Amr S. Khaled

Morphological spectrum of polyoma virus disease in renal allografts: diagnostic accuracy of urine cytology.

The morphological features of polyoma virus disease (PVDz) in 571 concurrent urine and biopsy samples from 413 patients are described. In 54 patients PV was found in both biopsy and urine samples. Histologically, PV presented as: (a) mild, viral cytopathic/cytolytic changes, with absent or minimal inflammation involving isolated tubules; (b) moderate and severe, cytopathic/cytolytic changes associated with patchy or diffuse tubulo‐interstitial inflammation and atrophy; (c) advanced, graft sclerosis with rare or absent viral cytopathic changes, indistinguishable from chronic allograft nephropathy. Histological progression from mild to moderate or severe disease was seen in 28 patients. The mean post‐transplantation time at diagnosis was similar in patients with mild or moderate‐severe renal involvement (1.05 and 1.3 years, respectively). All patients presented with similarly increased values of serum creatinine (mean 1.35 mg/dL). There was strong correlation between the number of PV infected cells in urine and the concurrent biopsies (p = 0.0001). In 13 patients PV was found only in urine; of these, two developed PVDz later. The positive predictive value of a positive urine was 90%, the negative predictive value of a negative urine was 99% and the accuracy of the test was 97%. We conclude that urine cytology is useful to evaluate renal transplant patients with PV reactivation because sloughed tubular cells are found in urine and positive urine samples are a consistent manifestation of PV renal involvement.

Treatment of established recurrent hepatitis C in liver-transplant recipients with pegylated interferon-alfa-2b and ribavirin therapy

Introduction. The management issues of transplant patients with hepatitis C virus (HCV) are complex, and interferon therapy is often ineffective. We present data from a retrospective review in liver-transplant recipients suffering from HCV recurrence that were treated with pegylated alpha-2b interferon and ribavirin. Methods. A retrospective review of transplant recipients that received combination pegylated alpha-2b interferon (1.5 mcg/kg/wk) and ribavirin (400–600 mg/day) therapy intended for at least 48 weeks. Complications were recorded and included neutropenia (<750 cells), anemia (hemoglobin <8 g) with and without treatment consisting of blood transfusions, erythropoietin, or dose reduction of ribavirin, and depression. The diagnosis of HCV recurrence was determined by an increase in liver chemistries, histopathologic findings with inflammation along with viral recurrence using the COBAS AMPLICOR HCV test. Results. Fifty-seven liver-transplant recipients were included, 29 naïve (group 1) to therapy and 28 nonresponders (group 2) to at least 6 months of interferon and ribavirin therapy. Eight (27.6%) patients in group 1 and six (21%) patients in group 2 were HCV nondetectable at the end of 48 weeks of therapy. Ribavirin therapy was decreased in 13 of 29 (45%) for group 1 and 11 of 28 (39%) in group 2. Therapeutic interventions were 4 of 57 (7%) blood transfusions, 23 of 57 (40%) erythropoietin, and 17 of 57 (30%) filgrastim. Conclusion. Combination pegylated interferon with ribavirin appears to effective therapy in HCV recurrence and in HCV nonresponsive to interferon and ribavirin. This data reveals the difficulty and caution that must be taken when treating HCV-R liver-transplant recipients with combination pegylated alpha-2b interferon and ribavirin therapy.

Reliability of histopathologic assessment for the differentiation of recurrent hepatitis C from acute rejection after liver transplantation

Histopathologic assessment is considered essential for the differentiation of recurrent hepatitis C (RHC) from acute cellular rejection (ACR) after liver transplantation (LT); however, there is limited information regarding its reliability. The aim of this study was to determine the interobserver and intraobserver agreement of the histopathologic diagnosis of RHC vs. ACR, and to determine the reliability of specific histopathologic features for the differentiation of RHC from ACR. Liver biopsy specimens from 105 consecutive patients transplanted for hepatitis C virus (HCV)‐related liver disease were studied retrospectively. All the biopsies were performed for evaluation of abnormal liver enzymes within the 1st year after LT. The slides were blindly coded and assessed by 5 liver‐transplant pathologists, practicing at 3 medical centers. The pathologists were asked to render a diagnosis, and determine the severity of the disease. Four of the pathologists were asked to determine the presence and severity of 36 histopathologic features. A total of 34 of the samples were then blindly resubmitted to each of the 4 pathologists to determine the intraobserver agreement. There was a slight agreement (κ = .12) among the 5 pathologists on the histopathologic diagnosis. All 5 pathologists were in agreement on the diagnosis of RHC in only 5 patients (5%) and on the diagnosis of ACR in only 2 patients (2%). The best agreement among any 4 pathologists was fair (κ = .20). Slight to moderate agreement occurred on the main histological features considered to be important in the diagnosis of ACR. Intraobserver agreement ranged from slight (κ = .19) to moderate (κ = .42) among 4 pathologists. In conclusion, the histopathologic differentiation of RHC from ACR after LT had relatively low interobserver and intraobserver agreement rates, and hence showed low reliability. Histopathologic assessment should be used cautiously for the differentiation of RHC from ACR post‐LT. (Liver Transpl 2004;10:1233–1239.)

