Abnormal Results of Newborn Screening for SCID After Azathioprine Exposure In Utero: Benefit of TPMT Genotyping in Both Mother and Child

Journal of Clinical Immunology -     

Human herpesvirus 8 load in matched serum and plasma samples of patients with AIDS-associated Kaposi's sarcoma

Human herpesvirus 8 (HHV-8) (or Kaposi's sarcoma [KS]-associated herpesvirus) is associated with all forms of KS. HHV-8 DNA load in peripheral blood mononuclear cells (PBMCs) of KS patients has been shown to correlate with the clinical stage of the disease. Studies have been done to assess the HHV-8 viral load in different sample types from KS patients and its clinical relevance. This paper describes the design and evaluation of a quantitative real-time (TaqMan) PCR assay for routine diagnosis of HHV-8 infection. The linear dynamic range was 5 to 5 x 10(6) copies of HHV-8 DNA (r(2) > 0.99). The assay is very sensitive, specific, and easily reproducible (less than 2% variability) and can be used for different clinical samples, such as serum, plasma, and PBMCs. The question of which clinical sample, serum or plasma, is preferable for HHV8 DNA testing was addressed, using this newly developed real-time PCR assay. From 85 patients with diagnosed AIDS-KS, matched plasma and serum samples were collected. Of the 85 patients tested, 35 were positive for HHV-8 DNA in both plasma and serum (41%), 8 were positive in serum but not plasma, and 7 had detectable HHV-8 DNA only in plasma. The HHV-8 load was similar in both plasma and serum, and no significant difference was found. However, more inhibition was seen in the plasma samples with the use of a system quality control, seal herpesvirus type 1. Therefore, our results suggest that serum is the preferred material for HHV-8 load testing, since there is less possible hindrance in the amplification than with plasma.     

Prophylactic window therapy with the clinical poly(ADP‐ribose) polymerase inhibitor olaparib delays BRCA1‐deficient mammary tumour formation in mice

Women with heterozygous germline mutations in the BRCA1 tumour suppressor gene are strongly predisposed to developing early‐onset breast cancer through loss of the remaining wild‐type BRCA1 allele and inactivation of TP53. Although tumour prevention strategies in BRCA1‐mutation carriers are still limited to prophylactic surgery, several therapeutic strategies have been developed to target the DNA repair defects (also known as ‘BRCAness’) of BRCA1‐deficient tumours. In particular, DNA‐damaging agents such as platinum drugs and poly(ADP‐ribose) polymerase (PARP) inhibitors show strong activity against BRCA1‐mutated tumours. However, it is unclear whether drugs that target BRCAness can also be used to prevent tumour formation in BRCA1‐mutation carriers, especially as loss of wild‐type BRCA1 may not be the first event in BRCA1‐associated tumourigenesis. We performed prophylactic treatments in a genetically engineered mouse model in which de novo development of BRCA1‐deficient mammary tumours is induced by stochastic loss of BRCA1 and p53. We found that prophylactic window therapy with nimustine, cisplatin or olaparib reduced the amount and size of mammary gland lesions, and significantly increased the median tumour latency. Similar results were obtained with intermittent prophylactic treatment with olaparib. Importantly, prophylactic window therapy with nimustine and cisplatin resulted in an increased fraction of BRCA1‐proficient mammary tumours, suggesting selective survival and malignant transformation of BRCA1‐proficient lesions upon prophylactic treatment with DNA‐damaging agents. Prophylactic therapy with olaparib significantly prolonged mammary tumour‐free survival without any significant increase in the fraction of BRCA1‐proficient tumours, warranting the evaluation of this PARP inhibitor in prophylactic trials in BRCA1‐mutation carriers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Phase I immunotherapeutic trial with long peptides spanning the E6 and E7 sequences of high-risk human papillomavirus 16 in end-stage cervical cancer patients shows low toxicity and robust immunogenicity.

PURPOSE: To determine the toxicity, safety, and immunogenicity of a human papillomavirus 16 (HPV16) E6 and E7 long peptide vaccine administered to end-stage cervical cancer patients. EXPERIMENTAL DESIGN: Three groups of end-stage cervical cancer patients (in total n = 35) were s.c. vaccinated with HPV16 E6 combined with or separated from HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant, four times at 3-week intervals. Group 1 received 300 microg/peptide at a single site and group 2 received 100 microg/peptide of the E6 peptides in one limb and 300 microg/peptide of the E7 peptides in a second limb. Group 3 received separate injections of E6 and E7 peptides, each at a dose of 50 microg/peptide. The primary end point was to determine safety and toxicity of the HPV16 long peptides vaccine. In addition, the vaccine-induced T-cell response was assessed by IFN gamma enzyme-linked immunospot. RESULTS: No toxicity beyond grade 2 was observed during and after four vaccinations. In a few patients, transient flu-like symptoms were observed. Enzyme-linked immunospot analysis of the vaccine-induced immune response revealed that coinjection of the E6 and E7 peptides resulted in a strong and broad T-cell response dominated by immunity against E6. Injection of the E6 and E7 peptides at two different sites increased the E7 response but did not affect the magnitude of the E6-induced immune response. CONCLUSIONS: The HPV16 E6 and E7 long peptide-based vaccine is well tolerated and capable of inducing a broad IFN gamma-associated T-cell response even in end-stage cervical cancer patients.     

