Metastatic alveolar rhabdomyosarcoma on the fine-needle aspiration cytology of cervical lymph node in an elderly patient, with FISH confirmation: A case report.

INTRODUCTION: Alveolar rhabdomyosarcoma (RMS) usually occurs in adolescents and young adults, and most frequently arises in the extremities. CASE REPORT: We present a rare case of metastatic alveolar RMS from a nasal primary to cervical lymph nodes (LNs) in an elderly patient, diagnosed on the fine-needle aspiration (FNA) biopsy. Smears showed malignant round cells featuring focal rhabdoid appearance, with rhabdomyoblastic differentiation further supported by immunocytochemical stains. Diagnosis of alveolar RMS was confirmed by fluorescence in situ hybridization (FISH) identifying FOXO1 gene involvement with dual colour break-apart probes at locus 13q14. DISCUSSION: The differential diagnosis for a small round blue cell tumour in the elderly generally includes metastatic small cell carcinoma, lymphoma, malignant melanoma, RMS, desmoplastic small round cell tumour and Ewing's sarcoma/primitive neuroectodermal tumour. Subtle morphological analysis and expression pattern of immunostaining for skeletal muscle differentiation led to the diagnosis of RMS. Cytogenetic testing on the FOXO1 gene rearrangement helps definite subtyping of alveolar RMS.

The incidence and patterns of BCR/ABL rearrangements in chronic myeloid leukaemia (CML) using fluorescence in situ hybridisation (FISH).

INTRODUCTION: Chronic myeloid leukaemia (CML) is characterised by the formation of the BCR/ABL fusion gene, usually as a result of the Philadelphia (Ph) translocation between chromosomes 9 and 22. MATERIALS AND METHODS: The incidence of both typical and atypical BCR/ ABL gene rearrangements was determined in 110 patients suspected of CML using dual fusion fluorescence in situ hybridisation (DF-FISH) probes. RESULTS: Eighty-seven per cent of CML patients showed Ph translocation while 13% were negative for the Ph chromosome. About 71.9% of Ph-positive patients displayed the typical DF-FISH signal pattern. Atypical patterns among the Ph-positive patients included the concurrent loss of residual proximal 9q and distal 22q (10.4%), complex translocation with additional partners (9.4%), supernumerary Ph (3.1%), loss of residual 9q sequences proximal to breakpoint (3.1%), and deletion of distal derivative 22q signal (2.1%). Cryptic genetic alterations with loss of proximal 9q sequences were found in 13.5% of CML Ph-positive patients, which is associated with poor prognosis. Fusion signals were detected in 57.1% of CML Ph-negative patients, indicating cryptic BCR/ABL rearrangements (i.e., masked Ph). CONCLUSION: FISH is able to detect BCR/ABL fusion in CML with masked or variant Ph not apparent with conventional karyotyping. Establishment of signal patterns with FISH is important as atypical patterns may have clinical prognostic implications.

The importance of high resolution chromosome analysis in the diagnosis of birth defects: case reports of holoproscencephaly and cystic hygroma.

INTRODUCTION: The goal of cytogenetics is the detection of chromosomal abnormalities, achieved by the analysis of adequate numbers of metaphases at the appropriate bands per haploid set (BPHS). CLINICAL PICTURE: Two cases presented here include a foetal blood sample (FBS) of a 33-week-old referred with holoproscencephaly by ultrasonography, and an amniotic fluid (AF) specimen of a 14-week-old foetus with cystic hygroma, cardiac and renal defects. OUTCOME: The FBS had a deletion at 18p11.31. Another laboratory had earlier given a normal cytogenetic result on its AF sample. In the second case, an unbalanced 46,XY,der(5)ins(5;3) (q33.1;q26.2q27)mat karyotype was obtained with the AF sample. In both cases, the abnormalities were more obvious when band levels were > or =450 BPHS. CONCLUSION: This report underscores the importance of obtaining longer chromosome preparations above the current recommended 400 BPHS for prenatal specimens. This is particularly important in cases with abnormal ultrasound findings suggestive of an underlying chromosomal pathology.

Sperm segregation patterns by fluorescence in situ hybridization studies of a 46,XY,t(2;6) heterozygote giving rise to a rare triploid product of conception with a 69,XXY,t(2;6)(p12;q24)der(6)t(2;6)(p12;q24)pat karyotype.

