R Perego

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells

Summary Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

Genome-wide screening of copy number alterations and LOH events in renal cell carcinomas and integration with gene expression profile.

BackgroundClear cell renal carcinoma (RCC) is the most common and invasive adult renal cancer. For the purpose of identifying RCC biomarkers, we investigated chromosomal regions and individual genes modulated in RCC pathology. We applied the dual strategy of assessing and integrating genomic and transcriptomic data, today considered the most effective approach for understanding genetic mechanisms of cancer and the most sensitive for identifying cancer-related genes.ResultsWe performed the first integrated analysis of DNA and RNA profiles of RCC samples using Affymetrix technology. Using 100K SNP mapping arrays, we assembled a genome-wide map of DNA copy number alterations and LOH areas. We thus confirmed the typical genetic signature of RCC but also identified other amplified regions (e.g. on chr. 4, 11, 12), deleted regions (chr. 1, 9, 22) and LOH areas (chr. 1, 2, 9, 13). Simultaneously, using HG-U133 Plus 2.0 arrays, we identified differentially expressed genes (DEGs) in tumor vs. normal samples. Combining genomic and transcriptomic data, we identified 71 DEGs in aberrant chromosomal regions and observed, in amplified regions, a predominance of up-regulated genes (27 of 37 DEGs) and a trend to clustering. Functional annotation of these genes revealed some already implicated in RCC pathology and other cancers, as well as others that may be novel tumor biomarkers.ConclusionBy combining genomic and transcriptomic profiles from a collection of RCC samples, we identified specific genomic regions with concordant alterations in DNA and RNA profiles and focused on regions with increased DNA copy number. Since the transcriptional modulation of up-regulated genes in amplified regions may be attributed to the genomic alterations characteristic of RCC, these genes may encode novel RCC biomarkers actively involved in tumor initiation and progression and useful in clinical applications.

The possible influences of B2A2 and B3A2 BCR/ABL protein structure on thrombopoiesis in chronic myeloid leukaemia

The Philadelphia chromosome, t(9;22)(q34;q11) gives rise more frequently, in chronic myeloid leukaemia (CML), to two BCR/ABL chimeric transcripts differing only by the absence of 75 nucleotides and defined as b2a2 and b3a2 types, encoding two 210-kDa tyrosine kinase proteins differing only by the absence of 25 amino acids coded by the b3 exon. In the present study the two transcripts, detected by RT-PCR in 88 consecutive unselected CML patients, were correlated with haematological findings at diagnosis and with the megakaryocyte size and frequency by morphometric evaluation of 45 bone marrow biopsies. The secondary structure prediction and hydrophobicity of the b2a2 and b3a2 type BCR/ABL protein were also obtained. The prediction results for the b3 exon amino acids using GOR IV and NnPredict methods showed a short beta strand corresponding to the hydrophobic portion of the peptide. Significantly higher values were found in the platelet count of patients carrying b3a2 transcripts. The megakaryocyte size and frequency in bone marrow biopsies did not show significant differences between the two groups of patients. Stratifying the patients on the basis of white blood cell (WBC) count below or above 100x10(9)/l we still had, in both groups, a significant difference in the platelet count between the b2a2 and b3a2 patients. The possible relationships between the structure of b2a2 and b3a2 types of BCR/ABL fused protein and thrombopoiesis are discussed.

Human urine biomarkers of renal cell carcinoma evaluated by ClinProt

Renal cell carcinoma (RCC) is one of the major causes of cancer death and is radio‐ and chemoresistant. Urine of 29 healthy subjects and 39 clear cell RCC patients were analyzed using the ClinProt technique to search for possible biomarkers for early RCC diagnosis. A cluster of three signals (marker A= at m/z 1827 ± 8 Da, marker B = 1914 ± 8 Da and marker C = 1968 ± 8 Da) was able to discriminate patients from controls. A receiver operating characteristic curve analysis showed values of area under the curve (AUC) higher than 0.9 for marker A and B, corresponding to a sensitivity of 85–90% and a specificity of 90%, while marker C gave a lower AUC (0.84) corresponding to sensitivity of 70% and specificity of 100%. The combination of three markers lead to an improvement in diagnostic efficacy, with specificity and sensitivity of 100% and 95%, respectively, in the training test and of 100% and of 85% in the test experiment. The efficacy of this cluster of signals to distinguish RCC patients grouped by tumor stage showed a sensibility of 100% for patients at the primary tumor 1 stage. One of the signals present in the cluster was identified as a fragment of Tamm‐Horsfall protein.

