Clinical Relevance of Absolute BK Polyoma Viral Load Kinetics in Patients With Biopsy Proven BK Polyomavirus Associated Nephropathy.

Introduction: The absolute BK viral load is an important diagnostic surrogate for BK polyomavirus associated nephropathy (PyVAN) after renal transplant (KTX) and serial assessment of BK viremia is recommended. However, there is no data indicating which particular viral load change, i.e., absolute vs. relative viral load changes (copies/ml; percentage of the preceding viremia) is associated with worse renal graft outcomes. Materials and Methods: In this retrospective study of 91 biopsy proven PyVAN, we analyzed the interplay of exposure time, absolute and relative viral load kinetics, baseline risk, and treatment strategies as risk factors for graft loss after 2 years using a multivariable Poisson-model. Results: We compared two major treatment strategies: standardized immunosuppression (IS) reduction (n = 53) and leflunomide (n = 30). The median viral load at the index biopsy was 2.15E+04 copies/ml (interquartile range [IQR] 1.70E+03-1.77E+05) and median peak viremia was 3.6E+04 copies/ml (IQR 2.7E+03-3.3E+05). Treatment strategies and IS-levels were not related to graft loss. After correction for baseline viral load and estimated glomerular filtration rate (eGFR), absolute viral load decrease/unit remained an independent risk factor for graft loss [incidence rate ratios [IRR] = 0.77, (95% CI 0.61-0.96), p = 0.02]. Conclusion: This study provides evidence for the prognostic importance of absolute BK viremia kinetics as a dynamic parameter indicating short-term graft survival independently of other established risk factors.

Primary External Stenting of an Autogenous Brachial-Basilic Upper Arm Transposition.

High-volume shunt flow after arteriovenous fistula (AVF) creation for hemodialysis can cause high-output heart failure. We used the Frame™ (Vascular Graft Solutions Ltd., Tel Aviv, Israel) external support, a stent, to limit vein dilatation and consecutive high-volume shunt in a 62-old female who underwent brachial-basilic upper arm transposition. After maturation, the shunt was used for dialysis and showed a plateauing flow volume 3 months after the operation. This case illustrates the safety and feasibility of this intervention when performed during AVF formation.

Bone matrix mineralization and osteocyte lacunae characteristics in patients with chronic kidney disease - mineral bone disorder (CKD-MBD).

OBJECTIVES: Little is known about bone mineralization and osteocyte lacunae properties in chronic kidney disease mineral bone disorder (CKD-MBD). METHODS: In this retrospective study, we measured the bone mineralization density distribution (BMDD) and osteocyte lacunar section (OLS) 2D-characteristics by quantitative backscatter electron imaging in Straumann drill biopsy samples from n=58 patients with CKD-MBD. Outcomes were studied in relation to serum parathyroid hormone (PTH), alkaline phosphatase (APH), histomorphometric bone turnover and treatment with cinacalcet or phosphate binders. RESULTS: Lower calcium concentrations in bone from high turnover (average degree of bone mineralization -6.2%, p<0.001) versus low turnover patients were observed. OLS-characteristics were distinctly different (p<0.01 to p<0.05) in patients with highest compared to those with lowest turnover. Patients with cinacalcet had different OLS-characteristics (p<0.05) compared to those without cinacalcet. Furthermore, patients with phosphate binders had differences in BMDD and OLS-characteristics (p<0.05) compared to patients without phosphate binders. CONCLUSIONS: Our findings suggest that in patients with CKD-MBD secondary hyperparathyroidism and increased bone turnover decrease the average degree of bone matrix mineralization. Conversely, density and lacunar size of the osteocytes are increased compared to adynamic bone disease pointing at distinct patterns of bone mineralization and osteocyte lacunar properties in these two disease entities.

Effects of angiotensin-converting-enzyme inhibitor therapy on the regulation of the plasma and cardiac tissue renin-angiotensin system in heart transplant patients.

BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors (ACEis) are beneficial in patients with heart failure, yet their role after heart transplantation (HTx) remains ambiguous. Particularly, the effects of ACEis on plasma and cardiac metabolites of the "classical" and "alternative" renin-angiotensin system (RAS) in HTx patients are unknown. METHODS: This cross-sectional study used a novel mass spectrometry-based approach to analyze plasma and tissue RAS regulation in homogenates of heart biopsy specimens from 10 stable HTx patients without RAS blockade and in 15 patients with ACEi therapy. Angiotensin (Ang) levels in plasma and Ang formation rates in biopsy tissue homogenates were measured. RESULTS: Plasma Ang II formation is exclusively ACE dependent, whereas cardiac Ang II formation is primarily chymase dependent in HTx patients. ACEi therapy substantially increased plasma Ang-(1-7), the key effector of the alternative RAS, leaving plasma Ang II largely intact. Importantly, neprilysin and prolyl-carboxypeptidase but not angiotensin converting enzyme 2 are essential for cardiac tissue Ang-(1-7) formation. CONCLUSION: ACE is the key enzyme for the generation of plasma Ang II, whereas chymase is responsible for cardiac tissue production of Ang II. Furthermore, our findings reveal that neprilysin and prolyl-carboxypeptidase are the essential cardiac enzymes for the alternative RAS after HTx. These novel insights into the versatile regulation of the RAS in HTx patients might affect future therapeutic avenues, such as chymase and neprilysin inhibition, beyond classical Ang II blockade.

Hereditary amyloidosis caused by R554L fibrinogen Aα-chain mutation in a Spanish family and review of the literature.

BACKGROUND: Hereditary amyloidosis with predominant renal disease can be caused by mutations in the gene encoding the fibrinogen Aα-chain (AFib). Here, we describe the clinical course of AFib amyloidosis associated with the rare R554L mutation, and the significance of extrarenal amyloid deposits and their possible influence on cardiovascular morbidity. METHODS: We report on 101 members of a family after having conducted patient interviews, chart review, genetic testing, renal biopsies and assessment for extrarenal amyloid deposition. RESULTS: Ten family members had chronic kidney disease with late-onset gross proteinuria and a variable course of declining renal function, starting in the fourth decade of life. In two affected living members, we identified the AFib R554L mutation. Renal biopsies from two affected members revealed almost complete obliteration of the mesangial glomerular architecture, although kidney function was only moderately impaired. There was neither evidence of extrarenal amyloidosis nor accelerated atherosclerosis. CONCLUSIONS: Renal amyloidosis associated with the R554L AFib variant dominated the clinical picture in this family, which was similar to that associated with the much more prevalent E526V mutation. Although it has been hypothesized that vascular deposits of fibrinogen amyloid may be associated with accelerated atherosclerosis, there was no suggestion of this in this particular kindred.

Gastric cancer growth control by BEZ235 in vivo does not correlate with PI3K/mTOR target inhibition but with [18F]FLT uptake.

PURPOSE: In this study, we tested the antitumor activity of the dual phosphoinositide 3-kinase (PI3K)/mTOR inhibitor BEZ235 against gastric cancer in vitro and in vivo. EXPERIMENTAL DESIGN: Gastric cancer cell lines (N87, MKN45, and MKN28) were incubated with BEZ235 and assessed for cell viability, cell cycle, and PI3K/mTOR target inhibition. In vivo, athymic nude mice were inoculated with N87, MKN28, or MKN45 cells and treated daily with BEZ235. 3'-Deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) uptake was measured via small animal positron emission tomography (PET). RESULTS: In vitro, BEZ235 dose dependently decreased the cell viability of gastric cancer cell lines. The antiproliferative activity of BEZ235 was linked to a G(1) cell-cycle arrest. In vivo, BEZ235 treatment resulted in PI3K/mTOR target inhibition as shown by dephosphorylation of AKT and S6 protein in all xenograft models. However, BEZ235 treatment only inhibited tumor growth of N87 xenografts, whereas no antitumor effect was observed in the MKN28 and MKN45 xenograft models. Sensitivity to BEZ235 in vivo correlated with downregulation of the proliferation marker thymidine kinase 1. Accordingly, [(18)F]FLT uptake was only significantly reduced in the BEZ235-sensitive N87 xenograft model as measured by PET. CONCLUSION: In conclusion, in vivo sensitivity of gastric cancer xenografts to BEZ235 did not correlate with in vitro antiproliferative activity or in vivo PI3K/mTOR target inhibition by BEZ235. In contrast, [(18)F]FLT uptake was linked to BEZ235 in vivo sensitivity. Noninvasive [(18)F]FLT PET imaging might qualify as a novel marker for optimizing future clinical testing of dual PI3K/mTOR inhibitors.