The Role of Donor Bone Marrow Infusions in Withdrawal of Immunosuppression in Adult Liver Allotransplantation

We investigated the role of donor bone marrow cell (DBMC) infusions in immunosuppression withdrawal in adult liver transplantation. Patients enrolled were at least 3 years post‐transplantation, with stable graft function. Forty‐five (study group: G1) received DBMC, and 59 (control group: G2) did not. Immunosuppression was reduced by one third upon enrollment, by another third the second year of the study and was completely withdrawn the third year.

Bax Deficiency Partially Corrects Interleukin-7 Receptor α Deficiency

The requirement for cytokines in hematopoiesis is partly attributable to the protection of cells from apoptosis. Since IL-7 is required for normal T cell development, we evaluated the role of Bax in vivo by generating mice deficient in both Bax and the IL-7 receptor α chain (IL-7R). Starting at birth, we observed complete recovery of all stages of αβ thymocyte development up to 4 weeks of age. However, by 12 weeks of age, thymic cellularity had reverted to that of mice deficient in IL-7R alone. The BH3 only proteins, Bad and Bim, were also part of the death pathway repressed by IL-7. Thus, in young mice, Bax emerges as an essential protein in the death pathway induced by IL-7 deficiency.

Prediction of sustained virological response in liver transplant recipients with recurrent hepatitis C virus following combination pegylated interferon alfa‐2b and ribavirin therapy using tissue hepatitis C virus reverse transcriptase polymerase chain reaction testing

The optimal duration of therapy for pegylated interferon combined with ribavirin in recurrent Hepatitis C virus (HCV) following liver transplantation is not known. We wanted to determine if testing for HCV in liver tissue by reverse transcriptase polymerase chain reaction (RT‐PCR) was superior in predicting sustained virological response (SVR) in comparison to standard HCV ribonucleic acid (RNA) detection in the serum. All recipients received combination pegylated alpha‐2b interferon (1.5 mcg / kg) and ribavirin (200–600mg / d) therapy for at least 48 weeks of therapy and were found to have nondetectable HCV RNA by PCR serum testing at the end of therapy. Sustained virological response (SVR) was defined as nondetectable serum HCV RNA at 6 months post treatment withdrawal. Ten liver transplant recipients were included in the study; mean time from transplantation was 29.2 months. All had nondetectable serum HCV RNA by RT‐PCR. In hepatic tissue 7/10 patients HCV RNA was found to be positive by RT‐PCR while 3/10 had nondetectable HCV RNA in their liver by RT‐PCR. SVR was attained in all 3/10 that were hepatic tissue HCV PCR negative after 12 months of combination therapy. In conclusion, direct detection of HCV RNA by RT‐PCR of liver tissue appears to more effectively predict SVR following pegylated interferon and ribavirin therapy than the conventional use of serum. (Liver Transpl 2004;10:595–598.)

Kaposi's sarcoma of the thyroid gland in an HIV-negative woman : A case report

BACKGROUND Kaposis sarcoma (KS) is a neoplastic disease that affects primarily the skin, but visceral involvement is not uncommon. Most of the cases are seen in AIDS patients and transplant recipients; however, rare HIV-negative cases have also been reported. Involvement of the thyroid is exceedingly rare, with only a fw cases reported, all of them associated with AIDS. CASE A 45-year-old, black, Haitian woman presented with a slowly enlarging left side of the thyroid. Computed tomography showed multiple thyroid nodules, and there was no uptake of iodine on the nuclear scan. Fine needle aspiration of the lesion was performed. The smears were composed of spindle and plasmacytoid cells, which raised the possibility of medullary carcinoma. The patient underwent left hemithyroidectomy. Histologic examination showed KS in the thyroid. CONCLUSION We present the first case of KS of the thyroid in a HIV-negative patient. Familiarity with the cytologic features can be useful in making the diagnosis.

