Use of RNA-Seq and a Transgenic Mouse Model to Identify Genes Which May Contribute to Mutant p53-Driven Prostate Cancer Initiation.

We previously demonstrated that the Trp53-R270H mutation can drive prostate cancer (CaP) initiation using the FVB.129S4 (Trp53tm3Tyj/wt); FVB.129S (Nkx3-1tm3(cre)Mmswt) genetically engineered mouse model (GEM). We now validate this finding in a different model (B6.129S4-Trp53tm3.1Tyj/J mice) and use RNA-sequencing (RNA-Seq) to identify genes which may contribute to Trp53 R270H-mediated prostate carcinogenesis. Wildtype (Trp53WT/WT), heterozygous (Trp53R270H/WT), and homozygous mice (Trp53R270H/R270H) were exposed to 5 Gy irradiation to activate and stabilize p53, and thereby enhance our ability to identify differences in transcriptional activity between the three groups of mice. Mouse prostates were harvested 6 h post-irradiation and processed for histological/immunohistochemistry (IHC) analysis or were snap-frozen for RNA extraction and transcriptome profiling. IHC analyses determined that presence of the Trp53-R270H mutation impacts apoptosis (lower caspase 3 activity) but not cell proliferation (Ki67). RNA-Seq analysis identified 1378 differentially expressed genes, including wildtype p53 target genes (E.g., Cdkn1a, Bax, Bcl2, Kras, Mdm2), p53 gain-of-function (GOF)-related genes (Mgmt, Id4), and CaP-related genes (Cav-1, Raf1, Kras). Further understanding the mechanisms which contribute to prostate carcinogenesis could allow for the development of improved preventive methods, diagnostics, and treatments for CaP.

Predictive assessment of kidney functional recovery following ischemic injury using optical spectroscopy.

Functional changes in rat kidneys during the induced ischemic injury and recovery phases were explored using multimodal autofluorescence and light scattering imaging. The aim is to evaluate the use of noncontact optical signatures for rapid assessment of tissue function and viability. Specifically, autofluorescence images were acquired in vivo under 355, 325, and 266 nm illumination while light scattering images were collected at the excitation wavelengths as well as using relatively narrowband light centered at 500 nm. The images were simultaneously recorded using a multimodal optical imaging system. The signals were analyzed to obtain time constants, which were correlated to kidney dysfunction as determined by a subsequent survival study and histopathological analysis. Analysis of both the light scattering and autofluorescence images suggests that changes in tissue microstructure, fluorophore emission, and blood absorption spectral characteristics, coupled with vascular response, contribute to the behavior of the observed signal, which may be used to obtain tissue functional information and offer the ability to predict posttransplant kidney function.

Diagnosis and differential diagnosis of hepatic graft versus host disease (GVHD).

Graft versus host disease (GVHD) is a common complication following allogeneic hematopoietic cell transplantation (HCT) that typically manifests as injury to the skin, gastrointestinal mucosa, and liver. In some cases, hepatic GVHD may be histologically indistinguishable from other disorders such as infection and drug-induced liver injury (DILI). Additionally, clinical signs and symptoms are frequently confounded by the superimposed effects of pretransplant chemoradiotherapy, immunotherapy (IT) (targeted to the underlying malignancy), GVHD prophylaxis, and infection. Thus, careful attention to and correlation with clinical findings, laboratory values, and histologic features is essential for diagnosis. This review, aimed at the practicing pathologist, will discuss current clinical and histologic criteria for GVHD, the approach to diagnosis of hepatic GVHD, and features helpful for distinguishing it from other entities in the differential diagnosis.

Clonogenically Culturing and Expanding CD34+ Liver Cancer Stem Cells in Vitro.