Targeting the dysregulated mammalian target of rapamycin pathway in organ transplantation: killing 2 birds with 1 stone.

Dysregulation and hyperactivation of the mammalian target of rapamycin (mTOR) pathway define the molecular basis of the hamartoma syndromes, including Cowden syndrome, tuberous sclerosis complex (TSC)/lymphangioleiomyomatosis, and Peutz-Jeghers syndrome. Loss of the tumor suppressors phosphatase and tensin homolog (PTEN), TSC1, TSC2, and LKB1 results in uncontrolled growth of usually benign tumors in various organs that, however, frequently lead to organ failure. Therefore, organ transplantation is a common therapeutic option in distinct patients with hamartoma syndromes, especially those with TSC/lymphangioleiomyomatosis. mTOR inhibitors are currently used in allogeneic transplantation as immunosuppressants and for the treatment of a growing number of cancers with dysregulated mTOR/phosphoinositide 3-kinase pathway. This dual targeting provides the unique opportunity for mTOR inhibitors to affect hamartoma syndromes at the molecular level along with potent immunosuppression in transplanted individuals. Here, we review the molecular mechanisms of hamartoma syndromes and discuss the recent clinical progress in transplant patients with hamartomas. Combining the identification of novel molecular targets of the phosphoinositide 3-kinase/mTOR pathway with insights into the clinical effectiveness of current therapeutic strategies sets the stage for a broader translational potential essential for further progress both in the treatment of cancer and for transplantation.

Vertical inhibition of the mTORC1/mTORC2/PI3K pathway shows synergistic effects against melanoma in vitro and in vivo.

The phosphatidyl inositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway has been shown to be involved in the development of melanoma. PI-103 is a kinase inhibitor blocking PI3K class IA and mTOR complex 1 and 2. Here, we studied the effect of targeting the PI3K/mTORC1/mTORC2 pathway by PI-103 and rapamycin in melanoma cells and in a melanoma mouse model. Dual targeting of PI3K and mTOR by PI-103 induced apoptosis and cell-cycle arrest, and inhibited viability of melanoma cells in vitro. Combined treatment with PI-103 and the prototypic mTORC1 inhibitor rapamycin led to the synergistic suppression of AKT and ribosomal S6 protein phosphorylation and to the induction of apoptosis. In vivo, PI-103 and rapamycin displayed only modest single-agent activity, but the combination significantly reduced the tumor growth compared with both single agents. These data show that blocking the PI3K/mTORC1/mTORC2 pathway using the combination of two distinct small-molecule inhibitors ("vertical inhibition") leads to superior efficacy against malignant melanoma in vitro and in vivo.

Bortezomib for the treatment of chronic antibody-mediated kidney allograft rejection: a case report.

