Safety and tolerability of bone marrow-derived mesenchymal stem cells in lupus animal models and a phase I clinical trial in humans.

OBJECTIVE: Several clinical trials aimed at treating various autoimmune diseases, including systemic lupus erythematosus (SLE), by introducing mesenchymal stem cells (MSCs) have been conducted. However, with refractory lupus nephritis (LN), the outcomes of MSC transplantation are not well known, and further validation is required. In particular, data concerning the safety and efficacy of LN treatment using bone marrow-derived MSCs (BM-MSCs) are still lacking. METHODS: We identified characteristics of BM-MSCs in terms of cell morphology, chromosomal stability, differentiation capacity, and phenotype through cell passages. The in vivo stability of BM-MSCs was evaluated by single-dose and repeated-dose toxicity tests, tumorigenicity tests, and biodistribution tests using lupus mouse models. Based on the encouraging nonclinical results, we conducted a nonrandomized, open-label, single-arm phase I clinical trial to evaluate the tolerability and safety of a single administration of haploidentical allogeneic BM-MSCs (CS20AT04) in seven LN patients (NCT03174587). We used a classical three + three design to find the optimal dosage. The starting dose was 2.0×106 cells/kg and escalated to 3.0×106 cells/kg if there was no dose-limiting toxicity (DLT). Evaluation of the safety and tolerability was assessed 28 days after the infusion, and the maximum tolerated dose was determined. RESULTS: Properly cultured BM-MSCs showed high proliferation and multipotency, but chromosomal changes were not found. There were two deaths by a rapid administration rate in the high-dose group (2.0×106 cells/head) in a single administration test. BM-MSCs were distributed in the kidneys until Day 7. In the phase I clinical trial, seven LN patients were enrolled. Participants received BM-MSCs through intravenous infusion. There was no DLT at both initial dose (2.0×106 cells/kg) and escalated dose (3.0×106 cells/kg). One patient was not administered the full 2.0×106 cells/kg dose because of a technical error during infusion. This patient did not show DLT. Three adverse events were reported, namely, one diarrhea, one toothache, and one arthralgia, and all were considered NCI-CTC grade I events. CONCLUSION: We defined the characteristics of BM-MSCs and identified their safety and tolerability in both animal models and a phase I clinical trial. The maximum tolerated dose was determined to be 3.0×106 cells/kg in patients with LN.

Human pancreatic cancer cell exosomes, but not human normal cell exosomes, act as an initiator in cell transformation.

Cancer evolves through a multistep process that occurs by the temporal accumulation of genetic mutations. Tumor-derived exosomes are emerging contributors to tumorigenesis. To understand how exosomes might contribute to cell transformation, we utilized the classic two-step NIH/3T3 cell transformation assay and observed that exosomes isolated from pancreatic cancer cells, but not normal human cells, can initiate malignant cell transformation and these transformed cells formed tumors in vivo. However, cancer cell exosomes are unable to transform cells alone or to act as a promoter of cell transformation. Utilizing proteomics and exome sequencing, we discovered cancer cell exosomes act as an initiator by inducing random mutations in recipient cells. Cells from the pool of randomly mutated cells are driven to transformation by a classic promoter resulting in foci, each of which encode a unique genetic profile. Our studies describe a novel molecular understanding of how cancer cell exosomes contribute to cell transformation. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that major issues remain unresolved (see decision letter).

Comparison of postoperative voice outcomes after postauricular facelift robotic hemithyroidectomy and conventional transcervical hemithyroidectomy.

BACKGROUND: The purpose of this study was to determine whether postauricular robotic and conventional hemithyroidectomy result in significantly different voice outcomes. METHODS: We prospectively compared the voice handicap index (VHI)-10 and acoustic parameters of a postauricular facelift robotic group and a conventional group preoperatively, 1 week, 1 month, and 6 months after surgery. RESULTS: Forty-two patients in the postauricular group and 68 patients in the conventional group completed the VHI-10 questionnaire and acoustic analysis. The postoperative VHI-10 scores were not significantly different between the two groups. In female patients, the highest frequency was higher and the frequency range was wider in the postauricular group compared to the conventional group postoperatively until 1 month after surgery. CONCLUSION: Postauricular facelift robotic thyroidectomy has advantages over conventional thyroidectomy in terms of postoperative voice pitch.

HORMAD1 Is a Negative Prognostic Indicator in Lung Adenocarcinoma and Specifies Resistance to Oxidative and Genotoxic Stress.

