Molecular Gene Expression Profiling Research Articles

(1335) - Cardiac Magnetic Resonance Imaging can Differentiate Rejection on Molecular Gene Expression Profiling in Pediatric Heart Transplant Recipients

(1335) - Cardiac Magnetic Resonance Imaging can Differentiate Rejection on Molecular Gene Expression Profiling in Pediatric Heart Transplant Recipients

Identification of MYC-Driven High-Grade B-Cell Lymphoma Using Deep Learning-Based Whole Slide Image Analysis

Introduction High-grade B-cell lymphoma (HGBL) is an aggressive lymphoma that often harbors MYC rearrangements ( MYC-R) and molecular signatures attributed to aberrant MYC activation (Olszewski et al. Blood 2022). Identifying and classifying HGBL is challenging; current classification systems recognize diffuse large B cell lymphoma/HGBL with MYC and BCL2 rearrangements ( MYC-R/ BCL2-R; double-hit; defined molecularly) and HGBL-not otherwise specified (defined morphologically) in which MYC-R occur in up to 45% of cases and the double-hit signature (DHITsig) occurs in 54% of cases (Alaggio et al. Leukemia 2022, Campo et al. Blood 2022, Olszewski et al. Blood 2022). Existing methods for molecular classification, such as fluorescence in situ hybridization (FISH), are expensive, time-consuming and not widely available, and morphological classification is subjective and associated with high inter-reader variability (Natkunam et al. Histopathology 2023). Therefore, we applied a deep-learning approach to identify MYC-driven HGBL from whole-slide images (WSIs) of ubiquitously available hematoxylin and eosin-stained slides. Methods The model was trained on a real-world data set containing 600 WSIs from multiple sites using MYC-R status determined by FISH as a label. The model was evaluated on an independent test set comprised of 287 WSIs from the GOYA study (ClinicalTrials.gov identifier: NCT01287741). WSIs were scanned with a Roche Ventana DP200 scanner at 0.25 μm/px. Model architecture extracted cytological features from single cells and architectural features from larger tissue regions, enabling the quantification of high-grade morphology characterized by monomorphic sheets of dense cells with round, intermediately sized nuclei and finely dispersed chromatin. Model predictions were compared with MYC-R and BCL2-R with the immunoglobulin heavy chain locus (IGH), gene expression signatures (including DHITsig and cell of origin [COO]), gene mutation signatures obtained by next generation sequencing, computationally derived morphological measurements, and morphological evaluation by hematopathologists. MCD ( MYD88/CD79B mutations) and A53 ( TP53 mutations) genetic subtypes were determined by LymphGen. Statistical comparisons were made using Kendall's tau-b (τb) correlation for continuous variables and Fisher's exact test with conditional maximum likelihood estimated odds ratios (ORs) for categorical variables. Results In the test set, the model distinguished between cases with and without MYC-R (IGH) with an area under the receiver operating characteristic curve of 0.85, sensitivity of 0.95, specificity of 0.59, positive predictive value of 0.16, and negative predictive value of 0.99. The model correctly classified 21/22 MYC-R (IGH) cases and all four MYC-R/ BCL2-R (IGH) double-hit cases. Of DHITsig cases, the model classified 25/33 as MYC-driven HGBL. Model scores correlated with MYC expression (τb = 0.25; p < 0.0001) and Ki67 expression (τb = 0.15, p < 0.0001) ( Figure 1A). Tumors classified as MYC-driven HGBL were enriched for the non-germinal center B-cell-like COO (OR 2.0; p < 0.01) and the MCD genetic subtype (OR 1.9; p < 0.05), and depleted for the A53 genetic subtype (OR 0.3; p < 0.001). Additionally, enrichment was observed for MCD hallmark mutations in MYD88 (OR 1.9; p < 0.05), CD79b (OR 4.6; p < 0.001) and CDKN2A (OR 3.6; p < 0.0001) ( Figure 1A). Cell density (τb = 0.30; p < 0.0001), mean nuclei roundness (τb = 0.15; p < 0.001), coefficient of variation of nucleus roundness (τb = −0.19; p < 0.0001) and size (τb = −0.29; p < 0.0001), and mean nuclear pixel intensity (τb = −0.36; p < 0.0001) were associated with positive model predictions ( Figure 1A) corresponding to high-grade morphology. WSIs labeled as having high-grade cytomorphology by hematopathologists also had high model scores (p = 0.07; OR 3.5). Conclusions Our deep learning model, trained using MYC-R status, identified MYC-driven HGBL by enriching for cases with MYC-R, MYC overexpression, molecular gene expression profiles associated with aberrant MYC activation and cell proliferation, and high-grade morphology. The model also identified molecular characteristics other than MYC-R, such as MCD genetic subtype, associated with increased MYC expression (Schmitz et al. N Engl J Med 2018, Lacy et al. Blood 2020, Varano et al. Nature 2017), which converge on a common phenotype and high-grade morphology ( Figure 1B).

