DataSheet_1_CD34+DNAM-1brightCXCR4+ haemopoietic precursors circulate after chemotherapy, seed lung tissue and generate functional innate-like T cells and NK cells.docx

BackgroundThere is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation.ObjectiveTo study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients, to characterize their entry into involved lung tissue and to characterize their progenies.MethodsFlow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments.ResultsSignificant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1, CXCR3, CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic, can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production.ConclusionIn advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment, enter involved tissues, generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment.</p

Image_2_CD34+DNAM-1brightCXCR4+ haemopoietic precursors circulate after chemotherapy, seed lung tissue and generate functional innate-like T cells and NK cells.jpeg

BackgroundThere is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation.ObjectiveTo study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients, to characterize their entry into involved lung tissue and to characterize their progenies.MethodsFlow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments.ResultsSignificant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1, CXCR3, CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic, can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production.ConclusionIn advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment, enter involved tissues, generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment.</p

A] c-Myc iperexpression (2+); B] absence of expression of c-Myc.

<p>A] c-Myc iperexpression (2+); B] absence of expression of c-Myc.</p

Image_1_CD34+DNAM-1brightCXCR4+ haemopoietic precursors circulate after chemotherapy, seed lung tissue and generate functional innate-like T cells and NK cells.jpeg

BackgroundThere is little information on the trajectory and developmental fate of Lin-CD34+DNAM-1bright CXCR4+ progenitors exiting bone marrow during systemic inflammation.ObjectiveTo study Lin-CD34+DNAM-1bright CXCR4+ cell circulation in cancer patients, to characterize their entry into involved lung tissue and to characterize their progenies.MethodsFlow cytometric analysis of PBMC from 18 patients with lung cancer on samples collected immediately before the first and the second treatment was performed to study Lin-CD34+DNAM-1bright CXCR4+ precursors. Precursors were purified (>99%) and cultured in vitro from all patients. Paired PBMC and tissue samples from patients undergoing tumor resection were analyzed by flow cytometry to assess tissue entry and compare phenotype and developmental potential of Lin-CD34+DNAM-1bright CXCR4+ cells in both compartments.ResultsSignificant circulation of Lin-CD34+DNAM-1bright CXCR4+ precursors was observed 20d after the first treatment. Precursors express CXC3CR1, CXCR3, CXCR1 consistent with travel towards inflamed tissues. Flowcytometric analysis of lung tissue samples showed precursor presence in all patients in tumor and neighboring uninvolved areas. Successful purification and in vitro culture from both blood and lung tissue generates a minor proportion of maturing NK cells (85%) of α/β T-progenies with innate-like phenotype expressing NKG2D,NKp30,DNAM-1. Innate-like maturing T-cells in vitro are cytotoxic, can be triggered via NKR/TCR co-stimulation and display broad spectrum Th1,Th2 and Th1/Th17 cytokine production.ConclusionIn advanced stage lung cancer CD34+DNAM-1brightCXCR4+ inflammatory precursors increase upon treatment, enter involved tissues, generate functional progenies and may thus represent an additional player contributing to immune balance in the highly SDF-1/CXCR4-biased pro-metastatic tumor microenvironment.</p

Patients’ characteristics, according to clinicopathological and molecular variables.

<p>G: grade; TUR: trans-urethral resection; IHC: immunohistochemistry. Among 46 cancer, 36 showed urothelial, 2 squamous, 2 sarcomatoid, 2 micropapillary, 1 squamo-glandular, 1 small cell, 1 nested, 1 plasmocytoid differentiation.</p><p>Patients’ characteristics, according to clinicopathological and molecular variables.</p

Univariate analysis for Overall Survival (OS).

<p>vs: versus; G: grade; IHC: immunohistochemistry.</p><p>Univariate analysis for Overall Survival (OS).</p

A] strong positive beta-3-tubulin expression versus absence cores B) 2+ beta-3-tubulin expression.

<p>A] strong positive beta-3-tubulin expression versus absence cores B) 2+ beta-3-tubulin expression.</p

Univariate analysis for Event Free Survival (EFS).

