotherapy, Cx46 expression was the only marker that correlated with overall survival within these subgroups. CONCLUSION: Our results suggest that Cx46 and Cx26 expression in breast cancer may improve the assessment of pathological response and refine intermediate prognostic subgroups of residual tumour classifications used after neoadjuvant chemotherapy.

Genome-wide screening of genes regulated by DNA methylation in colon cancer development.

Tumorigenesis is accompanied by changes in the DNA methylation pattern. Our aim was to test a novel approach for identification of transcripts at whole transcript level which are regulated by DNA methylation. Our approach is based on comparison of data obtained from transcriptome profiling of primary human samples and in vitro cell culture models. Epithelial cells were collected by LCM from normal, adenoma, and tumorous colonic samples. Using gene expression analysis, we identified downregulated genes in the tumors compared to normal tissues. In parallel 3000 upregulated genes were determined in HT-29 colon adenocarcinoma cell culture model after DNA demethylation treatment. Of the 2533 transcripts showing reduced expression in the tumorous samples, 154 had increased expression as a result of DNA demethylation treatment. Approximately 2/3 of these genes had decreased expression already in the adenoma samples. Expression of five genes (GCG, NMES-1, LRMP, FAM161B and PTGDR), was validated using RT-PCR. PTGDR showed ambiguous results, therefore it was further studied to verify the extent of DNA methylation and its effect on the protein level. Results confirmed that our approach is suitable for genome-wide screening of genes which are regulated or inactivated by DNA methylation. Activity of these genes possibly interferes with tumor progression, therefore genes identified can be key factors in the formation and in the progression of the disease.

Increase of α-SMA(+) and CK (+) cells as an early sign of epithelial-mesenchymal transition during colorectal carcinogenesis.

Our aim was to examine cell transition events by detecting the frequency of intrapithelial α-smooth muscle actin (SMA)(+)/cytokeratin (CK)(+) cells during colorectal adenoma-carcinoma sequence, in relation to E-cadherin expression. Our further aim was to determine the proliferative activity of intraepithelial α-SMA(+) cells. Histologically healthy, adenoma, and colorectal cancer (CRC) biopsy samples were taken during routine colonoscopy and were included into tissue microarrays (TMAs). Slides immunostained for Ki-67, α-SMA, E-cadherin and pan-cytokeratin were digitalized and analyzed by using a digital microscope software. The proportion of α-SMA(+)/CK(+) cells was significantly higher in CRC samples (3.34 ± 1.01%) compared to healthy (1.94 ± 0.69%) or adenoma (1.62 ± 0.78%) samples (p < 0.01). E-cadherin expression negatively correlated with the number of α-SMA(+) cells. The majority of intraepithelial α-SMA(+) cells were in the proliferative phase. During tumor progression, the appearance of dot-like α-SMA staining in CK positive cells may indicate the initial phase of the epithelial-to-mesenchymal transition (EMT). The high proportion of intraepithelial α-SMA(+) proliferating cells may refer to their increased plasticity compared to differentiated cells. The negative correlation between E-cadherin and intraepithelial α-SMA expression suggests that EMT is facilitated by a loss of epithelial cell contact.

Higher serum DPP-4 enzyme activity and decreased lymphocyte CD26 expression in type 1 diabetes.

Dipeptidyl peptidase-4 (DPP-4) is involved in the metabolism of peptide hormones, T-cell activation and proliferation. In type 1 diabetes mellitus (T1DM) ß-cell destruction involves a number of dysregulated T-cells. Our aim was to assess the serum DPP-4 activity and the lymphocyte membrane bound CD26 expression in patients with type 1 diabetes and healthy controls. Ninety-eight (T1DM: 48, F/M = 20/28, mean age: 34.4y; control: 50, F/M = 39/11 mean age: 32,4y) individuals were included. Fasting serum DPP-4 enzymatic activity, plasma glucose (FPG), CD26 expression on CD3+, CD4+ and CD8+ lymphocytes, HbA1c and body mass index (BMI) were assessed. ICA and GAD antibodies were assessed in the T1DM group. DPP-4 enzymatic activity was determined by kinetic enzyme assay, ICA and GAD were assessed by ELISA. Determination of the CD26 expression on CD3+, CD4+ and CD8+ lymphocytes was performed by flow-cytometric analysis. We found higher serum DPP-4 activity (Mean: T1DM: 30.069 U/L, control: 22.62 U/L, p < 0.0001) and decreased CD26 lymphocyte expression on all lymphocyte subpopulations in T1DM. Fasting serum DPP-4 activity was independent from the ICA or GAD status of patients with T1DM. Here we first present that the serum DPP-4 activity is increased and the lymphocyte membrane bound CD26 expression is decreased in type 1 diabetes. Decreased lymphocyte membrane bound CD26 expression in T1DM might be a novel part of the T-lymphocyte regulatory dysfunction observed in type 1 diabetes mellitus. These results might provide some basis for the clinical implication of DPP-4 inhibition in patients with T1DM.

Serum dipeptidyl peptidase-4 activity in insulin resistant patients with non-alcoholic fatty liver disease: a novel liver disease biomarker.

BACKGROUND: In a cross-sectional study we studied the fasting serum DPP-4 enzymatic activity (sDPP-4) and the insulin resistance index (HOMA2-IR) in gliptin naïve patients with type 2 diabetes and in non-alcoholic fatty liver disease (NAFLD) and in healthy controls (CNTRL). METHODS AND FINDINGS: sDPP-4 was measured by kinetic assay in 39 NAFLD (F/M:19/20, mean age: 47.42 yrs) and 82 type 2 diabetes (F/M:48/34, 62.8 yrs) patients and 26 (F/M:14/12, 35.3 yrs) controls. Definition of T2D group as patients with type 2 diabetes but without clinically obvious liver disease created non-overlapping study groups. Diagnosis of NAFLD was based on ultrasonography and the exclusion of other etiololgy. Patients in T2D and NAFLD groups were similarly obese. 75 g CH OGTT in 39 NAFLD patients: 24-NGT, 4-IGT or IFG ("prediabetes"), 11-type 2 diabetes. HOMA2-IR: CNTRL: 1.44; T2D-group: 2.62 (p = 0.046 vs CNTRL, parametric tests); NAFLD(NGTonly): 3.23 (p = 0.0013 vs CNTRL); NAFLD(IFG/IGT/type 2 diabetes): 3.82 (p<0.001 vs CNTRL, p = 0.049 vs 2TD group). sDPP-4 activity was higher in NAFLD both with NGT (mean:33.08U/L) and abnormal glucose metabolism (30.38U/L) than in CNTRL (25.89U/L, p<0.001 and p = 0.013) or in T2D groups (23.97U/L, p<0.001 and p = 0.004). Correlations in NAFLD among sDPP-4 and ALT: r = 0.4637,p = 0.0038 and gammaGT: r = 0.4991,p = 0.0017 and HOMA2-IR: r = 0.5295,p = 0.0026 and among HOMA2-IR and ALT: r = 0.4340,p = 0.0147 and gammaGT: r = 0.4128,p = 0.0210. CONCLUSIONS: The fasting serum DPP-4 activity was not increased in T2D provided that patients with liver disease were intentionally excluded. The high serum DPP-4 activities in NAFLD were correlated with liver tests but not with the fasting plasma glucose or HbA1C supporting that the excess is of hepatic origin and it might contribute to the speedup of metabolic deterioration. The correlation among gammaGT, ALT and serum DPP-4 activity and also between serum DPP-4 activity and HOMA2-IR in NAFLD strongly suggests that serum DPP-4 activity should be considered as a novel liver disease biomarker.