Effect of Prolonged Cold Ischemia on Intragraft Gene Expression Profiles

Background Deceased-donor (DD) kidneys are at higher risk for ischemia/reperfusion injury leading to increased inflammatory mediators and innate immune response. We aimed to investigate the effects of prolonged cold ischemia on intragraft molecular gene expression profiles of DD kidneys comparing to living-donors (LD) pre-implantation biopsies using microarrays. Methods There were 48 pre-implantation kidney biopsy samples (29 LD and 19 DD). The cold ischemia time (CIT) was < 16 hours in 10 and > 16 hours in 9 DD kidneys. The gene expression profiles were studied by Affymetrix HuGene 1.0 ST expression arrays. Results There were 198 differentially expressed genes when pre-implantation DD biopsies were compared to LD biopsies (Both False discovery rate p<0.05 and fold change >2). Pathogenesis based transcripts (PBT) showed increased transcripts associated with injury and response (IRIT), Constitutive Macrophage (CMAT), and Gamma Interferon (GRIT) in DD samples compared to pre-implantation LD biopsies. There was no difference in Cytotoxic T-cell (CAT), Natural-Killer cell (NKAT), B-cell (BAT), and Endothelial cell (ENDAT) associated gene transcripts between 2 groups. However, there was no statistically significant difference in expression of any PBTs studied when biopsies with CIT > 16 hours compared to biopsies with CIT < 16 hours. Gene Ontology analysis showed that DD preimplantation biopsies had increased expression of pathways related to acute inflammatory response, lymphocyte mediated immunity, innate and humoral immune response, complement activation and regulation of immune response and IL-6 activation. Conclusions Pre-implantation DD kidney biopsies showed increased expression of gene transcripts associated with inflammatory mediators, cytokines, macrophages, innate immune response and response to injury compared to LD kidneys. However, increased cold ischemia time does not change the intragraft gene expression profiles.

Dietary values of Forsythia suspensa extract in Penaeus monodon under normal rearing and Vibrio parahaemolyticus 3HP (VP3HP) challenge conditions: Effect on growth, intestinal barrier function, immune response and immune related gene expression