We report on a patient with chronic C4d-positive antibody-mediated rejection, who was subjected to treatment with bortezomib. Despite initial treatment with CD20 antibody rituximab and intravenous immunoglobulin, the patient presented with a steady increase in serum creatinine and de novo proteinuria. In an effort to directly target alloantibody-producing plasma cells and to prevent ongoing antibody-mediated graft injury, we applied treatment with a single cycle of bortezomib combined with dexamethasone. Treatment was associated with a > 50% decrease in DSA levels and disappearance of capillary C4d staining as detected in a follow-up biopsy. However, there were still profound glomerulitis, an unchanged degree of transplant glomerulopathy and a persistent discrete infiltration of the interstitium by CD138+ plasma cells. The clinical course was unfavorable: despite some decrease in urinary protein excretion, a further deterioration of kidney allograft function was noted. In summary, this case suggests distinct antihumoral efficacy of bortezomib also in the context of chronic AMR. Nevertheless, a major observation was that treatment failed to prevent deterioration of graft function. We speculate that, despite modulation of (complement-activating) DSA, advanced irreversible tissue injury in this late stage of rejection may have precluded a relevant clinical response. Together with other case studies, our results may provide a valuable basis for prospective trials designed to evaluate the efficacy of bortezomib in the prevention and treatment of earlier stages of chronic AMR, e.g. based on the results of early (protocol) biopsies and/or early post-transplant antibody monitoring.

Everolimus (RAD001) and anti-angiogenic cyclophosphamide show long-term control of gastric cancer growth in vivo.

Metronomic dosing of cytotoxic drugs such as cyclophosphamide has shown anti-angiogenic activity, most likely by inducing hypoxia in tumors. Hypoxia leads to activation of escape mechanisms allowing tumor cell survival. This potentially limits the activity of anti-angiogenic strategies. We hypothesized that mTORC1 inhibition by everolimus (RAD001) leads to suppression of HIF-1alpha and VEGF resulting in synergistic anti-tumor activity in combination with anti-angiogenically dosed cyclophosphamide. In vitro, effects of everolimus on mTORC1 signaling, proliferation, cell cycle, HIF-1alpha expression and VEGF secretion were evaluated in two gastric cancer cell lines. In vivo, anti-tumor activity of everolimus in combination with metronomic cyclophosphamide was studied in a NCI-N87 human gastric cancer SCID mouse xenograft model. Expression of Ki-67 and HIF-1alpha, activated caspase 3, microvascular density (MVD) and tumor necrotic area assessed. Everolimus decreased proliferation and attenuated production of HIF-1alpha as well as VEGF in gastric cancer cells in vitro. In vivo, everolimus significantly inhibited tumor growth. This anti-tumor activity was linked to a significant increase in tumor necrotic area (p < 0.02) and trends for decreased proliferation, increased apoptosis, decreased HIF-1alpha and lower tumor MVD (p = n.s.). The combination of everolimus and cyclophosphamide resulted in a striking and highly significant long-term tumor growth control compared to monotherapy (p < 0.001), which was associated with a sharp increase in central tumor necrosis (p < 0.001). In conclusion, the combination of everolimus and metronomic cyclophosphamide showed synergistic anti-tumor activity. Depriving cancer cells by everolimus of factors necessary for their survival under hypoxia induced by anti-angiogenic chemotherapy appears to be a promising approach for treatment of gastric cancer.

mTOR inhibition sensitizes gastric cancer to alkylating chemotherapy in vivo.

