The novel human MOST-1 (C8orf17) gene exhibits tissue specific expression, maps to chromosome 8q24.2, and is overexpressed/amplified in high grade cancers of the breast and prostate.

AIMS: To elucidate genes that participate in the process of oncogenesis, primers based on the E6 genes of genital human papillomaviruses (HPVs) were used to amplify potential expressed sequence tags (ESTs) from the MOLT-4 T lymphoblastic leukaemia cell line. METHODS: Using the polymerase chain reaction (PCR) with human papillomavirus E6 gene primers, an EST from the MOLT-4 T lymphoblastic leukaemia cell line was amplified. Via rapid amplification of cDNA ends (RACE) and cycle sequencing from MOLT-4 and fetal lung cDNA libraries, overlapping cDNAs of 2786 bp and 2054 bp of the corresponding novel human intronless gene designated MOST-1 (for MOLT-4 sequence tag-1) were characterised and assigned the symbol C8orf17 by the HUGO Nomenclature Committee. RESULTS: Both cDNAs contained a potential open reading frame (ORF) of 297 bp incorporating a methionine codon with an ideal Kozak consensus sequence for translation initiation, and encoding a putative hydrophilic polypeptide of 99 amino acids. Although reverse transcription PCR (RT-PCR) demonstrated MOST-1 expression in all 19 cancer and two normal cell lines tested, differential expression was seen in only nine of 16 normal tissues tested (heart, kidney, liver, pancreas, small intestine, ovary, testis, prostate, and thymus). A 388 bp fragment was amplified from the NS-1 mouse myeloma cell line, the sequence of which was identical to that within the MOST-1 ORF. The MOST-1 gene was mapped by fluorescent in situ hybridisation to chromosome 8q24.2, a region amplified in many breast cancers and prostate cancers, which is also the candidate site of potential oncogene(s) other than c-myc located at 8q24.1. Analysis of paired biopsies of invasive ductal breast cancer and adjacent normal tissue by semiquantitative and real time RT-PCR revealed average tumour to normal ratios of MOST-1 expression that were two times greater in grade 3 cancers than in grade 1 and 2 cancers. Quantitative real time PCR of archival prostatic biopsies displayed MOST-1 DNA values that were 9.9, 7.5, 4.2, and 1.4 times higher in high grade carcinomas, intermediate grade carcinomas, low grade carcinomas, and benign hyperplasias, respectively, than in normal samples. CONCLUSIONS: These data suggest a role for MOST-1 in cellular differentiation, proliferation, and carcinogenesis.

Corneal transmissibility of diode versus argon lasers and their photothermal effects on the cornea and iris.

PURPOSE: Diode laser (810 nm) may possess theoretical advantages over the argon blue-green laser (488 nm) for iridotomy/iridoplasty in an eye with oedematous cornea, such as the acute angle-closure glaucoma (AACG) patient, because of better diode laser tissue penetration in opaque media. We assessed the transmissibility of diode and argon lasers through corneas of varying clarity and evaluated the histopathological features of cornea and iris burns produced by these lasers. METHODS: The transmission of diode and argon lasers through human donor corneal buttons of three grades of clarity--clear, intermediate, and hazy--were compared. Corneal buttons of these varying levels of clarity were also treated with argon and diode lasers, with the beams deliberately focused onto the mid-stroma to assess their photothermal effects. Exposed pigmented irides from whole human eyes were treated directly with argon and diode lasers. The lasers were delivered via slit-lamp systems and the energy settings used were 1000 mW for argon and 980 mW for diode; spot sizes for both lasers were 100 microm, with exposure durations of 0.1 s. Light microscopy studies of these tissues were performed. RESULTS: Transmissibility of diode laser in clear, intermediate, and hazy corneas were 89, 87 and 85% respectively and was significantly superior to argon laser (78, 73 and 70% respectively; P < 0.001, paired Student's t-test). Diode laser did not produce morphological changes in all three grades of corneas whereas argon-laser-treated hazy corneas showed photothermal damage. Both lasers produced deep iris burns, with the diode laser tending to produce deeper burns. CONCLUSION: Our findings suggest that diode laser may be the ideal laser for iridotomy/iridoplasty in the AACG patient with hazy cornea.