The effects of sex and method of blood pressure measurement on genetic associations with blood pressure in the PAMELA study.

Background Phenotypic accuracy and specificity are essential for a successful genetic association study. Blood pressure (BP) measurements show heterogeneity depending on the method and time of measurement, sexual dimorphism and measurement errors, making genetic dissection difficult. Methods and results We studied 1550 adults aged 25–74 years, not on any antihypertensive treatment, resident in Monza, Italy (PAMELA study) all of whom had home, clinic and ambulatory BPs measured. We analysed 3705 single nucleotide polymorphisms (SNPs) (1324 typed and 2381 imputed) across 168 genes for association with these traits. No SNP achieved an experiment wide significance level of P less than 3 × 10−4 for any of the phenotypes studied. We selected 28 top candidate SNPs for further analysis of phenotypic heterogeneity and sexual dimorphism using a gene-centric strategy calculating empirical P values by permutations within each gene by including genic SNPs with an r2 less than 0.5. The association signals were not consistent across all the BP phenotypes, whether compared by genes or by physiological pathways. The top SNPs in WNK1, ADRA1A, ADRA1B, DRD1, NOS1 and PON3 showed significant sex interaction for BP and when analysed separately by sex showed evidence of dimorphism with opposite direction of effect for the same allele in the two sexes. Conclusion In the largest study of its kind, we show that sex and BP measurement methods have a significant impact on association signals. These findings might explain previous inconsistencies in studies on cardiovascular candidate genes and should have major implications for the design and interpretation of association studies.

Primary Cell Cultures from Human Renal Cortex and Renal-Cell Carcinoma Evidence a Differential Expression of Two Spliced Isoforms of Annexin A3

Primary cell cultures from renal cell carcinoma (RCC) and normal renal cortex tissue of 60 patients have been established, with high efficiency (more than 70%) and reproducibility, and extensively characterized. These cultures composed of more than 90% of normal or tumor tubular cells have been instrumental for molecular characterization of Annexin A3 (AnxA3), never extensively studied before in RCC cells although AnxA3 has a prognostic relevance in some cancer and it has been suggested to be involved in the hypoxia-inducible factor-1 pathway. Western blot analysis of 20 matched cortex/RCC culture lysates showed two AnxA3 protein bands of 36 and 33 kDa, and two-dimensional Western blot evidenced several specific protein spots. In RCC cultures the 36-kDa isoform was significantly down-regulated and the 33-kDa isoform up-regulated. Furthermore, the inversion of the quantitative expression pattern of two AnxA3 isoforms in tumor cultures correlate with hypoxia-inducible factor-1alpha expression. The total AnxA3 protein is down-regulated in RCC cultures as confirmed also in tissues by tissue microarray. Two AnxA3 transcripts that differ for alternative splicing of exon III have been also detected. Real-time PCR quantification in 19 matched cortex/RCC cultures confirms the down-regulation of longer isoform in RCC cells. The characteristic expression pattern of AnxA3 in normal and tumor renal cells, documented in our primary cultures, may open new insight in RCC management.