Two trials were conducted to determine the effects of dietary Forsythia suspensa extract (FSE) on shrimp, Penaeus monodon, first on growth performance, second on the immune response and immune related gene expression of shrimp. In trial 1, shrimp (mean initial wet weight about 3.02 g) were fed with five diets containing 0% (basal diet), 0.01%, 0.02%, 0.04% and 0.06% FSE in triplicate for 60 days. Growth performance (final body wet weight, FBW; weight gain, WG; biomass gain, BG) of shrimp fed FSE diets were higher (P < 0.05) than that of shrimp fed the basal diet. The survival among all the diets treatments were above 90% and no significant difference was revealed among them (P > 0.05). The antioxidant capacity (total antioxidant status, TAS; glutathione peroxidase, GSH-Px) appears in the trend of firstly increasing then decreasing with the increasing of dietary FSE levels. The highest value of TAS and GSH-Px were found in shrimp fed 0.02% FSE diet and were significantly higher than that of shrimp fed the basal and 0.06% FSE diets (P < 0.05). Hepatopancreas malondialdehyde (MDA) of shrimp fed FSE diets were lower (P < 0.05) than that of shrimp fed the basal diet. Total haemocyte count of shrimp fed the basal diet was lower (P < 0.05) than that of shrimp fed FSE diets. Haemolymph clotting time of shrimp had the opposite trend with the total haemocyte count of shrimp. No significant differences were found in haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) and in molecular gene expression profiles of heat shock protein 70 (Hsp 70) mRNA and hypoxia inducible factor-1α (HIF-1α) mRNA in haemolymph of shrimp among all diet treatments (P > 0.05). In trial 2, a pathogenic strain of Vibrio parahaemolyticus 3HP (VP3HP) injection challenge test was conducted for 6-day after the rearing trial and shrimp survival were also compared among treatments. Survival of shrimp fed diets supplemented with 0.01%–0.02% FSE were higher than that of shrimp fed the basal and 0.06% FSE diets (P < 0.05). Dietary FSE supplementation produced stronger hepatopancreas antioxidant capacity (TAS, GSH-Px) (P < 0.05) and higher glutathione (GSH) level (P < 0.05), lower superoxide dismutase activity (SOD) (P < 0.05), higher total haemocyte count (P < 0.05), lower haemolymph clotting time (P < 0.05), lower MDA and carbonyl protein concentration (P < 0.05), lower haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) (P < 0.05), generated lower molecular gene expression profiles of HSP 70 mRNA and higher HIF-1α mRNA (P < 0.05) than the basal diet. The immune response were characterized by lower TAS and higher antioxidant enzyme activities (SOD, GSH-Px) and higher oxidative stress level (MDA and carbonyl protein) and higher haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) compared to levels found in trail 1. However, the total haemocyte counts and haemolymph clotting times were not changed in 0.01%–0.02% FSE diets treatments between trial 1 and trial 2 (P > 0.05). The molecular gene expression profile of Hsp 70 mRNA was increased while HIF-1α mRNA was decreased when compared to trial 1. In conclusion, results suggested that dietary intake containing FSE could enhance the growth performance and antioxidant capacity of P. monodon and furthermore reduce oxidative stress and immune depression challenged by a pathogenic strain of Vibrio parahaemolyticus stress. Considering the effect of FSE on both growth performance and immune response of P. monodon, the level of FSE supplemented in the diet should be between 0.01% and 0.02%.

Rapid On Site Evaluation (ROSE) in Uveal Melanomas for Diagnostic Adequacy and Optimal Triaging for Molecular Gene Expression Profile (GEP) Testing: A Series Analysis of 67 Cases

Rapid On Site Evaluation (ROSE) in Uveal Melanomas for Diagnostic Adequacy and Optimal Triaging for Molecular Gene Expression Profile (GEP) Testing: A Series Analysis of 67 Cases

Systemic Monocytic-MDSCs Are Generated from Monocytes and Correlate with Disease Progression in Breast Cancer Patients.

Myeloid-derived suppressor cells (MDSCs) are highly immunosuppressive myeloid cells, which increase in cancer patients. The molecular mechanism behind their generation and function is unclear. Whereas granulocytic-MDSCs correlate with poor overall survival in breast cancer, the presence and relevance of monocytic-MDSCs (Mo-MDSCs) is unknown. Here we report for the first time an enrichment of functional blood Mo-MDSCs in breast cancer patients before they acquire a typical Mo-MDSC surface phenotype. A clear population of Mo-MDSCs with the typical cell surface phenotype (CD14+HLA-DRlow/-CD86low/-CD80low/-CD163low/-) increased significantly first during disease progression and correlated to metastasis to lymph nodes and visceral organs. Furthermore, monocytes, comprising the Mo-MDSC population, from patients with metastatic breast cancer resemble the reprogrammed immunosuppressive monocytes in patients with severe infections, both by their surface and functional phenotype but also at their molecular gene expression profile. Our data suggest that monitoring the Mo-MDSC levels in breast cancer patients may represent a novel and simple biomarker for assessing disease progression.

Effects of Prolonged Cold Ischemia On Intragraft Gene Expression Profiles.