A blighted ovum diagnosed initially by ultrasound was determined to be a partial hydatidiform mole with a 69,XXY,t(2;6)(p12;q24)der(6)t(2;6)(p12;q24)pat karyotype by cytogenetic analysis. The triploid state arose through dispermy in which both spermatozoa carried rearranged chromosomes, one carrying a balanced translocation through alternate segregation and the other an unbalanced derivative chromosome 6 through adjacent 1 segregation. Segregation analysis of 7,000 spermatozoa from the father was performed with a three-color fluorescence in situ hybridization (FISH) protocol using alpha-satellite 6, telomeric 2p, and telomeric 6q probes. Segregation frequencies of normal and balanced products (alternate segregation), adjacent 1, adjacent 2, and 3:1 were 49.9%, 42.4%, 2.5%, and 4.2%, respectively. The high percentage of alternate segregation is consistent with the knowledge of their preferential outcome. However, the high incidence of adjacent 1 sperm highlights the abnormality risk. Alternate and adjacent 1 segregations (92.3%) accounted for the observed rearranged chromosomes in the triploid. The most viable imbalanced combination would be the one carrying the der(6) chromosome, but since the unbalanced segment comprises 3.6% of the haploid autosomal length (HAL), no risk of a viable imbalanced offspring is indicated. However, an increased likelihood of recurrent miscarriages is likely, and this is confirmed by the couple's two earlier miscarriages. Sperm segregation patterns of translocation carriers determined by FISH can help in ascertaining expected and unexpected karyotypes. The high frequency of adjacent 1 products shows that the presence of the additional derivative chromosome in the partial mole, though rare in occurrence, should be less surprising.

Rapid one-day fluorescence in situ hybridisation in prenatal diagnosis using uncultured amniocytes and chorionic villi.

Rapid assays for the detection of the common chromosomal aneuploidies using DNA probes to chromosomes 13, 18, 21, X and Y have been developed in recent years. In this study, we report our experience using fluorescence in situ hybridisation (FISH) with uncultured amniocytes from amniotic fluids (AF) and cells from chorionic villus sampling (CVS) for prenatal diagnosis in 239 assays. Only 2 ml of AF or 5 mg of chorionic villi were required for FISH analysis using region-specific probes to chromosome 13, 18, 21, X and Y. Results were informative in all the assays conducted in this study. The average time to obtain a result was 24 hours. However, the results for chromosome X, Y and 18 were ready within four hours. The criteria employed for result interpretation were stringent. In a normal finding, at least 80% of all nuclei observed must have 2 discrete signals per cell. In a suspected abnormal case, a minimum of 60% of all nuclei observed must have one (monosomy X) or 3 signals (trisomy) per cell. One trisomy 13 and two trisomy 21 cases were detected. All FISH results were confirmed by karyotyping. Subsequent karyotyping of the resultant abortuses gave further confirmation of the results. The results obtained showed 100% positive and negative predictive values. This study shows that FISH can be used as a rapid routine assay in conjunction with full karyotyping analysis. Its prognostic value is especially important for patients with late referrals, parental anxiety, high-risk pregnancies identified by maternal serological screening and suspected foetal abnormalities detected by ultrasonographic examination.

Effect of pupillary dilation on retinal nerve fiber layer thickness as measured by scanning laser polarimetry in eyes with and without cataract.

PURPOSE: This study was conducted to evaluate the effect of pupillary dilation on retinal nerve fiber layer (RNFL) thickness as measured by scanning laser polarimetry (SLP) in cataractous and noncataractous eyes. METHODS: The study included 31 eyes of 31 consecutive patients (mean age, 62.5 +/- 14.0 years; range, 30-76 years). Eyes with refractive error exceeding 5.0 D sphere or 2.0 D cylinder, nonlenticular media opacity, cup-to-disc ratio > 0.9, corneal disease, ocular inflammation, or previous intraocular surgery were excluded. A standard reticule was used to measure pupillary diameter. Cataract grade was evaluated by a single observer using the Lens Opacities Classification System (LOCS III). RNFL thickness measurements were obtained by means of SLP before and after pupillary dilation. RESULTS: Of the patients, 10 had clear lenses and 21 had variable degrees of lenticular opacification. In four eyes, imaging could not be performed because of dense cataracts. Mean pupillary diameters before and after dilation were 2.5 +/- 0.7 mm and 7.3 +/- 1.1 mm, respectively. There were no significant differences in global RNFL thickness before and after dilation in noncataractous and cataractous eyes. Among cataractous eyes in which imaging was possible, there was no correlation between difference in RNFL thickness before and after dilation and nuclear opalescence, nuclear color, and cortical and posterior subcapsular grading of the LOCS III score. Six of 27 eyes (22.2%) had a change of more than 10% in RNFL thickness after pupillary dilation. CONCLUSION: Although pharmacologic mydriasis does not statistically alter RNFL thickness as measured by SLP, approximately one fifth of such eyes will have a change of more than 10% in retardation. Uniformity in pupil size is recommended when longitudinally evaluating RNFL measurements.