Cancer testis antigens (CTA) are expressed in testis and placenta and anomalously activated in a variety of tumors. The mechanistic contribution of CTAs to neoplastic phenotypes remains largely unknown. Using a chemigenomics approach, we find that the CTA HORMAD1 correlates with resistance to the mitochondrial complex I inhibitor piericidin A in non-small cell lung cancer (NSCLC). Resistance was due to a reductive intracellular environment that attenuated the accumulation of free radicals. In human lung adenocarcinoma (LUAD) tumors, patients expressing high HORMAD1 exhibited elevated mutational burden and reduced survival. HORMAD1 tumors were enriched for genes essential for homologous recombination (HR), and HORMAD1 promoted RAD51-filament formation, but not DNA resection, during HR. Accordingly, HORMAD1 loss enhanced sensitivity to γ-irradiation and PARP inhibition, and HORMAD1 depletion significantly reduced tumor growth in vivo These results suggest that HORMAD1 expression specifies a novel subtype of LUAD, which has adapted to mitigate DNA damage. In this setting, HORMAD1 could represent a direct target for intervention to enhance sensitivity to DNA-damaging agents or as an immunotherapeutic target in patients.Significance: This study uses a chemigenomics approach to demonstrate that anomalous expression of the CTA HORMAD1 specifies resistance to oxidative stress and promotes HR to support tumor cell survival in NSCLC. Cancer Res; 78(21); 6196-208. ©2018 AACR.

An Empirical Approach Leveraging Tumorgrafts to Dissect the Tumor Microenvironment in Renal Cell Carcinoma Identifies Missing Link to Prognostic Inflammatory Factors.

By leveraging tumorgraft (patient-derived xenograft) RNA-sequencing data, we developed an empirical approach, DisHet, to dissect the tumor microenvironment (eTME). We found that 65% of previously defined immune signature genes are not abundantly expressed in renal cell carcinoma (RCC) and identified 610 novel immune/stromal transcripts. Using eTME, genomics, pathology, and medical record data involving >1,000 patients, we established an inflamed pan-RCC subtype (IS) enriched for regulatory T cells, natural killer cells, TH1 cells, neutrophils, macrophages, B cells, and CD8+ T cells. IS is enriched for aggressive RCCs, including BAP1-deficient clear-cell and type 2 papillary tumors. The IS subtype correlated with systemic manifestations of inflammation such as thrombocytosis and anemia, which are enigmatic predictors of poor prognosis. Furthermore, IS was a strong predictor of poor survival. Our analyses suggest that tumor cells drive the stromal immune response. These data provide a missing link between tumor cells, the TME, and systemic factors.Significance: We undertook a novel empirical approach to dissect the renal cell carcinoma TME by leveraging tumorgrafts. The dissection and downstream analyses uncovered missing links between tumor cells, the TME, systemic manifestations of inflammation, and poor prognosis. Cancer Discov; 8(9); 1142-55. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.

Mutational analysis of DNMT3A gene in acute leukemias and common solid cancers.

DNMT3A, a DNA methyltransferase that functions for de novo methylation, is important in development and many cellular processes related to tumorigenesis. Somatic mutations of DNMT3A gene, including recurrent mutations in its Arg-882, were recently reported in acute myelogenous leukemia (AML), strongly suggesting its role in development of AML. To see whether DNMT3A mutation occurs in other malignancies as well, we analyzed DNMT3A in 916 cancer tissues from 401 hematologic malignancies (AML, acute lymphoblastic leukemias (ALL), multiple myelomas and lymphomas) and 515 carcinomas (lung, breast, prostate, colorectal and gastric carcinomas) using a single-strand conformation polymorphism (SSCP) assay. We identified DNMT3A mutations, especially the Arg-882 mutations, in adulthood AML (9.4%). In addition, we found DNMT3A mutations in pre-B-ALL and three lung cancers at lower frequencies. Allelic loss of DNMT3A was frequently observed in most cancer types analyzed, including lymphomas (48.1%), gastric cancers (23.5%) and lung cancers (18.3%) irrespective of DNMT3A mutation. Also, loss of DNMT3A expression was common in lung cancers (46.4%), and was associated with the allelic loss. Our data indicate that DNMT3A gene is mutated mainly in AML, but it occurs in other cancers, such as ALL and lung cancer, despite the lower incidences. Also, the data suggest that DNMT3A is altered in many cancer types by various ways, including somatic mutations, allelic loss and loss of expression that might play roles in tumorigenesis.

Mutational and expressional analyses of SPOP, a candidate tumor suppressor gene, in prostate, gastric and colorectal cancers.

