Evaluation of different real time PCRs for the detection of Pneumocystis jirovecii DNA in formalin-fixed paraffin-embedded bronchoalveolar lavage samples.

The presence of Pneumocystis jirovecii in fresh clinical materials can be detected by PCR with high sensitivity and is thus preferred over microscopic methods. However, fresh materials are not always available, and on formalin-fixed paraffin-embedded materials, PCR may result in reduced detection rates. In this study the diagnostic sensitivity of P. jirovecii real time PCR on DNA isolated from fresh bronchoalveolar lavage (BAL) samples versus that from matched FFPE derived DNA is analyzed. Our results indicate that when targeting a small DNA fragment P. jirovecii PCR can be performed on FFPE BAL samples with acceptable sensitivity (up to 83.3%). This is considerably higher than the 33.3% positives observed by classical staining of these samples.

Evaluation of Borrelia real time PCR DNA targeting OspA, FlaB and 5S-23S IGS and Borrelia 16S rRNA RT-qPCR.

Borrelia burgdorferi non-sensu lato (s.l.) strains occurred in the Netherlands. A multiplex OspA, FlaB, IGS real time PCR was compared to 16S rRNA/rDNA RT-qPCR with lower average Cycle threshold (Ct) and LOD on strain dilutions. Multiplexing increased sensitivity on CSF samples (n=74), distinguishing B. burgdorferi s.l. from non-s.l. strains.

Gonadal tumours and DSD.

Disorders of sex development (DSD), previously referred to as intersex, has been recognised as one of the main risk factors for development of type II germ cell tumours (GCTs), that is, seminomas/dysgerminomas and non-seminomas (e.g., embryonal carcinoma, yolk sac tumour, choriocarcinoma and teratoma). Within the testis, this type of cancer is the most frequent malignancy in adolescent and young adult Caucasian males. Although these males are not known to have dysgenetic gonads, the similarities in the resulting tumours suggest a common aetiological mechanism(s),--genetically, environmentally or a combination of both. Within the group of DSD patients, being in fact congenital conditions, the risk of malignant transformation of germ cells is highly heterogeneous, depending on a number of parameters, some of which have only recently been identified. Understanding of these recent insights will stimulate further research, with the final aim to develop an informative clinical decision tree for DSD patients, which includes optimal (early) diagnosis without overtreatment, such as prophylactic gonadectomy in the case of a low tumour risk.

Distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines.

Breast cancer has for long been recognized as a highly diverse tumor group, but the underlying genetic basis has been elusive. Here, we report an extensive molecular characterization of a collection of 41 human breast cancer cell lines. Protein and gene expression analyses indicated that the collection of breast cancer cell lines has retained most, if not all, molecular characteristics that are typical for clinical breast cancers. Gene mutation analyses identified 146 oncogenic mutations among 27 well-known cancer genes, amounting to an average of 3.6 mutations per cell line. Mutations in genes from the p53, RB and PI3K tumor suppressor pathways were widespread among all breast cancer cell lines. Most important, we have identified two gene mutation profiles that are specifically associated with luminal-type and basal-type breast cancer cell lines. The luminal mutation profile involved E-cadherin and MAP2K4 gene mutations and amplifications of Cyclin D1, ERBB2 and HDM2, whereas the basal mutation profile involved BRCA1, RB1, RAS and BRAF gene mutations and deletions of p16 and p14ARF. These subtype-specific gene mutation profiles constitute a genetic basis for the heterogeneity observed among human breast cancers, providing clues for their underlying biology and providing guidance for targeted pharmacogenetic intervention in breast cancer patients.

A novel SRY missense mutation affecting nuclear import in a 46,XY female patient with bilateral gonadoblastoma.

Patients with disorders of sex development (DSD), especially those with gonadal dysgenesis and hypovirilization, are at risk of developing the so-called type II germ cell tumors (GCTs). Both carcinoma in situ and gonadoblastoma (GB) can be the precursor lesion, resulting in a seminomatous or non-seminomatous invasive cancer. SRY mutations residing in the HMG domain are found in 10-15% of 46,XY gonadal dysgenesis cases. This domain contains two nuclear localization signals (NLSs). In this study, we report a unique case of a phenotypical normal woman, diagnosed as a patient with 46,XY gonadal dysgenesis, with an NLS missense mutation, on the basis of the histological diagnosis of a unilateral GB. The normal role of SRY in gonadal development is the upregulation of SOX9 expression. The premalignant lesion of the initially removed gonad was positive for OCT3/4, TSPY and stem cell factor in germ cells, and for FOXL2 in the stromal component (ie, granulosa cells), but not for SOX9. On the basis of these findings, prophylactical gonadectomy of the other gonad was performed, also showing a GB lesion positive for both FOXL2 (ovary) and SOX9 (testis). The identified W70L mutation in the SRY gene resulted in a 50% reduction in the nuclear accumulation of the mutant protein compared with wild type. This likely explains the diminished SOX9 expression, and therefore the lack of proper Sertoli cell differentiation during development. This case shows the value of the proper diagnosis of human GCTs in identification of patients with DSD, which allows subsequent early diagnosis and prevention of the development of an invasive cancer, likely to be treated by chemotherapy at young age.