Serum biomarkers of Renal Cell Carcinoma assessed using a protein profiling approach based on ClinProt technique

OBJECTIVES To investigate the possibility of using the ClinProt technique to find serum cancer related diagnostic markers that are able to better discriminate healthy subjects from patients affected by renal cell carcinoma (ccRCC). Renal cell carcinoma is the most common malignancy of the kidney. Biomarkers for early detection, prognosis, follow-up, and differential diagnosis of ccRCC from benign renal lesions are needed in daily clinical practice when imaging is not helpful. METHODS Serum of 29 healthy subjects and 33 ccRCC patients was analyzed by the ClinProt/MALDI-ToF technique. RESULTS A cluster of 3 peptides (A = m/z 1083 +/- 8 Da, B = m/z 1445 +/- 8 Da and C = m/z 6879 +/- 8 Da) was able to discriminate patients from control subjects. Cross-validation analysis using the whole casistic showed 88% and 96% of sensitivity and specificity, respectively. Moreover, the cluster showed 100% sensitivity for the identification of patients at pT2 (n = 5) and pT3 (n = 8) and 85% for pT1 patients (n = 20). The intensity of peaks A and C continuously decreased from pT1 to pT3, whereas peak B increased in pT1 and pT2. CONCLUSIONS These results may be useful to set up new diagnostic or prognostic tools.

PKHhigh cells within clonal human nephrospheres provide a purified adult renal stem cell population

The existence and identification of adult renal stem cells is a controversial issue. In this study, renal stem cells were identified from cultures of clonal human nephrospheres. The cultured nephrospheres exhibited the activation of stem cell pathways and contained cells at different levels of maturation. In each nephrosphere the presence of 1.12-1.25 cells mirroring stem cell properties was calculated. The nephrosphere cells were able to generate three-dimensional tubular structures in 3D cultures and in vivo. In clonal human nephrospheres a PKH(high) phenotype was isolated using PKH26 epifluorescence, which can identify quiescent cells within the nephrospheres. The PKH(high) cells, capable of self-renewal and of generating a differentiated epithelial, endothelial and podocytic progeny, can also survive in vivo maintaining the undifferentiated status. The PKH(high) status, together with a CD133(+)/CD24(-) phenotype, identified a homogeneous cell population displaying in vitro self-renewal and multipotency capacity. The resident adult renal stem cell population isolated from nephrospheres can be used for the study of mechanisms that regulate self-renewal and differentiation in adult renal tissue as well as in renal pathological conditions.

Concentration and microsatellite status of plasma DNA for monitoring patients with renal carcinoma

We verified the feasibility of plasma bound method for detecting renal cell carcinoma (RCC) combining the study of plasma DNA concentration and microsatellite alterations (LOH). Plasma DNA concentration was evaluated with real-time PCR in 54 patients with renal neoplasm before surgery and in 20 of these patients during a 26-64 month follow-up. Microsatellite study was performed on tumour tissue DNA of 33 RCC clear cell (RCCcc) and on plasma DNA of 14 RCCcc patients during preoperative and/or follow-up period. Patients had a significantly high (26.4+/-48.3 ng/ml versus controls 3.2+/-1.5 ng/ml; p=0.003) preoperative plasma DNA concentration that decreased after nephrectomy. During follow-up, plasma DNA increased in 12 patients without evidence of neoplasia; 3 patients successively relapsed. Tumour tissue DNA of 25 RCCcc patients (75.8%) displayed microsatellite LOH. Preoperative plasma DNA of 9 patients harboured LOH in 5 cases (55.6%). Augmented plasma DNA of 7 patients displayed LOH in 3 cases (42.9%) at follow-up, and in 1 case preceded the recurrence of disease. Plasma DNA concentration combined with microsatellite LOH in plasma DNA may predict disease recurrence in RCC patients.

Drop of connexin 43 in replicative senescence of human fibroblasts HEL-299 as a possible biomarker of senescence

The expression of connexin 43 (cx43) and cell-cell communication were studied in replicative senescence of cultured HEL-299 fibroblasts. A progressive decrease in fluorescent dye transfer was detected by a scrape-loading technique in aging fibroblasts. This change was accounted for by a marked decrease in the amount of cx43 in aging cells, as detected by western blot analysis (cell extracts) and indirect fluorescence (cells in culture). However, semiquantitative RT-PCR assays of cx43 mRNA did not reveal appreciable changes, which suggests several possible explanations for the mechanism(s) underlying the decrease of cx43 in aging cells. These findings support the idea that the reduced expression of cx43 might be a biomarker of cell senescence.