A large number of cancer stem cells (CSCs) have been isolated and identified; however, none has been cultured in an unlimited manner in vitro without losing tumorigenicity and multipotency. In this study, we successfully clonogenically cultured a newly identified CD34+ liver CSC (LCSC) on feeder cells up to 22 passages (to date) without losing CSC property. Cloned CD34+ LCSC formed a round packed morphology and it could also be cryopreserved and recultured. Stem cell markers, CD34, CD117, and SOX2; normal liver stem cell markers, alpha fetoprotein, CK19, CK18, and OV6; putative CSC markers, CD44, CD133, EpCAM, and CD90; as well as CD31 were expressed in cloned CD34+ LCSC. SOX2 was the major factor in maintaining this LCSC before colonization, and interestingly, OCT4, SOX2, NAONG, Klf4, c-Myc, and Lin28 were upregulated in association with symmetric self-renewal for colony growth of CD34+ LCSC on feeder cells. Gene expression patterns of in vitro differentiation were consistent with our in vivo finding; furthermore, the tumorigenicity of cloned CD34+ LCSC was not different from uncloned CD34+ LCSC sorted from parental PLC. These results show that our cloned CD34+ LCSC maintained CSC property, including self-renewal, bipotency, and tumorigenicity after long-term culture, demonstrating that this LCSC can be cultured in an unlimited manner in vitro. Thus, establishing pure population of CSCs isolated from the patients will provide an opportunity to explore the mechanisms of tumorigenesis and cancer development, and to identify unique biomarkers presenting potential indicators of drug efficacy against CSCs for establishment of a novel strategy for cancer therapy.

Identification of cancer stem cell subpopulations of CD34(+) PLC/PRF/5 that result in three types of human liver carcinomas.

CD34(+) stem cells play an important role during liver development and regeneration. Thus, we hypothesized that some human liver carcinomas (HLCs) might be derived from transformed CD34(+) stem cells. Here, we determined that a population of CD34(+) cells isolated from PLC/PRF/5 hepatoma cells (PLC) appears to function as liver cancer stem cells (LCSCs) by forming HLCs in immunodeficient mice with as few as 100 cells. Moreover, the CD34(+) PLC subpopulation cells had an advantage over CD34(-) PLCs at initiating tumors. Three types of HLCs were generated from CD34(+) PLC: hepatocellular carcinomas (HCCs); cholangiocarcinomas (CC); and combined hepatocellular cholangiocarcinomas (CHCs). Tumors formed in mice transplanted with 12 subpopulations and 6 progeny subpopulations of CD34(+) PLC cells. Interestingly, progenies with certain surface antigens (CD133, CD44, CD90, or EPCAM) predominantly yielded HCCs. CD34(+) PLCs that also expressed OV6 and their progeny OV6(+) cells primarily produced CHC and CC. This represents the first experiment to demonstrate that the OV6(+) antigen is associated with human CHC and CC. CD34(+) PLCs that also expressed CD31 and their progeny CD31(+) cells formed CHCs. Gene expression patterns and tumor cell populations from all xenografts exhibited diverse patterns, indicating that tumor-initiating cells (TICs) with distinct antigenic profiles contribute to cancer cell heterogeneity. Therefore, we identified CD34(+) PLC cells functioning as LCSCs generating three types of HLCs. Eighteen subpopulations from one origin had the capacity independently to initiate tumors, thus functioning as TICs. This finding has broad implications for better understanding of the multistep model of tumor initiation and progression. Our finding also indicates that CD34(+) PLCs that also express OV6 or CD31 result in types of HLCs. This is the first report that PLC/PRF/5 subpopulations expressing CD34 in combination with particular antigens defines categories of HLCs, implicating a diversity of origins for HLC.

Is segmental enhancement inversion on enhanced biphasic MDCT a reliable sign for the noninvasive diagnosis of renal oncocytomas?

OBJECTIVE: The objective of our study was to retrospectively determine whether segmental enhancement inversion or other CT patterns seen at enhanced biphasic MDCT are predictive for the diagnosis of renal oncocytoma. MATERIALS AND METHODS: Twenty-nine patients with 32 oncocytomas diagnosed by either biopsy or resection who had undergone enhanced biphasic CT between January 2004 and March 2010 were included in this study. Biphasic CT scans were analyzed for the presence of segmental enhancement inversion. Segmental enhancement inversion was defined as a renal mass with two distinctive segments in which the attenuation of the segments changed between the two phases of CT. The masses were further characterized on imaging to determine if any imaging feature is predictive of renal oncocytoma. RESULTS: Of the 32 renal oncocytomas, 16 oncocytomas were eliminated from analysis. These masses were eliminated because they were larger than 4 cm (n = 4), the CT examinations were inadequate (n = 10), or the pathology results were questionable (n = 2). The remaining 16 tumors (mean size, 2.6 cm; range, 1.8-3.9 cm) were included in our study. Only two tumors showed distinct segments of variable degrees of enhancement, with one of those tumors having segmental enhancement inversion. Three masses had a central region of low density. The most common feature, identified in eight of the 16 oncocytomas, was a slightly heterogeneous mass that became homogeneous on the later phase of CT. Three oncocytomas had a homogeneous appearance on both phases. CONCLUSION: Contrary to a prior report, we did not find segmental enhancement inversion to be a characteristic enhancement pattern of small renal oncocytomas on biphasic MDCT. We found no specific features on biphasic CT that could be considered reliable and could strongly suggest the diagnosis of renal oncocytoma.

