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 new paraneoplastic encephalomyelitis autoantibody reactive with the axon initial segment.

Serum from a patient with paraneoplastic encephalomyelitis (PEM) and small cell lung cancer (SCLC) showed high titer immunohistochemical staining of the axon initial segment (AIS) on rat and human brain sections. EM studies showed that the antigen was localized in close proximity of the microtubules in the AIS. Double labeling experiments and absence of staining at the nodes of Ranvier excluded the previously identified betaIV spectrin as autoantigen. Screening a rat hippocampal cDNA library resulted in the isolation of ubiquitin-conjugating enzyme E2E1 (UBE2E1). However, blocking and elution experiments excluded UBE2E1 as the AIS autoantigen.

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.

Targeting malignant gliomas with a glial fibrillary acidic protein (GFAP)-selective oncolytic adenovirus.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein abundantly expressed in malignant gliomas. We have constructed a novel oncolytic adenovirus, Ad5-gfa2(B)3-E1, for treatment of these tumors. In this construct, the E1 region is under control of the tissue-specific GFAP promoter (gfa2) with three additional copies of the glial specific 'B' enhancer. Infection of a GFAP-positive cell line with Ad5-gfa2(B)3-E1 resulted in E1A and E1B expression at 75% and 30% of the levels obtained after wtAd5 infection. Q-PCR showed that Ad5-gfa2(B)3-E1 replicated 4.5 times more efficiently in the GFAP-positive than in the GFAP-negative cell lines. Cell viability assays showed efficient elimination of GFAP-positive cells by Ad5-gfa2(B)3-E1, in some cell lines as efficiently as wtAd5, while the elimination was attenuated in GFAP-negative cell lines. When tested in human tumor xenografts in nude mice, Ad5-gfa2(B)3-E1 effectively suppressed the growth of GFAP-positive SNB-19 glial tumors but not of GFAP-negative A549 lung tumors. In Ad5-gfa2(B)3-E1, the E3 region was deleted to create space for future insertion of heterologous therapeutic genes. Experiments with dl7001, an E3-deleted variant of wtAd5, confirmed that the specificity of Ad5-gfa2(B)3-E1 replication was based on the promoter driving E1 and not on the E3 deletion. Strategies to further improve the efficacy of Ad5-gfa2(B)3-E1 for the treatment of malignant gliomas include the insertion of therapeutic genes in E3 or retargeting to receptors that are more abundantly expressed on primary glioma cells than CAR.

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.

Increased glia-specific transgene expression with glial fibrillary acidic protein promoters containing multiple enhancer elements.

The ability to direct transgene expression to astrocytes has become increasingly important as the roles for these cells continue to expand. Promoters consisting of the 5'-flanking region of the human or mouse glial fibrillary acidic protein (GFAP) gene have generally proved satisfactory. However, a more powerful promoter would be advantageous for several applications, such as expression of dominant negative RNAs or proteins, or for gene therapy. We investigated the possibility of increasing the transcriptional activity of the human GFAP promoter by inserting into it one or three additional copies of putative GFAP enhancer regions. The promoters enhanced with three additional copies gave 75-fold higher LacZ expression levels upon plasmid transfection into GFAP-expressing U251 cells than the parental gfa2 promoter. Surprisingly, in a transgenic mouse model, the enhanced promoters resulted in no or only very low expression of marker genes, probably caused by toxicity. When various cell lines were infected with replication-deficient adenoviral vectors, the enhanced promoters gave LacZ expression levels that were approximately 10-fold higher than those with the parental gfa2 promoter, while retaining specificity for GFAP-expressing cells. Injection of the adenoviral vectors carrying the enhanced promoters into nude mouse brain showed that LacZ expression was limited to GFAP-positive cells. We conclude that gfa2 enhanced promoters are useful for production of short-term, glia-specific, high expression levels of genes in an adenoviral context. Adenoviral vectors containing these enhanced promoters may be useful in glioma gene therapy.

Paraneoplastic cerebellar degeneration associated with antineuronal antibodies: analysis of 50 patients.