Activity patterns of proteasome subunits reflect bortezomib sensitivity of hematologic malignancies and are variable in primary human leukemia cells.

Proteasomal proteolysis relies on the activity of six catalytically active proteasomal subunits (beta1, beta2, beta5, beta1i, beta2i and beta5i). Applying a functional proteomics approach, we used a recently developed activity-based, cell-permeable proteasome-specific probe that for the first time allows differential visualization of individual active proteasomal subunits in intact primary cells. In primary leukemia samples, we observed remarkable variability in the amounts of active beta1/1i-, beta2/2i- and beta5/5i-type of subunits, contrasting with their constant protein expression. Bortezomib inhibited beta5- and beta1-type, but to a lesser extend beta2-type of subunits in live primary cells in vitro and in vivo. When we adapted the bortezomib-sensitive human acute myeloid leukemia cell line HL-60 to bortezomib 40 nM (HL-60a), proteasomal activity profiling revealed an upregulation of active subunits, and residual beta1/beta5-type of activity could be visualized in the presence of bortezomib 20 nM, in contrast to control cells. In a panel of cell lines from hematologic malignancies, the ratio between beta2-type and (beta1 + beta5)-type of active proteasomal polypeptides mirrored different degrees of bortezomib sensitivity. We thus conclude that the proteasomal activity profile varies in primary leukemia cells, and that the pattern of proteasomal subunit activity influences the sensitivity of hematologic malignancies toward bortezomib.     

Relationship between the exposure to cisplatin, DNA-adduct formation in leucocytes and tumour response in patients with solid tumours.

The study was designed to investigate possible relationships between tumour response and exposure to cisplatin (area under the curve of unbound cisplatin in plasma, AUC) and DNA-adduct formation in leucocytes (WBC) in patients with solid tumours. Patients were treated with six weekly courses of cisplatin at a dose of 70 or 80 mg m-2. The AUC was determined during the first course and DNA-adduct levels in WBC during all courses at baseline, 1 h (A(max)) and 15 h after a 3 h infusion of cisplatin. The area under the DNA-adduct-time curve (AUA) was calculated. The tumour response was determined after six courses. Forty-five evaluable patients received 237 courses of cisplatin. Sixteen patients with head and neck cancer received a dose of 80 mg m-2 and 29 with various other tumour types received 70 mg m-2 plus daily 50 mg oral etoposide. There were 20 responders (partial and complete) and 25 non-responders (stable and progressive disease). The AUC was highly variable (mean +/- s.d. = 2.48 +/- 0.51 micrograms h-1 ml-1; range 1.10-3.82) and was closely correlated with the AUA (r = 0.78, P < 0.0001) and A(max) (r = 0.73, P < 0.0001). The AUC, AUA and A(max) were significantly higher in responders than in non-responders in the total population (P < 0.0001) and in the two subgroups treated at 70 or 80 mg m-2. In logistic regression analysis AUC, AUA and A(max) were important predictors of response. The magnitude of exposure to cisplatin is, through DNA-adduct formation, the major determinant of the response rate in this population. Hence, individualised dosing of cisplatin using AUC or DNA-adducts should lead to increased response rates.     

Docetaxel and paclitaxel inhibit DNA-adduct formation and intracellular accumulation of cisplatin in human leukocytes

 The purpose of this study was to determine the mechanism of the pharmacodynamic interaction between docetaxel/paclitaxel and cisplatin. Cisplatin-induced DNA-adducts and cisplatin accumulation were quantitated in peripheral blood leukocytes (WBC). The WBC were obtained from patients treated with docetaxel or paclitaxel in phase I/II studies and were incubated in vitro with cisplatin. In addition, blank whole-blood samples were obtained from patients and healthy subjects and incubated in vitro with cisplatin or docetaxel/paclitaxel and cisplatin. The cisplatin-induced DNA-adduct levels measured in WBC after treatment with docetaxel or paclitaxel were significantly lower than those determined in non-pretreated WBC. Docetaxel and paclitaxel reduced the intracellular accumulation of cisplatin in WBC by 46–47%. If the pharmacodynamic interaction between docetaxel/paclitaxel and cisplatin also occurs in other normal tissues such as bone marrow, it may well contribute to the sequence dependent toxicity that has been observed in clinical studies. Received: 15 February 1995/Accepted: 6 June 1995     

Glutathione conjugates and their synthetic derivatives as inhibitors of glutathione-dependent enzymes involved in cancer and drug resistance

Alterations in levels of glutathione (GSH) and glutathione-dependent enzymes have been implicated in cancer and multidrug resistance of tumor cells. The activity of a number of these, the multidrug resistance-associated protein 1, glutathione S-transferase, DNA-dependent protein kinase, glyoxalase I, and gamma-glutamyl transpeptidase, can be inhibited by GSH-conjugates and synthetic analogs thereof. In this review we focus on the function of these enzymes and carriers in cancer and anti-cancer drug resistance, in relation to their inhibition by GSH-conjugate analogs.