Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance.

BACKGROUND: Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. METHODS: A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. RESULTS: Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a 'methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. CONCLUSION: Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.

Global microRNA profiles in cervical squamous cell carcinoma depend on Drosha expression levels.

Gain of chromosome 5p is seen in over 50% of advanced cervical squamous cell carcinomas (SCCs), although the genes responsible for the selective advantage provided by this abnormality are poorly understood. In the W12 cervical carcinogenesis model, we observed that 5p gain was rapidly selected over approximately 15 population doublings and was associated with the acquisition of a growth advantage and invasiveness. The most significantly upregulated transcript following 5p gain was the microRNA (miRNA) processor Drosha. In clinically progressed cervical SCC, Drosha copy-number gain was seen in 21/36 clinical samples and 8/10 cell lines and there was a significant association between Drosha transcript levels and copy-number gain. Other genes in the miRNA processing pathway, DGCR8, XPO5 and Dicer, showed infrequent copy-number gain and over-expression. Drosha copy-number and expression were not elevated in pre-malignant cervical squamous intraepithelial lesions. Importantly, global miRNA profiling showed that Drosha over-expression in cervical SCC appears to be of functional significance. Unsupervised principal component analysis of a mixed panel of cervical SCC cell lines and clinical specimens showed clear separation according to Drosha over-expression. miRNAs most significantly associated with Drosha over-expression are implicated in carcinogenesis in other tissues, suggesting that they regulate fundamental processes in neoplastic progression. Our evidence suggests that copy-number driven over-expression of Drosha and consequent changes in miRNAs are likely to be important contributors to the selective advantage provided by 5p gain in cervical neoplastic progression.

Malignant germ cell tumours of childhood: new associations of genomic imbalance.

Malignant germ cell tumours (MGCTs) of childhood are a rare group of neoplasms that comprise many histological subtypes and arise at numerous different sites. Genomic imbalances have been described in these tumours but, largely because of the paucity of cases reported in the literature, it is unclear how they relate to abnormalities in adult MGCTs and impact on potential systems for classifying GCTs. We have used metaphase-based comparative genomic hybridisation to analyse the largest series of paediatric MGCTs reported to date, representing 34 primary tumours (22 yolk sac tumours (YSTs), 11 germinomatous tumours and one metastatic embryonal carcinoma) occurring in children from birth to age 16, including 17 ovarian MGCTs. The large dataset enabled us to undertake statistical analysis, with the aim of identifying associations worthy of further investigation between patterns of genomic imbalance and clinicopathological parameters. The YSTs showed an increased frequency of 1p- (P=0.003), 3p+ (P=0.02), 4q- (P=0.07) and 6q- (P=0.004) compared to germinomatous tumours. Gain of 12p, which is invariably seen in adult MGCTs, was present in 53% of primary MGCTs of children aged 5-16 and was also observed in four of 14 YSTs affecting children less than 5. Two of these cases (14% of MGCTs in children less than 5) showed gain of the 12p11 locus considered to be particularly relevant in adult MGCTs. Gain of 12p showed a significant association with gain of 12q. Conversely, MGCTs without 12p gain displayed a significantly increased frequency of loss on 16p (P=0.04), suggesting that this imbalance may contribute to tumour development in such cases. This data provides new insight into the biology of this under-investigated tumour group and will direct future studies on the significance of specific genetic abnormalities.

Do "Shufflebottoms" bottom shuffle?

AIMS: To investigate anecdotal evidence that the name "Shufflebottom" originates from the dominantly inherited characteristic of bottom shuffling. METHODS: A questionnaire based retrospective study to determine the incidence of bottom shuffling and age of first walking among those named "Shufflebottom" and a control population, of those named "Walker". RESULTS: There was no statistically significant difference in incidence of bottom shuffling or age at first walking, between the two groups. The incidence of bottom shuffling (21.4%) was generally higher than has been described previously and Walkers were more likely to walk later than Shufflebottoms. CONCLUSION: Shufflebottoms are no more likely to bottom shuffle than other children. The origin of the surname as representing this physical characteristic cannot be confirmed.

A randomized pilot study of SRL172 (Mycobacterium vaccae) in patients with small cell lung cancer (SCLC) treated with chemotherapy.

BACKGROUND: SRL172 is a suspension of heat killed Mycobacterium vaccae, that has been found to be a potent immunological adjuvant when used with autologous cells in animal models. This is a phase II study to test the clinical activity, feasibility and safety of combining SRL172 with chemotherapy to treat patients with small cell lung cancer (SCLC). METHODS: Patients were randomized to receive chemotherapy with (n=14) or without (n=14) SRL172. The chemotherapy was either platinum-based (MVP, n=10) or anthracycline-based (ACE, n=18). SRL172 was given intradermally on day 0, weeks 4, 8 and then 3-6 monthly. RESULTS: The treatment arms were well balanced for disease extent (43% with limited stage in each arm). The toxicity of chemotherapy and overall response at 12-15 weeks (57%) was the same for both treatment regimens. Median survival was 8.6 months and 12.9 for patients treated with chemotherapy alone and with the combination respectively (P=0.10). The survival trend was similar for both disease extent and chemotherapy regimen employed in favour of combination chemotherapy with SRL172. CONCLUSIONS: There is a trend to improved median survival in SCLC with the combination of chemotherapy and SRL172 with no increased toxicity and irrespective of drug regimen. A phase III study examining chemotherapy in combination with SRL172 in SCLC is now underway.