Paraneoplastic cerebellar degeneration (PCD) is a heterogeneous group of disorders characterized by subacute cerebellar ataxia, specific tumour types and (often) associated antineuronal antibodies. Nine specific antineuronal antibodies are associated with PCD. We examined the relative frequency of the antineuronal antibodies associated with PCD and compared the neurological symptoms and signs, associated tumours, disability and survival between groups of PCD with different antibodies. Also, we attempted to identify patient-, tumour- and treatment-related characteristics associated with functional outcome and survival. In a 12-year period, we examined >5000 samples for the presence of antineuronal antibodies. A total of 137 patients were identified with a paraneoplastic neurological syndrome and high titre (> or =400) antineuronal antibodies. Fifty (36%) of these patients had antibody-associated PCD, including 19 anti-Yo, 16 anti-Hu, seven anti-Tr, six anti-Ri and two anti-mGluR1. Because of the low number, the anti-mGluR1 patients were excluded from the statistical analysis. While 100% of patients with anti-Yo, anti-Tr and anti-mGluR1 antibodies suffered PCD, 86% of anti-Ri and only 18% of anti-Hu patients had PCD. All patients presented with subacute cerebellar ataxia progressive over weeks to months and stabilized within 6 months. The majority of patients in all antibody groups had both truncal and appendicular ataxia. The frequency of nystagmus and dysarthria was lower in anti-Ri patients (33 and 0%). Later in the course of the disease, involvement of non-cerebellar structures occurred most frequently in anti-Hu patients (94%). In 42 patients (84%), a tumour was detected. The most commonly associated tumours were gynaecological and breast cancer (anti-Yo and anti-Ri), lung cancer (anti-Hu) and Hodgkin's lymphoma (anti-Tr and anti-mGluR1). In one anti-Hu patient, a suspect lung lesion on CT scan disappeared while the PCD evolved. Seven patients improved by at least 1 point on the Rankin scale, while 16 remained stable and 27 deteriorated. All seven patients that improved received antitumour treatment for their underlying cancer, resulting in complete remission. The functional outcome was best in the anti-Ri patients, with three out of six improving neurologically and five were able to walk at the time of last follow-up or death. Only four out of 19 anti-Yo and four out of 16 anti-Hu patients remained ambulatory. Also, survival from time of diagnosis was significantly worse in the anti-Yo (median 13 months) and anti-Hu (median 7 months) patients compared with anti-Tr (median >113 months) and anti-Ri (median >69 months). Patients receiving antitumour treatment (with or without immunosuppressive therapy) lived significantly longer [hazard ratio (HR) 0.3; 95% confidence interval (CI) 0.1-0.6; P = 0.004]. Patients > or =60 years old lived somewhat shorter from time of diagnosis, although statistically not significant (HR 2.9; CI 1.0-8.5; P = 0.06).

Survival and outcome in 73 anti-Hu positive patients with paraneoplastic encephalomyelitis/sensory neuronopathy.

In a retrospective study, we determined clinical and serological findings, associated tumours, outcome and prognostic factors in 73 Hu-Ab positive patients detected in a Dutch reference laboratory. The most frequent signs and symptoms at presentation were sensory neuropathy (55 %), cerebellar degeneration (22 %), limbic encephalitis (15 %) and brainstem encephalitis (16 %). 23 % developed autonomic dysfunction including gastro-intestinal motility disorders in 14 %. In 85 % a tumour was detected, which was a lung tumour in 77 %. Signs, symptoms and associated tumours did not differ in six patients with additional neuronal antibodies (anti-amphiphysine, anti-CV2, anti-Ri). The overall 3 months, one-year and three-year survival rates from the time of diagnosis were 64 %, 40 % and 22 %. Rankin Scale Score (RS) at diagnosis and presence of tumour at the time of diagnosis predicted mortality with hazard ratios (95 % CI) of 2.6 (1.5-4.6) and 1.5 (1.1-2). The median delay between onset of symptoms and Hu-Ab diagnosis was 4 months. There was a negative association between delay RS at diagnosis (P=0.03). In a logistic regression analysis, only older age (OR=0.15; 0.02-0.63) and a higher RS at diagnosis (OR=0.29; 0.11-0.73) were associated with a lower probability of successful functional outcome. Adjusted for these factors, antitumour therapy showed a higher but statistically not significant probability of successful outcome (OR=3.5; 0.87-14.3). Our study underlines the importance of early diagnosis and start of antitumour treatment when the patient is still in a better functional state. The delay between onset of symptoms and diagnosis of PEM/SN suggests a window for improving outcome in these patients.

Anti-Ri antibody positive opsoclonus-myoclonus in a male patient with breast carcinoma.

A 65-year-old male patient developed truncal ataxia, opsoclonus and myoclonus. In the serum anti-Ri antibodies were found, which led to the detection of a small adenocarcinoma of the breast. Other prominent clinical features were an excessive startle response and behavioral disorders, such as anxiety and impatience. These features suggest an immune response against both Nova-1 and Nova-2 antigens throughout the central nervous system.