PSMA-Targeted Theranostic Nanocarrier for Prostate Cancer

Herein, we report the use of a theranostic nanocarrier (Folate-HBPE(CT20p)) to deliver a therapeutic peptide to prostate cancer tumors that express PSMA (folate hydrolase 1). The therapeutic peptide (CT20p) targets and inhibits the chaperonin-containing TCP-1 (CCT) protein-folding complex, is selectively cytotoxic to cancer cells, and is non-toxic to normal tissue. With the delivery of CT20p to prostate cancer cells via PSMA, a dual level of cancer specificity is achieved: (1) selective targeting to PSMA-expressing prostate tumors, and (2) specific cytotoxicity to cancer cells with minimal toxicity to normal cells. The PSMA-targeting theranostic nanocarrier can image PSMA-expressing cells and tumors when a near infrared dye is used as cargo. Meanwhile, it can be used to treat PSMA-expressing tumors when a therapeutic, such as the CT20p peptide, is encapsulated within the nanocarrier. Even when these PSMA-targeting nanocarriers are taken up by macrophages, minimal cell death is observed in these cells, in contrast with doxorubicin-based therapeutics that result in significant macrophage death. Incubation of PSMA-expressing prostate cancer cells with the Folate-HBPE(CT20p) nanocarriers induces considerable changes in cell morphology, reduction in the levels of integrin β1, and lower cell adhesion, eventually resulting in cell death. These results are relevant as integrin β1 plays a key role in prostate cancer invasion and metastatic potential. In addition, the use of the developed PSMA-targeting nanocarrier facilitates the selective in vivo delivery of CT20p to PSMA-positive tumor, inducing significant reduction in tumor size.

Targeting chaperonin containing TCP1 (CCT) as a molecular therapeutic for small cell lung cancer

Identifying new druggable targets is desired to meet the needs for effective cancer treatments. To this end, we previously reported the efficacy of a therapeutic peptide called CT20p that displays selective cytotoxicity through inhibition of a multi-subunit, protein-folding complex called Chaperonin-Containing TCP-1 (CCT). To investigate the role of CCT in cancer progression, we examined protein levels of CCT subunits in liver, prostate, and lung cancer using human tissue microarrays. We found that these cancers expressed higher levels of CCT2 as compared to normal tissues. Small cell lung cancer (SCLC) stood out as having statistically significant difference in CCT2. Higher levels of CCT2 in tumors from lung cancer patients were also associated with decreased survival. Using SCLC cell lines, we observed detectable amounts of CCT subunits and cells were susceptible to killing by CT20p. Treatment with CT20p, delivered to cells using polymeric nanoparticles, was cytotoxic to all SCLC cell lines, decreasing the levels of CCT client proteins like STAT3. In contrast, treatment with a STAT3 inhibitor was effective in one of the SCLC cell lines. While we found that CCT levels could vary in cell lines, normal tissues had low levels of CCT and minimal toxicity to liver or kidney function was observed in mice treated with CT20p. These results indicate that in SCLC, changes in CCT levels could be used as a biomarker for diagnosis and that targeting CCT for inhibition with CT20p is a promising treatment approach for those cancers such as SCLC that currently lack targeted therapeutics.

Chaperonin Containing-TCP-1 Protein Level in Breast Cancer Cells Predicts Therapeutic Application of a Cytotoxic Peptide

Purpose: Metastatic disease is a leading cause of death for patients with breast cancer, driving the need for new therapies. CT20p is a peptide previously discovered by our group that displays cancer-specific cytotoxicity. To design the optimal therapeutic use of the peptide, we identified the intracellular target of CT20p in breast cancer cells, correlating expression patterns of the target with susceptibility to CT20p. Experimental Design: Using polymeric nanoparticles to deliver CT20p, we assessed cytoskeletal changes, cell migration, adhesion, and viability in cells treated with the peptide. Protein pull-down experiments, coupled to mass spectrometry, enabled identification of the peptides intracellular target. Biochemical and histologic techniques validated target identity in human cell lines and breast cancer tissue microarrays and revealed susceptibility patterns to CT20p. Results: Chaperonin containing TCP-1 (CCT) was identified as the intracellular target of CT20p. Cancer cells susceptible to CT20p had increased CCT, and overexpression of CCTβ, a subunit of the CCT complex, enhanced susceptibility to CT20p. Susceptible cells displayed reduced tubulin, a substrate of CCT, and inhibition of migration upon CT20p treatment. CCTβ levels were higher in invasive ductal carcinomas than in cancer adjacent tissues and increased with breast cancer stage. Decreased breast cancer patient survival correlated with genomic alternations in CCTβ and higher levels of the chaperone. Conclusions: Increased CCT protein in breast cancer cells underlies the cytotoxicity of CT20p. CCT is thus a potential target for therapeutic intervention and serves as a companion diagnostic to personalize the therapeutic use of CT20p for breast cancer treatment. Clin Cancer Res; 22(17); 4366–79. ©2016 AACR.