Background: Deceased-donor (DD) kidneys are at higher risk for ischemia/reperfusion injury leading to increased inflammatory mediators and innate immune response. We aimed to investigate the effects of prolonged cold ischemia on intragraft molecular gene expression profiles of DD kidneys comparing to living-donors (LD) before and after transplantation using microarrays. Methods: There were 20 pre-implantation kidney biopsy samples (10 LD and 10 DD). The cold ischemia time (CIT) was < 24 hours in 7 and > 24 hours in 3 DD kidneys. There were 15 post-transplant biopsy samples from DD (8 had CIT < 24 h and 7 had > 24 h) with the diagnosis of acute tubular necrosis (ATN) which were compared to 9 normal transplant biopsies. The gene expression profiles were studied by Affymetrix HuGene 1.0 ST expression arrays. Results: There were 336 differentially expressed genes when pre-implantation DD biopsies were compared to LD biopsies (Both False discovery rate p<0.05 and fold change >2). Pathogenesis based transcripts (PBT) showed increased transcripts associated with injury and response (IRIT), Cytotoxic T-cell (CAT), Natural-Killer cell (NKAT), Constitutive Macrophage (CMAT), and Gamma Interferon (GRIT) in DD samples compared to pre-implantation LD biopsies. There was no difference in T-regulatory cell (TREG), B-cell (BAT), and Endothelial cell (ENDAT) associated gene transcripts between 2 groups. However, there was no statistically significant difference in expression of any PBTs studied when biopsies with CIT > 24 hours compared to biopsies with CIT < 24 hours. There was also no difference in expression of any PBTs studied in post-transplant DD biopsies with the diagnosis of ATN compared to normal transplant biopsies (Table, P-value for significance <0.05). Conclusions: Pre-implantation DD biopsies showed increased expression of gene transcripts associated with inflammatory mediators (IRIT and GRIT) and innate immune response involving NKAT and CAT compared to LD kidneys. However, increased CIT does not change the intragraft gene expression profiles before and after transplantation.Table: No Caption available.

Clinical, genomic, and imaging predictors of malignancy: Analysis of the first U.S. cooperative group prospective clinical trial in asymptomatic monoclonal gammopathies (SWOG S0120).

8515 Background: Asymptomatic monoclonal gammopathies (AMG) are the most common plasma cell dyscrasia classified as either monoclonal gammopathy of undetermined significance (MGUS) or asymptomatic multiple myeloma (AMM). Clinical outcome in AMGs can be highly variable and there is an unmet need to identify newer clinical, genomic and imaging parameters from prospective studies to guide patient management. Methods: We analyzed clinical, genomic and imaging data from AMG patients (n=334) enrolled in a prospective observational clinical trial (S0120) conducted under the auspices of SWOG. Baseline data from clinical variables, as well as gene expression profiles of purified tumor cells and the findings on magnetic resonance imaging (MRI) were correlated with the risk of progression to symptomatic myeloma (MM) requiring therapy. Results: In addition to serum M spike, percent bone marrow plasma cells and the ratio of involved/uninvolved serum free light chains, the level of serum beta-2-microglobulin was associated with an increased risk of progression to clinical myeloma requiring therapy. Gene expression profiles (GEP) of purified tumor cells revealed that all of the known molecular GEP subtypes of human MM are also represented in the precursor phase. An increased risk score (&gt; - 0.26) based on a 70-gene signature (GEP70) was an independent predictor of the risk of progression to clinical MM requiring therapy. The presence of focal lesions on MRI also conferred an increased risk of disease progression but was not independent of other clinical and genomic features. Conclusions: These data represent the first comprehensive evaluation of clinical, genomic and imaging features of AMGs in the context of a prospective US-cooperative group trial, and demonstrate that while all genetic subtypes of MM have a precursor phase, genetic signatures previously associated with high risk myeloma also predict the risk of progression to clinical malignancy requiring therapy. These findings suggest the need to integrate both clinical assessment of tumor bulk and genomic properties of tumor cells in the clinical management of these patients. Clinical trial information: NCT00900263.