A randomized phase II study of SRL172 (Mycobacterium vaccae) combined with chemotherapy in patients with advanced inoperable non-small-cell lung cancer and mesothelioma.

Mycobacterial preparations have been used with limited success against cancer apart from superficial bladder cancer. Recently, a therapeutic vaccine derived from Mycobacterium vaccae has been given to patients with prostate cancer and melanoma indicating a possible beneficial effect on disease activity in such patients. We have recently initiated a series of randomized studies to test the feasibility and toxicity of combining a preparation of heat-killed Mycobacterium vaccae (designated SRL172) with a multidrug chemotherapy regimen to treat patients with inoperable non-small cell lung cancer (NSCLC) and mesothelioma. 28 evaluable patients with previously untreated symptomatic NSCLC and mesothelioma were randomized to receive either 3 weekly intravenous combination chemotherapy alone, or chemotherapy given with monthly intra-dermal injections of SRL172. Safety and tolerability were scored by common toxicity criteria and efficacy was evaluated by survival of patients and by tumour response assessed by CT scanning. The toxicity of chemotherapy was similar in the two groups. SRL172 caused mild inflammation at the injection site. In the group of patients randomized to receive chemotherapy combined with SRL172, there was a trend towards improved response rate (54% vs. 33%) with more patients in the combined arm receiving radical surgery and radiotherapy, improved median survival (9.7 months vs. 7.5 months) and improved 1 year survival (42% vs. 18%). SRL172 appeared to improve sleep (P = 0.08) and improved appetite (P = 0.01). There was no detectable change in serum cytokine levels for gamma-interferon and TNF-alpha before and after treatment. In patients with NSCLC and mesothelioma, there may be a beneficial interaction when chemotherapy is administered in combination with SRL172. Confirmation of this effect and further investigation is underway in a randomized phase III trial and in laboratory models.

Functional role of position 22 in the homeodomain of Brn-3 transcription factors.

The Brn-3a POU family transcription factor activates a number of neuronal promoters which are repressed by the closely related Brn-3b factor. Although transcriptional activation by mutant forms of Brn-3a/Brn-3b can occur with a number of different amino acids at position 22 of the POU homeodomain, an isoleucine at this position is of critical importance for transcriptional repression. In addition, this isoleucine residue can mediate an interaction with the herpes simplex virus Vmw65 regulatory protein whereas the valine found in Brn-3a cannot do so. Thus, position 22 in the homeodomain plays a critical role in the function of neuronally expressed POU factors by controlling their interaction with viral and cellular co-activator/co-repressor proteins.

Successful treatment of non-familial haemophagocytic lymphohistiocytosis with interferon and gammaglobulin.

Two cases of non-familial haemophagocytic lymphohistiocytosis (HLH) are presented in which treatment with interferon alfa and gammaglobulin was associated with complete clinical remission. In one case, serological evidence of recent Epstein-Barr virus infection was found. Natural killer cell activity was within normal limits in both children, compatible with a secondary form of HLH. The combination of interferon alfa and intravenous gammaglobulin requires further evaluation in the treatment of non-familial HLH.

Cellular localization of mRNA for cellular retinoic acid-binding protein II and nuclear retinoic acid receptor-gamma 1 in retinoic acid-treated human skin.

We have previously shown that cellular retinoic acid-binding protein II (CRABP-II), but not cellular retinoic acid-binding protein I (CRABP-I), mRNA expression is markedly induced in human skin by topical retinoic acid. In the present study, we have investigated the pattern of expression of CRABP-II transcripts in 4-d RA-treated human skin by non-radioactive in situ hybridization (n = 5) and Northern analysis (n = 4). RA induced accumulation of CRABP-II transcripts throughout the epidermis, dermal fibroblasts, and endothelial cells as determined by in situ hybridization. In skin treated with vehicle, a faint hybridization signal was observed only in basal layers of the epidermis consistent with low-level expression of CRABP-II mRNA. RA-mediated accumulation of CRABP-II transcripts in skin was also confirmed by Northern analysis. Neither RA nor vehicle induced significant changes in nuclear RA receptor-gamma 1 or keratin 5 gene expression in skin as determined by in situ or Northern hybridization. These results indicate that RA-induced CRABP-II mRNA accumulation is primarily localized to spinous and granular layers in epidermis, and in superficial dermis.

Patterns of sexually transmitted enteric diseases in a city.

In the past three years cases of shigellosis, amoebiasis, and viral hepatitis A and B have increased fourfold to tenfold in San Francisco. These diseases were most common in people who had adopted "alternative life-styles", especially in young men, In many cases there was a history of frequent orogenital and oral-anal sexual contact between men with no common source. Despite energetic public-health measures and intensive efforts by physicians treating these cases, the increase has not yet been brought under control. Other cities may experience similar difficulties with sexual transmission of enteric diseases.20

Cluster analysis of preference ratings of pictorial stimuli.

The present research attempted to utilize cluster analysis to develop a comprehensive array of objective pictorial scales that could be used to identify the distinguishing perceptual and personality characteristics of various medical and psychiatric hospital populations. A cluster analysis of individuals' preference ratings of a large number of picture stimuli was described.