Knockout of toll-like receptor-2 attenuates both the proinflammatory state of diabetes and incipient diabetic nephropathy.

OBJECTIVE: Type 1 diabetes (T1DM) is a proinflammatory state and confers an increased risk for vascular complications. Toll-like receptors (TLR) could participate in diabetic vasculopathies. Whether TLR activation contributes to the proinflammatory state of T1DM and the pathogenesis of diabetic nephropathy remains unknown. METHODS AND RESULTS: We induced T1DM in TLR2 knockout mice (TLR2-/-) and wild-type littermates (C57BL/6J-WT) using streptozotocin (STZ). Fasting blood, peritoneal macrophages, and kidneys were obtained for flow cytometry, Western blot, microscopy, and cytokine assays at 6 and 14 weeks after induction of diabetes. Macrophage TLR2 expression and MyD88-dependent signaling were increased in diabetic mice (WT+STZ) compared with nondiabetic WT mice. These biomarkers were attenuated in diabetic TLR2-/- macrophages. WT+STZ mice showed increased kidney:body weight ratio due to cell hypertrophy, increased albuminuria, decreased kidney nephrin, podocin, and podocyte number and increased transforming growth factor-ß and laminin compared with WT mice. Nephrin, podocin, and podocyte number and effacement were restored, and transforming growth factor-ß and laminin levels were decreased in TLR2-/-+ STZ mice kidneys versus WT+STZ. Peritoneal and kidney macrophages were predominantly M1 phenotype in WT+STZ mice; this was attenuated in TLR2-/-+STZ mice. CONCLUSIONS: These data support a role for TLR2 in promoting inflammation and early changes of incipient diabetic nephropathy, in addition to albuminuria and podocyte loss.

Increased soluble leptin receptor levels in morbidly obese patients with insulin resistance and nonalcoholic fatty liver disease.

The adipocyte hormone, leptin has been demonstrated to have profibrogenic actions in vitro and in animal models. However, no correlation was found between plasma leptin levels and fibrosis stage in humans. Thus, our aim was to study whether soluble leptin receptor (SLR) or free leptin index (FLI; calculated as the ratio of leptin to SLR), may correlate better with the features of metabolic syndrome and with the histological grade and stage of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). We studied a population (n = 104) of morbidly obese patients undergoing bariatric surgery. Data including BMI, type 2 diabetes mellitus, hypertension, and hyperlipidemia were obtained. Plasma fasting leptin and SLR, fasting glucose and insulin were measured, and homeostasis model of assessment insulin resistance (HOMA(IR)) index and FLI were calculated. All patients had intraoperative liver biopsies. Leptin levels correlated with the BMI. The multiple regression analysis indicated that increasing HOMA and decreasing FLI were predictors of steatosis in the liver (P < 0.0003). SLR levels were positively correlated with the presence of diabetes mellitus and the stage of fibrosis. In conclusion, increased SLR levels in morbidly obese patients with diabetes are correlated with the stage of liver fibrosis, and may reflect progressive liver disease.

Experience with tacrolimus in children with steroid-resistant nephrotic syndrome.