Blinded Comparator Study of Immunohistochemical Analysis versus a 92-Gene Cancer Classifier in the Diagnosis of the Primary Site in Metastatic Tumors

Accurate tumor classification is fundamental to inform predictive biomarker testing and optimize therapy. Gene expression-based tests are proposed as diagnostic aids in cases with uncertain diagnoses. This study directly compared the diagnostic accuracy of IHC analysis versus molecular classification using a 92-gene RT-PCR assay for determination of the primary tumor site. This prospectively defined blinded study of diagnostically challenging cases included 131 high-grade, primarily metastatic tumors. Cases were reviewed and reference diagnoses established through clinical correlation. Blinded FFPE sections were evaluated by either IHC/morphology analysis or the 92-gene assay. The final analysis included 122 cases. The 92-gene assay demonstrated overall accuracy of 79% (95% CI, 71% to 85%) for tumor classification versus 69% (95% CI, 60% to 76%) for IHC/morphology analysis (P = 0.019). Mean IHC use was 7.9 stains per case (median, 8; range, 2 to 15). IHC/morphology analysis accuracy was 79%, 80%, and 46% when 1 to 6 (n = 42), 7 to 9 (n = 41), and >9 (n = 39) IHC stains were used, respectively, versus 81%, 85%, and 69%, respectively, with the 92-gene assay. Results from this blinded series of high-grade metastatic cases demonstrate superior accuracy with the 92-gene assay versus standard-of-care IHC analysis and strongly support the diagnostic utility of molecular classification in difficult-to-diagnose metastatic cancer.

M2/M1 ratio of tumor associated macrophages and PPAR-gamma expression in uveal melanomas with class 1 and class 2 molecular profiles

Macrophages have been found to be negative predictors of outcome in patients with uveal melanoma. In particular, recent studies point toward a disease-progressing role of proangiogenic M2 macrophages in melanomas with monosomy 3. Although most studies implicate a protective effect of PPAR-gamma activation in tumors, PPAR-gamma has also been shown to promote the polarization of M1 macrophages toward the M2 phenotype. The purpose of this investigation was first, to characterize the phenotype of tumor infiltrating macrophages and second, to study PPAR-gamma expression in uveal melanomas with molecular gene expression profile as prognostic predictors for patients' outcome. Twenty specimens from patients with uveal melanoma were analyzed for clinical and histologic tumor characteristics. The molecular RNA profile (class 1 or class 2) was commercially determined. Using immunohistochemical techniques, the specimens were dual labeled for CD68 and CD163. CD68 + CD163− M1 macrophages and CD68 + CD163+ M2 macrophages were analyzed in ten high power fields sparing macrophage-poor areas and a mean value was calculated for each tumor. The tumors were immunostained for von Willebrand factor and the micro vascular density (MVD) was analyzed according to Foss. To assess the proliferative rate of each tumor, Ki67 expression was evaluated in ten high power fields followed by calculation of a mean value. Expression of PPAR-gamma was evaluated using a score from 0 (no staining) to 3 (tumor entirely stained). Statistical analysis and a respective correlation were made between histologic characteristics, molecular profile, type of tumor infiltrating macrophages (M1 vs. M2), MVD, proliferative rate, and PPAR-gamma expression. Our results showed a correlation between the ratio of M2/M1 macrophages and the molecular profile with a ratio of approximately 1 corresponding to molecular class 1 and a ratio of approximately 2 corresponding to molecular class 2 (p = 0.01). The ratio of M2/M1 macrophages was higher in tumors with extraocular extension (p = 0.01). PPAR-gamma was predominantly expressed in the cytoplasm of tumor cells. Its expression showed no association with the molecular RNA profile (p = 0.83). This study confirmed that the ratio of M2/M1 macrophages is another prognostic factor in uveal melanoma. Thus, polarization of macrophages plays an important role for patients' outcome. PPAR-gamma is expressed in uveal melanoma tumor cells and further studies are warranted to determine its role in tumor biology.