Bacillus Calmette-Guerin (BCG) in the treatment of superficial bladder cancer.

We evaluated the efficacy of immune and scarification Bacillus Calmette-Guerin (BCG) in the treatment of carcinoma in situ and prophylaxis against recurrence in patients with superficial transitional carcinoma of the bladder. A single-blind, randomised, comparative trial involving 43 patients with a median follow-up of 39 months was analysed. The end points were progression to muscle invasive disease or recurrence. The overall response rate was 93% after one to two courses. There was no difference between the two preparations and no statistically significant difference between the response or progression rates of the carcinoma in-situ or prophylactic groups. However, the response to BCG was found to be a significant prognostic indicator in a multivariate analysis.

EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif.

Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly alpha-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine "finger" motifs. The COOH-terminal fingers, Cys-X2-Cys-X12-Cys-X2-Cys and Cys-X2-Cys-X16-Cys-X2-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.

The c-erbA beta thyroid hormone receptor. Expression and cDNA sequence analysis of the hormone-binding domain in human cancer cell lines.

The human c-erbA beta protooncogene encodes a thyroid hormone receptor (comprising a hormone-binding domain and a DNA-binding domain) which modulates expression of specific genes, such as cell differentiation genes. Using the reverse transcription and polymerase chain reaction (RT-PCR) assay, significant expression of the c-erbA beta gene was detected in the SiHa, CaSki, HeLa cervical carcinoma; Hep3B, PLC/PRF/5, Mahlavu hepatocellular carcinoma; HT-1080 fibrosarcoma cell lines; as well as in normal MRC-5 embryo lung and FS-4 foreskin fibroblast cell lines. However, the Molt-4 leukaemia and Raji Burkitt's lymphoma cell lines exhibited very low levels of c-erbA beta expression. Single-strand conformation polymorphism analysis and direct sequencing of PCR products of the c-erbA beta hormone-binding domain cDNAs of these cell lines revealed identical sequences, but differed from the published human placental c-erbA beta sequence by five single base disparities. Sequencing of an aberrant fragment fortuitously amplified from the HT-1080 cDNA library demonstrated concordance with the cDNA of pregnancy-specific glycoprotein 4, which is related to the tumour marker, carcinoembryonic antigen.

The third zinc finger of the WT1 gene is mutated in Wilms' tumour but not in a broad range of other urogenital tumours.

Aberrations in the WT1 tumour suppressor gene have been documented in a fraction of Wilms' tumours (WTs). Encoding a protein with four zinc fingers, the WT1 gene is expressed in the developing kidney, gonads, uterus, spleen, mesothelium and brain. Using polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing, we analysed 156 diverse tumours for abnormalities of zinc finger 3 (ZF3), a mutational hotspot in WT1. Only one sample (WT) exhibited PCR-SSCP mobility shift. A CGA to TGA nonsense mutation at codon 390 with arginine being substituted with a stop codon was detected and predicted to encode a faulty WT1 protein in this WT, out of 8 WTs studied. Our results are consistent with the presence of WT1 ZF3 mutations in a subset of WTs, but not in other tumours of urogenital nor of WT1-related origin.

Alternative splicing of the p53 tumor suppressor gene in the Molt-4 T-lymphoblastic leukemia cell line.