Children with steroid-resistant nephrotic syndrome (SRNS) are at risk of developing renal failure. We report here the results of a single-center retrospective observational study of the remission rate in pediatric patients with SNRS receiving tacrolimus. Serial renal biopsies from children on tacrolimus therapy were evaluated for tubulointerstitial fibrosis and transforming growth factor-beta immunostaining. Of the 16 children with SRNS, 15 went into complete remission after a median of 120 days of therapy. Nine children were able to stop steroids, while the others were on tapering doses. Forty-seven percent had relapses, most of which were steroid-responsive. Serial renal biopsies were obtained from seven children after a median treatment duration of 24 months; two of these children had increased tubulointerstitial fibrosis and four showed increased transforming growth factor-beta tissue staining. Children with worsening histological findings were younger. There was no significant association between tacrolimus exposure and biopsy changes, although the average trough level was higher in those children with worsening histological findings. In conclusion, tacrolimus may be a safe and effective alternative agent for inducing remission in children with SRNS. However, caution needs to be taken when prescribing this agent due to its narrow therapeutic index. Serial renal biopsies are necessary to check for subclinical nephrotoxicity, especially in younger children and those with higher trough levels.

A prospective, blinded trial of touch prep analysis versus frozen section for intraoperative evaluation of sentinel lymph nodes in breast cancer.

BACKGROUND: Sentinel lymph node biopsy (SLNB) has largely replaced axillary dissection (ALND) for axillary staging in early breast cancer. However, intense pathologic evaluation is not routinely available intraoperatively; therefore, patients with SLN metastasis may require a second surgery for completion ALND. We hypothesized that a single-section approach (by either frozen section [FS] or touch preparation analysis [TPA]) could be accurate for intraoperative SLN evaluation. METHODS: We performed a prospective, blinded study of patients undergoing SLNB for breast cancer from September 2004 to July 2006. SLNs were bivalved along the long axis, underwent FS and TPA of the facing halves, followed by routine sentinel node processing (serial sectioning with hematoxylin/eosin staining). A single pathologist reviewed all study slides and was blinded to the permanent section interpretation. RESULTS: We analyzed 233 nodes from 118 patients. Overall, 21% of patients (N = 25) had SLN metastasis by serial-section histopathology. Single-section FS and TPA had similar sensitivities (0.67 and 0.66, P = .82) and specificities (0.995 and 0.995, P = 1.0) for detection of SLN metastasis, yielding equivalent accuracies (95%). All micrometastases (<2 mm; N = 4) were missed by both techniques. False positives were rare-only one in each group (2% overall). CONCLUSION: Single-section TPA and FS have similar accuracies and can be safely used to identify the majority of patients with SLN metastasis, sparing these patients a delayed ALND. False-negative results from TPA or FS occur in patients with micrometastatic disease, for which the role of completion ALND remains controversial.

A phase II trial of image-guided radiofrequency ablation of small invasive breast carcinomas: use of saline-cooled tip electrode.

BACKGROUND: Local ablative therapy of breast cancer represents the next frontier in the evolution of minimally-invasive breast conservation therapy. We performed this Phase II trial to determine the efficacy and safety of Radiofrequency (RF) ablation of small invasive breast carcinomas. METHODS: Seventeen patients with biopsy-proven invasive breast cancer, < or = 1.5 cm in diameter were enrolled in this trial. Under ultrasound guidance, the tumor and a 5 mm margin of surrounding breast tissue were ablated with saline-cooled RF electrode followed by surgical resection. Pathologic and immunohistochemical stains were performed to assess tumor viability. We examined whether loss of ER, PR receptor and pancytokeratin expression following RF ablation would correlate with non-viability. RESULTS: Fifteen patients completed the treatment. The mean tumor size was 1.28 cm. The mean ablation time was 21 minutes using a mean power of 35.5 watts. During ablation, the tumors became progressively echogenic that corresponded with the region of severe electrocautery injury at pathological examination. Of the 15 treated patients, NADPH viability staining was available for 14 patients and in 13 (92.8%), there was no evidence of viable malignant cells. ER, PR expression and pancytokeratin immunohistochemistry analysis were unreliable surrogates for determining non-viability. Following RF ablation, 2 patients developed skin puckering. CONCLUSIONS: RF ablation is a promising minimally invasive treatment of small breast carcinomas, as it can achieve effective cell killing with a low complication rate. Further research is necessary to optimize this image-guided technique and evaluate its future role as the sole local therapy.

Review of vascularized bone marrow transplantation: current status and future clinical applications.