Mounting evidence exists that alterations of ubiquitination processes are involved in cancer pathogenesis. Speckle-type POZ protein (SPOP) is a key adaptor for Cul3-based ubiquitination process. Recent studies reported that SPOP may be a tumor suppressor gene (TSG) and somatic mutation of SPOP was detected in prostate cancer (PCA). The aim of this study was to see whether alterations of SPOP protein expression and somatic mutation of SPOP gene are features of cancers. In this study, we analyzed SPOP somatic mutation in 45 gastric (GC), 45 colorectal cancer (CRC) and 45 PCA by single-strand conformation polymorphism (SSCP). Also, we analyzed SPOP protein expression in 60 GC, 60 CRC and 60 PCA by immunohistochemistry. Overall, we detected three somatic missense mutations of SPOP gene in the coding sequences (p.Ser14Leu, p.Tyr87Cys and p.Phe133Leu). The mutations were observed in two PCA and one CRC. Of note, the p.Phe133Leu was a recurrent mutation reported in an earlier study. In the immunohistochemistry, SPOP protein was expressed in normal gastric, colonic and prostate epithelial cells, whereas it was lost in 30% of GC, 20% of CRC and 37% of PCA. Our data indicate that loss of SPOP expression was common in GC, CRC and PCA, but somatic mutation of SPOP in this study was rare in these tumors. Also, the data provide a possibility that loss of expression of SPOP gene might play a role in cancer pathogenesis by altering TSG functions of SPOP.

Association between simple renal cysts and development of hypertension in healthy middle-aged men.

OBJECTIVES: The temporal relationship between simple renal cysts and incident hypertension is unknown. In this study, we assessed the time-dependent relationship between simple renal cysts and incident hypertension in a cohort of healthy middle-aged men. METHODS: A cohort study was conducted with data for 4516 healthy men with no evidence of hypertension at baseline. Study participants received a health checkup including abdominal ultrasonography between 2003 and 2004, and were followed in annual or biennial health examinations until May 2011. We matched groups with and without renal cysts by age. RESULTS: Renal cysts were found in 123 participants (2.7%). The age-matched control group included 1476 men. Mean age of the cyst group did not differ from that of the control group (42.3 ±â€Š6.6 and 42.2 ±â€Š6.8 years, respectively; P = 0.939). SBP was lower in the cyst group than in the control group (118.0 ±â€Š13.2 and 120.5 ±â€Š12.2, respectively; P = 0.044). During 10,731.5 person-years of follow-up, 169 participants developed hypertension (1.6/100 person-years). Cumulative incidence of hypertension was higher in the cyst group than in the control group (29.9 and 15.4%, respectively; P = 0.000). After adjusting for confounding factors, renal cysts still significantly increased risk for hypertension (hazard ratio, 3.28; 95% confidence interval 2.24-4.80; P = 0.000). Age, BMI, mean arterial pressure and a family history of hypertension were also risk factors. CONCLUSION: Simple renal cysts independently predicted incident hypertension in this cohort of middle-aged men. Further research is justified to test the causal role of renal cysts in the development of hypertension.

Somatic mutations and losses of expression of microRNA regulation-related genes AGO2 and TNRC6A in gastric and colorectal cancers.

Mounting evidence indicates that deregulation of microRNAs (miRNAs) are involved in development of many human diseases, including cancers. Regulation of miRNA is a complicated process and some components in the regulation are known to be altered in human cancers. Among the miRNA regulation-related genes, we found that AGO1, AGO2, TNRC6A, TNRC6C, TARBP2 and EXPORTIN5 genes have mononucleotide repeats in their coding sequences. To see whether these genes are mutated in cancers with microsatellite instability (MSI), we analysed the mononucleotide repeats in 27 gastric cancers (GCs) with high MSI (MSI-H), 18 GC with low MSI (MSI-L), 45 GC with stable MSI (MSS), 41 colorectal cancers (CRCs) with MSI-H, 14 CRCs with MSI-L and 45 CRCs with stable MSI (MSS) by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. We found AGO2, TNRC6A, TARBP2, TNRC6C and EXPORTIN5 mutations in 10, six, one, one and one cancer(s), respectively. They were detected in MSI-H but not in MSI-L or MSS cancers. The GCs and CRCs with MSI-H harboured one or more mutations of the genes in 22% and 27%, respectively. We also analysed Ago2 and TNRC6A protein expressions in GCs and CRCs with MSI-H. In cancers with MSI-H, loss of Ago2 expression was observed in 40% of GCs and 35% of CRCs, while loss of TNRC6A was observed in 52% of the GCs and 54% of the CRCs. Our data indicate that frameshift mutations in AGO2 and TNRC6A and their losses of expression are common in GCs and CRCs with MSI-H, and suggest that these alterations may contribute to the cancer development by deregulating miRNA regulation.