<p>vs: versus; G: grade; IHC: immunohistochemistry.</p><p>Univariate analysis for Event Free Survival (EFS).</p

Identification and Validation of Protein Biomarkers of Response to Neoadjuvant Platinum Chemotherapy in Muscle Invasive Urothelial Carcinoma

<div><p>Background</p><p>The 5-year cancer specific survival (CSS) for patients with muscle invasive urothelial carcinoma of the bladder (MIBC) treated with cystectomy alone is approximately 50%. Platinum based neoadjuvant chemotherapy (NAC) plus cystectomy results in a marginal 5-10% increase in 5-year CSS in MIBC. Interestingly, responders to NAC (</p><p>Methods</p><p>mRNA expression data from a prior report on a NAC-treated MIBC cohort were re-analyzed in conjunction with the antibody database of the Human Protein Atlas (HPA) to identify candidate protein based biomarkers detectable by immunohistochemistry (IHC). These candidate biomarkers were subsequently tested in tissue microarrays derived from an independent cohort of NAC naive MIBC biopsy specimens from whom the patients were treated with neoadjuvant gemcitabine cisplatin NAC and subsequent cystectomy. The clinical parameters that have been previously associated with NAC response were also examined in our cohort.</p><p>Results</p><p>Our analyses of the available mRNA gene expression data in a discovery cohort (n = 33) and the HPA resulted in 8 candidate protein biomarkers. The combination of GDPD3 and SPRED1 resulted in a multivariate classification tree that was significantly associated with NAC response status (Goodman-Kruskal γ = 0.85 p<0.0001) in our independent NAC treated MIBC cohort. This model was independent of the clinical factors of age and clinical tumor stage, which have been previously associated with NAC response by our group. The combination of both these protein biomarkers detected by IHC in biopsy specimens along with the relevant clinical parameters resulted in a prediction model able to significantly stratify the likelihood of NAC resistance in our cohort (n = 37) into two well separated halves: low-26% n = 19 and high-89% n = 18, Fisher’s exact p = 0.0002).</p><p>Conclusion</p><p>We illustrate the feasibility of translating a gene expression signature of NAC response from a discovery cohort into immunohistochemical markers readily applicable to MIBC biopsy specimens in our independent cohort. The results from this study are being characterized in additional validation cohorts. Additionally, we anticipate that emerging somatic mutations in MIBC will also be important for NAC response prediction. The relationship of the findings in this study to the current understanding of variant histologic subtypes of MIBC along with the evolving molecular subtypes of MIBC as it relates to NAC response remains to be fully characterized.</p></div

Global 5-Hydroxymethylcytosine Levels Are Profoundly Reduced in Multiple Genitourinary Malignancies

<div><p>Solid tumors are characterized by a plethora of epigenetic changes. In particular, patterns methylation of cytosines at the 5-position (5mC) in the context of CpGs are frequently altered in tumors. Recent evidence suggests that 5mC can get converted to 5-hydroxylmethylcytosine (5hmC) in an enzymatic process involving ten eleven translocation (TET) protein family members, and this process appears to be important in facilitating plasticity of cytosine methylation. Here we evaluated the global levels of 5hmC using a validated immunohistochemical staining method in a large series of clear cell renal cell carcinoma (n = 111), urothelial cell carcinoma (n = 55) and testicular germ cell tumors (n = 84) and matched adjacent benign tissues. Whereas tumor-adjacent benign tissues were mostly characterized by high levels of 5hmC, renal cell carcinoma and urothelial cell carcinoma showed dramatically reduced staining for 5hmC. 5hmC levels were low in both primary tumors and metastases of clear cell renal cell carcinoma and showed no association with disease outcomes. In normal testis, robust 5hmC staining was only observed in stroma and Sertoli cells. Seminoma showed greatly reduced 5hmC immunolabeling, whereas differentiated teratoma, embryonal and yolk sack tumors exhibited high 5hmC levels. The substantial tumor specific loss of 5hmC, particularly in clear cell renal cell carcinoma and urothelial cell carcinoma, suggests that alterations in pathways involved in establishing and maintaining 5hmC levels might be very common in cancer and could potentially be exploited for diagnosis and treatment.</p></div