In this review, we examine the applicability of the vascularized bone marrow transplant (VBMT) as an alternative to conventional bone marrow transplantation (BMT). As a new surgical approach, the VBMT is unique by transplantation of the stromal environment that eliminates the need for an engraftment period, provides critical signaling and modulatory functions, and may potentiate tolerance induction. Thus far, VBMT studies have demonstrated an absence of graft-versus-host disease (GVHD) and robust engraftment into nonmanipulated as well as irradiated recipients with evidence of immunological tolerance. Further investigation is needed to determine the applicability of VBMT as an alternative to BMT.

De novo nonalcoholic fatty liver disease after liver transplantation.

Hepatic steatosis is a recognized problem in patients after orthotopic liver transplant (OLT). However, de novo development of nonalcoholic fatty liver disease (NAFLD) has not been well described. The aim of this study was to determine the prevalence and predictors of de novo NAFLD after OLT. A retrospective analysis was performed on 68 OLT patients with donor liver biopsies and posttransplantation liver biopsies. Individual medical charts were reviewed for demographics, indication for OLT, serial histology reports, genotypes for hepatitis C, comorbid conditions, and medications. Liver biopsies were reviewed blindly and graded according to the Brunt Scoring System. Multivariate logistic regression analysis was used to study the risk factors for developing NAFLD. The interval time from OLT to subsequent follow-up liver biopsy was 28 +/- 18 months. A total of 12 patients (18%) developed de novo NAFLD, and 6 (9%) developed de novo NASH. The regression model indicated that the use of angiotensin-converting enzyme inhibitors (ACE-I) was associated with a reduced risk of developing NAFLD after OLT (odds ratio, 0.09; 95% confidence interval, 0.010-0.92; P = 0.042). Increase in body mass index (BMI) of greater than 10% after OLT was associated with a higher risk of developing NAFLD (odds ratio, 19.38; 95% confidence interval, 3.50-107.40; P = 0.001). In conclusion, de novo NAFLD is common in the post-OLT setting, with a significant association with weight gain after transplant. The use of an ACE-I may reduce the risk of developing post-OLT NAFLD.

Vascularized lymph node transplantation induces graft-versus-host disease in chimeric hosts.

BACKGROUND: The role of lymph nodes (LNs) in adaptive immune responses has been the subject of extensive research. In previous studies, the surgical removal of lymph nodes from rat hind limbs prevented the development of lethal graft-versus-host disease (GVHD) after allogeneic hind limb transplantation to chimeric recipient rats. The purpose of this study was to establish the role of the cellular fraction versus the microenvironment of LNs in the development of GVHD in this model. METHODS: A rat model for vascularized LN transplantation was developed and graft-versus-host responses were compared after: 1) naive ACI LN cells were infused into Wistar-Furth (WF) rats as chimeric recipients (e.g. [ACI-->WF]); 2) vascularized WF lymph nodes were transplanted to syngeneic WF recipients; 3) nonvascularized ACI lymph nodes were transplanted to [ACI-->WF] chimeric recipients; 4) vascularized ACI lymph nodes were transplanted to [ACI-->WF] chimeric recipients. RESULTS: Transplantation of vascularized ACI lymph nodes to [ACI-->WF] chimeric recipient rats resulted in severe and sometimes lethal GVHD. In contrast, neither the infusion of purified ACI LN cells nor the transplantation of nonvascularized LNs led to GVHD in chimeric recipients. CONCLUSIONS: When introducing allogeneic cells into chimeric recipients, concomitant transplantation of the vascularized LN microenvironment makes a manifest difference between induction and absence of GVHD. This illustrates the important role of the LN microenvironment in adaptive immune responses.

Liver hepcidin mRNA correlates with iron stores, but not inflammation, in patients with chronic hepatitis C.