New insights into type II germ cell tumor pathogenesis based on studies of patients with various forms of disorders of sex development (DSD).

Disorders of sex development (DSD), previously known as intersex, refer to congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical. Patients with specific variants of this disorder have an elevated risk for the development of so-called type II germ cell cancers, i.e., the seminomatous and nonseminatous tumors, referred to as germ cell tumors (GCTs). Specifically DSD patients with gonadal dysgenesis or hypovirilization are at risk. A prerequisite for type II GCT formation is the presence of a specific part of the Y chromosome (referred to as the GBY region), with the TSPY gene being the most likely candidate. Also the octamer binding transcription factor OCT3/4 is consistently expressed in all type II GCTs with pluripotent potential, as well as in the precursor lesions carcinoma in situ (CIS) in case of a testis and gonadoblastoma (GB) in the DSD gonad. The actual risk for malignant transformation in individual DSD patients is hard to predict, because of confusing terminology referring to the different forms of DSD, and unclear criteria for identification of the presence of malignant germ cells, especially in young patients. This is specifically due to the phenomenon of delay of germ cell maturation, which might result in over diagnosis. This review will give novel insight into the pathogenesis of the type II GCTs through the study of patients with various forms of DSD for which the underlying molecular defect is known. To allow optimal understanding of the pathogenesis of this type of cancers, first normal gonadal development, especially regarding the germ cell lineage, will be discussed, after which type II GCTs will be introduced. Subsequently, the relationship between type II GCTs and DSD will be described, resulting in a number of new insights into the development of the precursor lesions of these tumors.

Locoregional delivery of adenoviral vectors.

UNLABELLED: The overall median survival of patients with a malignant glioma is <1 y. Because malignant gliomas rarely metastasize outside the skull, locoregional treatment strategies, such as gene therapy, are under investigation. Recently, convection-enhanced delivery (CED) has been presented as a method to improve delivery of large molecules. The goal of this study was to evaluate whether CED improves intratumoral delivery of adenoviral vectors and compare it with single injection (SI) and multiple injection (4x, MI). METHODS: A replication-deficient adenoviral vector encoding the herpes simplex virus thymidine kinase (HSV-tk) and the human somatostatin receptor subtype 2 (sst(2)) was administered into nude mice bearing subcutaneous U87 xenografts. Tumors were injected with 1.5 x 10(9) plaque-forming units of Ad5.tk.sstr by CED, SI, or MI. Three days later, [(99m)Tc-N(4)(0-1),Asp(0),Tyr(3)]octreotate ((99m)Tc-Demotate 2) was injected intravenously to monitor the virus-induced sst(2) expression. gamma-Camera imaging was performed for in vivo imaging, and the tumor uptake of (99m)Tc-Demotate 2 was determined by gamma-counter. Furthermore, the tumor was sectioned and ex vivo autoradiography was performed. After decay of radioactivity, adjacent sections were submitted to in vitro autoradiography with (125)I-DOTA-Tyr(3)-octreotate, which was used to calculate the transduced areas. RESULTS: Transfected xenograft tissues showed high sst(2) expression and were clearly visualized with a gamma-camera. Accumulation of radioactivity was 2-fold higher in the tumors that were injected with MI compared with CED and SI (P = 0.01). CED and SI resulted in equal uptake of radioactivity in the tumors. The measured areas of transduction in ex vivo and in vitro autoradiographs showed a high concordance (r(2) = 0.89, P < 0.0001). The maximum area of transfection was significantly larger after MI than after CED (P < 0.05) or SI (P = 0.05). Also, the measured volume of distribution was twice as high after administration of Ad5.tk.sstr by MI (56.6 mm(3)) compared with SI (25.3 mm(3)) or CED (26.4 mm(3)). CONCLUSION: CED does not increase adenoviral vector distribution in a glioma xenograft model compared with SI. Therefore, in the clinic MI is probably the most effective delivery method for the large adenoviral particle (70 nm) in malignant gliomas.