Randomized clinical trial of a new dexamethasone delivery system (Surodex) for treatment of post-cataract surgery inflammation.

OBJECTIVE: To evaluate the safety of Surodex Drug Delivery System (Oculex Pharmaceuticals, Inc., Sunnyvale, CA) containing dexamethasone 60 micrograms, for use in cataract surgery, and to compare its anti-inflammatory efficacy with conventional dexamethasone 0.1% eyedrops. DESIGN: Randomized, masked, and partially controlled trial. PARTICIPANTS: Sixty eyes of 60 Asian patients undergoing extracapsular cataract extraction with intraocular lens implantation were examined. Of these, 28 eyes of 28 patients served as control eyes. Patients were stratified for age and presence of diabetes mellitus. INTERVENTION: Surodex was inserted in the anterior chamber of 32 eyes at the conclusion of surgery. These eyes received placebo eyedrops four times a day after surgery for 4 weeks. Control eyes received neither Surodex nor a placebo implant but were prescribed conventional 0.1% dexamethasone eyedrops four times a day for 4 weeks. MAIN OUTCOME MEASURES: Anterior chamber cells and flare were clinically graded at the slit lamp. Anterior chamber flare was objectively assessed with the Kowa FM500 Laser Flare Meter (Kowa Co. Ltd, Tokyo, Japan) for up to 3 months after surgery. Intraocular pressure and corneal endothelial specular microscopy with morphometric cell analysis were performed for up to 1 year after surgery. RESULTS: Clinical slit-lamp assessment of anterior chamber flare and cells showed no difference between Surodex-treated eyes and dexamethasone eyedrop-treated eyes. Flare meter readings showed lower flare levels in the Surodex group at all postoperative visits compared with the dexamethasone eyedrop group. Flare reduction in the Surodex group reached statistical significance at days 4, 8, 15, and 30 after surgery. At 3 months, flare was reduced to preoperative levels in the Surodex group but was still raised in the dexamethasone eyedrop group. Five eyes in the dexamethasone eyedrop group required augmentation of steroids and were deemed therapeutic failures as opposed to one eye in the Surodex group. One patient in the dexamethasone eyedrop group developed postoperative open-angle glaucoma with profound visual field loss and optic disc cupping, resulting in hand movements vision. No significant difference in endothelial cell loss was noted between Surodex-inserted eyes and dexamethasone eyedrop-treated eyes for up to 1 year after surgery. CONCLUSIONS: Intraocular placement of a single Surodex is a safe and effective treatment method to reduce intraocular inflammation after cataract surgery. There was no statistical difference in efficacy between Surodex and 0.1% dexamethasone eyedrops in reducing intraocular inflammation, as measured by clinical methods, while Surodex was clearly superior to eyedrops in reducing aqueous flare as objectively assessed with the laser flare meter.

Effect of pterygium morphology on pterygium recurrence in a controlled trial comparing conjunctival autografting with bare sclera excision.