BACKGROUND: Gastric cancer is a highly chemoresistant tumor. Previous studies suggest that cancer cells can be sensitized to standard chemotherapy, and especially alkylating agents, by inhibition of mammalian target of rapamycin (mTOR) signaling. The work presented here shows that the mTOR inhibitor everolimus, in combination with cyclophosphamide, exhibits synergistic antitumor activity in gastric cancer xenografts. MATERIALS AND METHODS: Treatment with everolimus at the minimal effective dose was studied in combination with cyclophosphamide at maximum tolerated dose in a human gastric cancer severe combined immunodeficient (SCID) mouse xenograft model. Besides tumor size, biomarker expression for proliferation (Ki-67), hypoxia (HIF-12alpha), apoptosis (activated caspase 3), angiogenesis (microvascular density, MVD) and levels of circulating endothelial progenitors (CEPs) were assessed. RESULTS: Everolimus single agent treatment significantly inhibited tumor growth relative to control and cyclophosphamide treatment (T/C 19%, p<0.01). This antitumor activity was linked to a significant decrease in tumor cell proliferation (p<0.01) and a trend towards lower tumor MVD, HIF-1alpha expression and levels of CEPs. Notably, the combination of everolimus with cyclophosphamide resulted in synergistic anti-tumor activity (T/C 9%, p<0.01). This antitumor activity coincided with a statistically significant decrease in MVD (p<0.01). Whereas treatment with everolimus was well tolerated, cyclophosphamide at maximum tolerated dose (MTD) showed significant toxicity both as monotherapy and in combination with everolimus. CONCLUSION: The antiangiogenic activity of everolimus combined with a high dose of cyclophosphamide shows synergistic antitumor activity against gastric cancer in vivo. In potential future clinical trials, the toxicity of cyclophosphamide in combination regimens with everolimus deserves careful evaluation.

Comparison of a treatment strategy combining CCI-779 plus DTIC versus DTIC monotreatment in human melanoma in SCID mice.

This study compares the antineoplastic potential of a novel treatment strategy combining cell cycle inhibitor-779 (CCI-779) plus dacarbazine (DTIC) versus DTIC monotreatment, the current chemotherapeutic mainstay in combating metastatic melanoma. A controlled four-group parallel study design comprising 24-40 mice per tumor cell line was used in a severe combined immunodeficiency (SCID)-mouse xenotransplantation model. SCID mice were injected with 518A2, Mel-JUSO, or 607B human melanoma cells. After they developed tumors, mice received daily CCI-779 or solvent over 14 days. From treatment day 4-8 mice were additionally injected with DTIC or saline. Treatment with CCI-779 plus DTIC was superior to single agent DTIC in two out of three cell lines (P<0.05). The tumor weight reduction was 44+/-17 and 61+/-6% compared with DTIC monotreatment in Mel-JUSO and 607B melanomas, respectively (P<0.05). In contrast, in 518A2 xenotransplants, CCI-779 plus DTIC treatment was as effective as DTIC monotreatment. CCI-779 monotherapy exerted no statistically significant antitumor effect. Collectively, these data indicate that CCI-779 has the potential to increase the chemotherapeutic efficacy, as the combination of CCI-779 plus DTIC proved to be more efficacious compared to DTIC monotherapy in two out of three melanoma cell lines in vivo.

Mammalian target of rapamycin pathway activity in hepatocellular carcinomas of patients undergoing liver transplantation.

BACKGROUND: Because mammalian target of rapamycin (mTOR) inhibitors combine anticancer and immunosuppressive properties we investigated: 1) the activation status and prognostic significance of the mTOR pathway in hepatocellular carcinoma (HCC) tissues of patients undergoing orthotopic liver transplantation (OLT) for HCC, and 2) the single and combinatorial efficacy of RAD001 in HCC cells. METHODS: PTEN, p-AKT, p-mTOR, p-p70S6K, and p-4EBP-1 were analyzed by immunohistochemistry in explanted HCCs of 166 patients undergoing OLT. Efficacy of RAD001 as mono- and combination therapy with doxorubicin was tested in Hep3B and SNU398 cells. RESULTS: The mTOR pathway is activated in about 40% of patients undergoing OLT for HCC but no direct correlation between up- and downstream proteins was observed. We found no influence of mTOR pathway protein expression on disease free survival (DFS) or overall survival (OS). There was a marked single agent and chemo-sensitizing effect of RAD001 against HCC cells in vitro. CONCLUSION: The mTOR pathway is active in 40% of patients with HCC undergoing OLT, but has no influence of DFS or OS. No direct correlation was observed between up- and downstream proteins limiting the use of upstream proteins to predict mTOR activity. Prospective clinical trials are needed to test whether the activation status of the mTOR pathway in HCCs predicts the antitumor effect of rapamycin derivative in the posttransplantation course.