The expression of the p53 tumor suppressor gene in ten human cell lines (nine cancers and one normal) was studied using reverse transcription, polymerase chain reaction (PCR) and direct sequencing. Using P53U and P53D primers for amplifying a 371-base pair (bp) target fragment spanning exons 7-10 of p53 cDNA, normal-sized PCR products were amplified from 9 cell lines but not from the Hep3B hepatocellular carcinoma (HCC) cell line. An additional larger band (504 bp) was observed for the Molt-4 T-lymphoblastic leukemia cell line. Employing P531 and P53D primers which flank a 76-bp p53 cDNA fragment, 76 bp as well as 209 bp products were generated by PCR of Molt-4 cDNA. Direct sequencing of the 504 bp and 209 bp bands confirmed the presence of a 133 bp insertion between exons 9 and 10 in the aberrant transcript. This insertion was homologous to a 130-bp sequence within the wild-type p53 intron 9, except for 2 point mutations and 3 base insertions. Sequencing of P53U/P53D PCR products of Molt-4 genomic DNA revealed an 8 bp deletion just downstream to the 133 bp insertion, creating a novel donor splicing site within intron 9. This site, coupled with an inherent acceptor splicing site just upstream to the 133 bp insertion, suggests that the 133 bp stretch represents an alternative exon. The occurrence of a termination signal within this alternative transcript is predicted to culminate in a truncated p53 translational product. The sequences of the 371 bp PCR products of Molt-4, HT-1080, SiHa, CaSki, HeLa and MRC-5 cell lines corresponded with the wild-type p53 cDNA. G-->T transversions at the third base of codon 249 of p53 were detected in Mahlavu and PLC/PRF/5 HCC lines, while a TAC to CAC mutation at codon 234 was observed in an allele of the Raji Burkitt lymphoma line.

Intravesical chemotherapy for superficial carcinoma of the bladder.

Intravesical chemotherapy has been shown to be of value in the treatment of superficial transitional cell carcinoma of the bladder, not only in the prevention of recurrence but possibly progression of the disease to higher stage as well. At the Department of Surgery, National University of Singapore from 1980 to 1986 we had used intravesical chemotherapy for multiple or recurrent superficial carcinoma of bladder in 45 patients. Of these, 21 patients had associated carcinoma in situ. Initially, thiotepa was used as the main intravesical chemotherapeutic agent. Since 1984, mitomycin C was introduced. The schedule used is 30 mg in 30 mg of water, and left in the bladder for 2 hours weekly for 4 weeks. Intermittent courses were given when deemed necessary on follow-up cystoscopy at 3 to 6 months. Patients were deemed to have good response if there was no evidence of tumour on cytology and biopsy at follow-up cystoscopy. Eleven patients had thiotepa only, of these 4 had good response, 4 were stable and 3 had progression of disease to higher stage. Thirty-four patients had mitomycin therapy. Thirteen of them following thiotepa treatment. Twenty-one patients (64%) had good response to therapy. Three patients (9%) had progression of disease, requiring cystectomy. Of those who responded to therapy, none had developed muscle invasive disease so far with mean follow-up of 43 months. Of the group of patients treated with mitomycin, no patient developed myelosuppression.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemorrhagic papillary cystadenoma of the seminal vesicle mimicking giant seminal vesicle cyst: MRI appearances.

Papillary cystadenoma of the seminal vesicle is very rare. We describe such a case presenting in a 58 year old man with bladder outlet obstruction. Investigations included magnetic resonance imaging (MRI), the usefulness of which in pre-operative diagnosis is highlighted in this case. Seminal vesicle cysts can usually be identified by conventional radiological imaging techniques such as ultrasound and computed tomography; however, identification would be difficult if the cyst is very large, causing distortion of the adjacent anatomy. In such cases, MRI, through coronal and sagittal scanning, can be helpful in localising the lesion, as in this patient. The precise pathological nature of the cyst can only be confirmed by biopsy.

Diagnostic sensitivity of polymerase chain reaction and Southern blot hybridization for the detection of human papillomavirus DNA in biopsy specimens from cervical lesions.

Human papillomaviruses (HPVs) are associated with benign and malignant neoplasms of the cervix. One of the criteria for their etiologic role requires an assessment of whether virtually all or only a small fraction of lesions contain viral genomes. DNA preparations from colposcopically directed punch biopsies of cervical lesions were analyzed by Southern blot hybridization and the polymerase chain reaction (PCR) for the presence of HPV DNA. The biopsy specimens represented different pathologic entities (koilocytosis, condyloma, cervical intraepithelial neoplasia, and invasive carcinoma). In Southern blot hybridization with radioactive probes for HPV 11, 16, 18, 31, and 33, HPV DNA was detected in 74% of the biopsy specimens (42 of 57 cases), with the predominant types being HPV 16 and HPV 18. In contrast, after PCR amplification with primers yielding fragments of characteristic size for HPV 11, 16, and 18, the analysis of the same 57 biopsy specimens revealed that all samples were positive for at least one HPV type. To exclude false-positive PCR results, controls without HPV DNA were interspersed at regular intervals, and results were evaluated only if these controls remained HPV negative. To exclude false-negative results due to failure of the reaction, a target sequence within the c-Ha-ras-1 gene was used as an internal control. All HPV typing results obtained by Southern blot hybridization were in agreement with HPV typing by PCR. The higher number of positive samples in the latter analysis stems from the increased sensitivity of PCR, which was which was effective in identifying as few as 10-100 HPV DNA molecules; in contrast, the sensitivity of Southern blot hybridization was 1 pg, or approximately 10(5) molecules of HPV DNA. The authors conclude that, with sufficiently sensitive diagnostic methods, HPV DNA can be detected in most, if not all, neoplastic cervical lesions.