Identification of Biomarkers to Assess Organ Quality and Predict Posttransplantation Outcomes

The increased disparity between organ supply and need has led to the use of extended criteria donors and donation after cardiac death donors with other comorbidities. We have examined the preimplantation transcriptome of 112 kidney transplant recipient samples from 100 deceased-donor kidneys by microarray profiling. Subject groups were segregated based on estimated glomerular filtration rate (eGFR) at 1 month after transplantation: the GFR-high group (n=74) included patients with eGFR 45 mL/min per 1.73 m(2), whereas the GFR-low group (n=35) included patients with eGFR 45 mL/min or less per 1.73 m(2). Gene expression profiling identified higher expression of 160 probe sets (140 genes) in the GFR-low group, whereas expression of 37 probe sets (33 genes) was higher in the GFR-high group (P<0.01, false discovery rate <0.2). Four genes (CCL5, CXCR4, ITGB2, and EGF) were selected based on fold change and P value and further validated using an independent set of samples. A random forest analysis identified three of these genes (CCL5, CXCR4, and ITGB2) as important predictors of graft function after transplantation. Inclusion of pretransplantation molecular gene expression profiles in donor quality assessment systems may provide the necessary information for better donor organ selection and function prediction. These biomarkers would further allow a more objective and complete assessment of procured renal allografts at pretransplantation time.

Molecular Gene Expression Profiling to Predict the Tissue of Origin and Direct Site-Specific Therapy in Patients With Carcinoma of Unknown Primary Site: A Prospective Trial of the Sarah Cannon Research Institute

Molecular tumor profiling is a promising diagnostic technique to determine the tissue of origin in patients with carcinoma of unknown primary site (CUP). However, the clinical value of these molecular predictions is unknown. We used tumor profiling results to direct site-specific therapy for patients with CUP. Tumor biopsy specimens from previously untreated patients with CUP were tested with a 92-gene reverse transcriptase polymerase chain reaction cancer classification assay. When a tissue of origin was predicted, patients who were treatment candidates received standard site-specific first-line therapy. Of 289 patients enrolled, 252 had successful assays performed, and 247 (98%) had a tissue of origin predicted. Sites most commonly predicted were biliary tract (18%), urothelium (11%), colorectal (10%), and non-small-cell lung (7%). Two hundred twenty-three patients were treatment candidates, and 194 patients received assay-directed site-specific treatment. In these 194 patients, the median survival time was 12.5 months (95% CI, 9.1 to 15.4 months). When the assay predicted tumor types that were clinically more responsive, the median survival was significantly improved when compared with predictions of more resistant tumors (13.4 v 7.6 months, respectively; P = .04). In this large prospective trial, molecular tumor profiling predicted a tissue of origin in most patients with CUP. The median survival time of 12.5 months for patients who received assay-directed site-specific therapy compares favorably with previous results using empiric CUP regimens. Patients with CUP predicted to have more responsive tumor types had longer survival compared with patients with less responsive tumor types. Molecular tumor profiling contributes to the management of patients with CUP and should be a part of their standard evaluation.

Molecular gene expression profiling to predict the tissue of origin and direct site-specific therapy in patients (pts) with carcinoma of unknown primary site (CUP): Results of a prospective Sarah Cannon Research Institute (SCRI) trial.

10530 Background: Tumor profiling is an emergent technique to determine tissue of origin in CUP patients. However, the value of these predictions in improving treatment efficacy is unknown. In this prospective trial, we used tumor profiling results to direct site-specific therapy for CUP pts. Methods: A 92-gene RT-PCR assay (CancerTYPE ID; bioTheranostics, Inc.) was performed on tumor biopsies from previously untreated CUP pts who consented. When a tissue of origin was predicted, pts who were treatment candidates were assigned standard site-specific first-line therapy. Results: Between 10/08 and 12/11, 289 pts were enrolled, 252 had successful assays performed, and 247 (98%) had a tissue of origin predicted. 224 pts were eligible for treatment; 197 pts received assay-directed treatment. 120 of 224 treated pts (54%) had assay diagnoses of tumor types known to derive substantial benefit from standard site-specific treatment (bladder 27, colorectal 26, NSCLC 24, breast 10, ovary 10, kidney 9, prostate 4, germ cell 4, others 6 (3 sites), while 104 pts (46%) had assay diagnoses of relatively resistant tumors (biliary tract 45, pancreas 12, gastroesophageal 10, liver 7, sarcoma 5, cervix 5, others 20 (8 sites). Median OS for all treated pts was 10.8 months (mos); OS for 197 pts with assay-directed treatment was 12.2 mos (versus 6.0 mos for 27 pts receiving empiric therapy). Median OS was better in the 120 pts with assay diagnoses of more responsive tumor types (12.8 vs 7.4 mos; p = .027). Median OS (mos) in specific subgroups: pancreas 9, kidney 12, colon 12, NSCLC 16, ovary 30. Conclusions: This is the first prospective trial in which molecular profiling has directed site-specific therapy in CUP pts. Assay-directed therapy in 197 pts produced a median OS (12.2 mos) that compares favorably with previous empiric CUP therapy. CUP pts predicted to have more responsive tumor types had longer survival compared to less responsive types, suggesting accurate identification by the assay. These results strengthen the rationale for molecular profiling in CUP management.