Prognostic effect of pathologic fracture in localized osteosarcoma: a cohort/case controlled study at a single institute.

BACKGROUND: The negative prognostic role of pathologic fracture in osteosarcoma is not determined, as previous case-control and retrospective cohort studies have produced contradictory results. METHODS: We conducted both cohort (n = 384) and case-control (n = 111) studies on 37 pathologically fractured localized osteosarcoma of extremity. RESULTS: In cohort study, patients with a fracture showed a tendency of poorer survival, but the difference did not reach the level of significance (5-year metastasis-free survival rates; 48% for cases vs. 61% for controls; P = 0.06). A case-control study on 37 fractured and 74 control recruited from 347 patients matched for tumor size and location showed no survival difference between the cases and controls (P = 0.12). CONCLUSIONS: Reported negative prognostic effect of a pathologic fracture is likely to be due to confounding by tumor size and location. The present study suggests that the presence of a pathologic fracture has no prognostic relevance.

Expressional and mutational analysis of pro-apoptotic Bcl-2 member PUMA in hepatocellular carcinomas.

Deregulation of apoptosis is involved in mechanisms of cancer development. PUMA is a pro-apoptotic member of the Bcl-2 family and mediates p53-dependent and -independent apoptosis. The aim of this study was to investigate whether alterations of PUMA protein expression and somatic mutations of PUMA gene are characteristics of human hepatocellular carcinoma (HCC). We analyzed expression of PUMA protein in 20 HCCs using immunohistochemistry. Also, we analyzed mutation of the Bcl-2 homology 3 (BH3) domain of PUMA gene, which is an important domain in apoptosis function of PUMA by single-strand conformation polymorphism (SSCP) in 69 HCCs. PUMA protein expression was detected in both HCC cells and non-tumor hepatocytes in all of the 20 HCCs. In 10 of these HCCs, cancer cells showed higher PUMA expression than non-tumor (cirrhotic) hepatocytes of the same patients; whereas in the remaining 10, cancer cells and non-tumor hepatocytes showed similar levels. Mutational analysis revealed no PUMA BH3 domain mutation in the 69 HCCs, suggesting that PUMA BH3 domain mutation is not a direct target of inactivation in hepatocellular cancer development. The increased expression of PUMA in malignant hepatocellular cells relative to that in non-tumor hepatocytes suggests that PUMA expression may play a role in HCC development.

Occult thyroid carcinoma detected by FDG-PET scan in elderly osteosarcoma patients: report of two cases.

Osteosarcoma is a rare tumor in elderly patients. Simultaneous osteosarcoma and thyroid papillary carcinoma are much rarer, especially in the absence of early radiation or cancer predisposition. We present two patients with concurrent osteosarcoma and thyroid cancer that were detected using positron-emission tomography.

Differential effect of 7,12-dimethylbenz[a]anthracene on human and mouse CYP1B1 from livers of castrated transgenic mice.

Humanized transgenic mice coexpressing tetracycline-controlled transactivator (tTA) and human cytochrome P450 1B1 (CYP1B1) (hCYP1B1) have been created by this group. The aims of this study was to determine if 7,12-dimethylbenz[a]anthracene (DMBA) functions as testosterone or doxycycline in its ability to induce or reduce expression of hCYP1B1 or endogenous mouse CYP1B1 (mCYP1B1). This was tested in the livers by treating castrated transgenic males and hCYP1B1/luciferase-transfected cells with DMBA. Herein, DMBA-treated group exhibited (i) gradual reduction of hCYP1B1 expression at the transcript, protein, and activity levels but gradually induced its transcript level during DMBA release; (ii) gradual reduction of hCYP1B1 at the transcript and protein levels, as in the case of doxycycline or testosterone; (iii) gradual induction of mCYP1B1 expression at the transcript and protein levels but gradually reduced its transcript level during DMBA release. In parallel, DMBA-treated transfected cells exhibited gradual increase in luciferase activity in a time-and dose-dependent manner. Thus, castrated transgenic males or in vitro system could be useful as models for the detection of polycyclic aromatic hydrocarbons (PAHs) or environmental toxicants by measuring either hCYP1B1 or mCYP1B1 expressions.