Effects of pulsed Nd:YAG laser on the surface of rabbit retina.

Six albino rabbits were subjected to pulsed Nd:YAG laser irradiation to the retinal surface to determine whether such treatment would lead to proliferative vitreoretinopathy. Choroidal, retinal, and preretinal hemorrhages, noted at the time of treatment, resolved after 7 to 10 days. Histological examination showed no signs of anterior segment damage or proliferative vitreoretinopathy. These preliminary findings suggest that more extensive experimentation is warranted to determine if pulsed Nd:YAG laser may in fact safely be used to separate vitreoretinal adhesions in the treatment of retinal detachment.

Ovarian cancer in Oriental women from Singapore: disease pattern and survival.

A retrospective analysis of 88 ovarian tumours diagnosed over five years (1980-1984) in multi-racial Singaporean Oriental women revealed 70 (79%) epithelial tumours (ET), 17 (8%) sex cordstromal (SCS), six (7%) germ cell (GCT) and five (6%) secondary (metastatic) (SEC) cancers. The racial proportions and histological types found in the study were very similar to those of all Singaporean women in the population. Of 70 ET, 33 (47%) were stage I, five (7%) stage II, 30 (43%) stage III and two (3%) stage IV. In 97% surgical resection of primary tumour with/without removal of the uterus and opposite ovary was performed, followed by adjuvant chemotherapy in 76% and complete follow-up in 98%. The 5-year actuarial survivals in ET were in stage I 84% (low malignant potential 100% and frank carcinomas 70%), stage II 60%, stage III 29% and 0% in stage IV, whereas of the others none with SCS but four with GCT and all five with SEC died of disease. Though the incidence of ovarian cancer is much lower in Oriental than Caucasian women the proportion of different histological types, stage at presentation and survival from ovarian cancer in Oriental women does not differ from that in Caucasians.

Carcinoma in situ of the urinary bladder--the Singapore experience.

Carcinoma in situ of the urinary bladder has been described since 1952. However, it was not well recognised as a clinical entity until recently when we are more aware of its clinical significance, that a large proportion progresses to muscle invasive cancer and becomes life threatening, and therefore more aggressive management is needed. From 1980 to 1984 over a period of 5 years, we studied the records of 130 patients with transitional cell carcinoma of the urinary bladder. Eighty-two (63%) were staged as superficial carcinoma while 48 (37%) were diagnosed as muscle invasive cancer (Tables II, III & IV). Out of the 82 cases of superficial carcinoma, 12 (11%) were found to have associated carcinoma in situ of the bladder. Diagnosis depends on a high index of suspicion, urinary cytology and biopsies of not only the obvious papillary or solid tumours but also any abnormal bladder mucosa and random bladder biopsy. Management is a problem and controversial. We have been using intensive intravesical chemotherapy with thiotepa and mitomycin, and if there is evidence of deep muscle invasion, then more aggressive therapy such as cystectomy would be advised. Management of these 12 cases and the problems we encountered are discussed.

Major salivary gland tumours.

Forty-one major salivary gland tumours and tumour-like glandular enlargements are reviewed. In nine patients the swellings could not be differentiated from a true neoplasm. The diagnostic and therapeutic approaches to these patients are discussed. The clinical features, diagnostic and surgical management of 31 salivary neoplasms involving the parotid and submandibular glands are correlated with their histological features.