Differential Hematopoietic Supportive Potential and Gene Expression of Stroma Cell Lines from Midgestation Mouse Placenta and Adult Bone Marrow

During mouse embryogenesis, hematopoietic development takes place in several distinct anatomic locations. The microenvironment of different hematopoietic organs plays an important role in the proliferation and maturation of the hematopoietic cells. We hypothesized that fetal stromal cells would be distinct to adult bone marrow (BM)-derived stromal cells because the BM contributes mainly to the homeostasis of hematopoietic stem cells (HSCs), while extensive expansion of HSCs occurs during fetal development. Here we report the establishment of stromal cell lines from fetal hematopoietic organs, namely aorta-gonad-mesonephros (AGM), midgestation placenta (PL), and fetal liver (FL) together with adult bone marrow (BM). The growth patterns and hematopoietic supportive potential were studied. Their phenotypic and molecular gene expression profiles were also determined. Stromal cell lines from each tissue were able to support cobblestone area formation of BM c-Kit(+)Sca-1(+) hematopoietic cells: 22 (22/47) from AGM, three (3/4) from PL, three (3/4) from FL, and three (3/3) from BM. There were similar levels of expansion of total mononuclear cells (TMNs) when HSCs were cocultured with fetal stroma and adult BM stroma. However, PL-derived stromal cells supported higher levels of generation of colony-forming progenitor cell (CFU-C), indicated by more colonies and colonies with significantly larger size. Flow cytometric analysis of the PL1 cells demonstrated a phenotype of CD45(-), CD105(+), Sca-1(+), CD34(+), and CD49d(+), compared to adult BM1 cells, which were CD45(-), CD105(+), Sca-1(+), CD34(-), and CD49d(-). Using Affymetrix microarray analysis, we identified that genes specifically express in endothelial cells, such as Tie1, Tek, Kdr, Flt4, Emcn, Pecam1, Icam2, Cdh5, Esam1, Prom1, Cd34, and Sele were highly expressed in stroma PL1, consistent with an endothelial phenotype, while BM1 expressed a mesenchymal stromal phenotype. In summary, these data demonstrate distinct characteristics of stromal cells that provide insights into the microenvironmental control of HSCs.

Abstract A103: Breast cancer molecular subtype analysis in Egypt reveals high prevalence of Luminal A: Implications for improving prognosis

Abstract The World Health Organization has ranked breast cancer as the most common type of cancer among women world-wide. Breast cancer represents 37% of cancer cases in Egypt, and although incidence in Egypt remains lower than in highly developed countries, incidence rates are steadily increasing. Breast cancer is a heterogeneous disease composed of a number of recognizable subtypes. These intrinsic tumor subtypes, based on molecular gene expression profiles, reflect underlying biological behavior of tumors. The distribution of molecular subtypes in women of North African descent is currently unknown. Although parity is a contributing factor to the breast cancer dilemma in Egypt, understanding the underlying and unique molecular genetic factors is critical for tackling this disease in Egypt. Accordingly, our study examined the prevalence of breast cancer subtypes in Egypt in specimens obtained from medical centers in Cairo and Minya. We classified tumors according to an accepted IHC panel of surrogate markers for intrinsic subtype. Our study results demonstrated that in spite of younger age, larger tumor size, high tumor grade and high percentage of node positive cases, the Luminal A subtype was found in over 55% of cases examined. There was a surprisingly low prevalence of the basal-like subtype which was found in less than 10% of cases. Luminal B, HER2+/ER-and non-basal triple negative tumors also each constituted less than 15% of cases examined. Thus, although Egyptians with breast cancer have more aggressive disease at diagnosis and dismal survival rates, they appear to have favorable underlying biology. We feel this study will help raise awareness of the breast cancer problem in Egypt and aid the clinician in making better treatment and prognostic decisions based on a set of already known prognostic and therapeutic markers. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A103.