PURPOSE: Liver iron is frequently elevated in chronic hepatitis C and may contribute to liver injury. The pathophysiology behind this phenomenon may involve hepcidin, a gene that is up-regulated in the liver by inflammation and iron. Inappropriately low hepcidin is important to the pathophysiology of hereditary hemochromatosis. However, the role of hepcidin in the iron loading of patients with hepatitis C is unknown. SUBJECTS AND METHODS: To determine whether liver hepcidin mRNA correlates with markers of hepatic inflammation and iron status in patients with hepatitis C, we extracted total RNA from liver biopsy specimens of patients with chronic hepatitis C and quantified hepcidin mRNA. Liver hepcidin mRNA levels were then correlated with aspartate aminotransferase, alanine aminotransferase, ferritin, viral load, fibrosis, hepatic iron concentration, and Hepatic Activity Index (HAI). RESULTS: Among patients with hepatitis C, there was a significant correlation of hepcidin mRNA expression in the liver with hepatic iron concentration and serum ferritin (r = 0.72, P = 0.006, and r = 0.60, P = 0.01, respectively). Hepcidin mRNA expression in the liver did not correlate with aspartate aminotransferase, alanine aminotransferase, HAI, or viral load. No differences in hepcidin mRNA were found based on viral genotype or the presence of fibrosis. CONCLUSION: In contrast to other inflammatory states, hepcidin mRNA expression in the liver was independent of markers of inflammation in hepatitis C. Instead, our results suggest that iron stores in patients with hepatitis C regulate hepcidin expression and that iron loading in chronic hepatitis C is not due to inappropriate hepcidin expression.

Occult pretransplantation systemic inflammation and posttransplantation vascular changes in a primate arterial allograft model.

BACKGROUND: Occult systemic inflammation, as manifested by increased levels of C-reactive protein (CRP), identify patients at increased risk for renal allograft rejection. The mechanisms linking occult systemic inflammation to these adverse outcomes remain unclear. The purpose of this study was to examine the anatomic and physiologic effects of occult pretransplantation systemic inflammation on posttransplantation allograft outcome in a nonhuman primate model. METHODS: Seventy-one healthy male Rhesus macaques were stratified into quartiles based on serum CRP. Five high quartile and six low quartile animals underwent common iliac artery transplantation from male donors. Duplex ultrasound measured graft flow at 3 weeks postoperatively; luminal narrowing was assessed by graft/femoral peak systolic velocity ratio. At 6 weeks, the grafts were harvested and morphometry studies were performed. Vessel wall changes were assessed by measuring the intimal medial area. RESULTS: The allografts placed in high CRP quartile animals had more luminal narrowing by 3 weeks than those placed in low quartile animals, as evidenced by a higher mean graft/femoral peak systolic velocity ratio (1.6 vs. 0.90, P=0.006). Morphometry studies after graft harvest showed increased vessel wall area in the high quartile group versus the low quartile group (1.39 mm vs. 1.03 mm, P=0.018). CONCLUSIONS: Occult pretransplantation systemic inflammation is associated with increased intimal thickening and stenosis after arterial allograft transplantation in a primate model. Additional studies are needed to confirm these results and to further investigate potential mechanisms linking pretransplantation systemic inflammation to adverse outcomes after transplantation.

Dermatofibrosarcoma protuberans: reappraisal of wide local excision and impact of inadequate initial treatment.

BACKGROUND: The extent of local invasion in dermatofibrosarcoma protuberans (DFSP) is often clinically difficult to appreciate, and this leads to inadequate resections. We examined the effect of inadequate initial treatment and the efficacy of wide resection. METHODS: We performed a retrospective analysis of the records of 35 patients with DFSP treated at our institution (1985 and 2001). Data were analyzed with Wilcoxon's ranked sum test and Fisher's exact test. RESULTS: Of the 24 patients eligible for analysis, 11 had definitive wide resection after diagnostic excisions elsewhere (primary group), and 13 had recurrent tumors after previous surgical treatment elsewhere (recurrent group). Twenty-three patients were treated with wide resection only, and adjuvant radiation was administered to one patient who had a fibrosarcoma. At a median follow-up of 54 months, patients definitively treated at our institution had a 100% local recurrence-free survival. In comparison to the primary group, recurrent DFSPs were significantly larger and deeper and occurred in the head and neck region. Five cases had bone involvement, and of these, 80% occurred in the recurrent group. CONCLUSIONS: Inadequate initial treatment results in larger, deeper recurrent lesions, but these can be managed by appropriate wide excision. Wide resection of DFSP (whether recurrent or primary) with negative histological margins predicts a superior local recurrence-free survival.