Relationship between prolonged QTc interval, cardiac allograft dysfunction and elevated molecular gene expression profiling test score after heart transplantation

Acute cellular cardiac allograft rejection after heart transplantation (HTx) is diagnosed histologically using endomyocardial biopsy (EMB), which is associated with limitations. 1 Marboe C.C. Billingham M. Eisen H. et al. Nodular endocardial infiltrates (Quilty lesions) cause significant variability in diagnosis of ISHLT Grade 2 and 3A rejection in cardiac allograft recipients. J Heart Lung Transplant. 2005; 24: S219-S226 Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar Using gene expression profiling (GEP) of peripheral blood mononuclear cells (PBMCs), a molecular classifier was developed and validated to rule out International Society of Heart and Lung Transplantation (ISHLT) moderate/severe (Grade ≥3A/2R) rejection, 2 Deng M.C. Eisen H.J. Mehra M.R. et al. Noninvasive discrimination of rejection in cardiac allograft recipients using gene expression profiling. Am J Transplant. 2006; 6: 150-160 Crossref PubMed Scopus (414) Google Scholar with a high negative predictive value of 99.6%. The corrected QT (QTc) interval has been described as a sensitive and specific tool to predict acute cellular cardiac allograft rejection. 3 Richartz B.M. Radovancevic B. Bologna M.T. et al. Usefulness of the QTc interval in predicting acute allograft rejection. Thorac Cardiovasc Surg. 1998; 46: 217-221 Crossref PubMed Scopus (18) Google Scholar We have recently shown that the GEP score correlates with the QTc interval during quiescence. 4 Cadeiras M. Shahzad K. John M.M. et al. Relationship between a validated molecular cardiac transplant rejection classifier and routine organ function parameters. Clin Transplant. 2009; (Aug 27. [Epub ahead of print]) PubMed Google Scholar In the present pilot study we hypothesized that both increased QTc interval and elevated GEP score are associated with allograft dysfunction, as defined by echocardiographic (TTE) criteria.

MRNA microarray analysis in lymphoma and leukemia.

We have reviewed the ability of gene expression microarrays to characterize subgroups of lymphoma and leukemia, identify expression profiles that correlate with known cytogenetic abnormalities, demonstrate that expression profiles can predict prognosis, new proteins identified for diagnosis and followup, and provided new therapeutic targets for chemotherapy. We can expect that new prognostic models will be designed and tested, incorporating the pathologic diagnosis based on morphology, the molecular gene expression profile, and the clinical assessment (e.g. International prognostic index). In addition, the gene expression profiles will be used to generate correlative and ultimately predictive data for response to particular chemotherapeutic regimens. Translation for clinical usage is likely in a diagnostic fashion in both lymphoma and leukemia.

In vivo analysis of cancerous gene expression by RNA-polymerase chain reaction.

An easy and routine procedure to amplify messenger RNA (mRNA) libraries from a few tissue cells can provide molecular gene expression profiles at high resolution. A novel PCR-like method, the RNA-PCR, was developed to generate high quality and quantity mRNAs from as few as 20 cells (2 pg mRNAs). The principle relies upon the cycling steps of promoter-linked double-stranded cDNA synthesis and promoter-driven transcription to amplify mRNAs up to 250-fold/cycle with good representation of high and low copy mRNAs. The amplified mRNA libraries were shown to possess high fidelity, purity, specificity and reproducibility for in vivo analyses of cancerous gene expression in human prostate cancers.