OBJECTIVES: To compare success rates of conjunctival autografting and bare sclera excision for primary and recurrent pterygium in the tropics and to evaluate risk factors for pterygium recurrence. METHODS: A prospective, controlled clinical trial was performed in which 123 primary and 34 recurrent pterygia, matched for age and pterygium morphology, were randomized in 2 separate studies to receive either bare sclera excision or conjunctival autograft. The surgical procedures were performed by one surgeon and reviewed at 1, 3, 6, and 12 months after surgery by an independent observer. Pterygium morphology was clinically graded as atrophic, intermediate, or fleshy according to an assessment of pterygium translucency. Risk factors were assessed using likelihood ratio tests. Weibull curves were used to estimate recurrence rates allowing for the interval censoring. RESULTS: In the group with primary pterygium (mean follow-up, 15.1 months), 38 (61%) of the 62 cases of bare sclera excision (heretofore referred to as the bare sclera group) had pterygium recur in contrast with 1 (2%) of the 61 cases of conjunctival autograft (heretofore referred to as the conjunctival autograph group) (P<.001, likelihood ratio X2 test). Nontranslucency, or fleshiness of the pterygium, and not age was a significant risk factor for recurrence in the bare sclera group (P<.001, likelihood ratio X2 test). In the group with recurrent pterygium (mean follow-up, 13.2 months), 14 (82%) of the 17 bare sclera group had pterygium recur, while no recurrences occurred among 17 cases in the conjunctival autograft group. Nontranslucency was again a highly significant factor for recurrence (P<.001, likelihood ratio X2 test). CONCLUSIONS: Pterygium recurrence is related to pterygium morphology and fleshiness of the pterygium is a significant risk factor for recurrence if bare sclera excision is performed. Conjunctival autografting for primary and recurrent pterygium is effective in reducing pterygium recurrence compared with bare sclera excision.

CD28-induced cytokine production and proliferation by thymocytes are differentially regulated by the p59fyn tyrosine kinase.

CD28 is a 44-kDa homodimeric receptor that is expressed on the majority of T cells. Engagement of the CD28 receptor by soluble anti-CD28 mAb in conjunction with phorbol ester (PMA) induces the production of cytokines and the proliferation of resting T cells via signal transduction pathways independent of the TCR. Evidence is provided herein that CD28 signals leading to cytokine production do not require the p59fyn (Fyn) tyrosine kinase, whereas CD28-mediated proliferation is dependent on the presence of the Fyn kinase in thymic, but not lymph node, cells. The defect in proliferation is not due to failure of IL-2R signaling, since addition of high concentrations of exogenous IL-2 can overcome the proliferative defect. Analysis of CD28-directed induction of the IL-2R alpha (CD25)-chain, which confers high affinity binding to IL-2, showed that Fyn-deficient thymocytes, but not lymph node cells, failed to up-regulate CD25 expression following anti-CD28 and PMA stimulation. Thus, the Fyn tyrosine kinase is critically required for thymic CD28-mediated CD25 expression and proliferation but not for CD28-mediated cytokine production.

Molecular studies of loss of heterozygosity in retinoblastoma.

Retinoblastoma (Rb) is a paediatric intraocular tumour in which predisposition can be inherited. Cases of Rb tumours can be divided into three types: familial cases, sporadic bilateral cases and sporadic unilateral cases. Familial and sporadic bilateral cases are usually categorised as hereditary while sporadic unilateral cases as non-hereditary. In both familial and non-familial forms of Rb, loss of heterozygosity of the Rb locus has been reported although its frequency in tumours has not so far been accurately determined. The cloning of the gene responsible for retinoblastoma (Rb1) has facilitated DNA studies and genetic counselling of patients. We have examined forty-five cases of retinoblastoma at five intragenic sites of the Rb1 locus, namely intron 1/BamH1, intron 17/Xba1, intron 24/TthIII1, intron 25/Dra1 and Rb1.20 VNTR. Thirty-six out of the forty-five cases (80%) were informative for these markers. Comparison of results between genomic DNA from peripheral blood and from tumours revealed that loss of heterozygosity of alleles could be detected in 50% of cases studied in which tumour samples were available. Investigation of parental origin of retained alleles showed that in all these cases, the paternal alleles were preferentially retained. The analysis of the genetic origin of mutations predisposing to retinoblastoma can facilitate new approaches for identifying recessive mutant genes that lead to cancer as well as to provide a conceptual basis for accurate prenatal predictions of cancer predisposition.

Factors influencing perfect surgical outcome.

With affluence and education, the population of Asia will be demanding quality surgical care. The energetic, affluent and educated Asian professionals and business communities in the cities demand the best; and in surgery, they seek perfect results. Perfect results require a combination of 3 factors: the skill, knowledge and experience of the surgeon. He must be a skilled surgeon with good basic surgical techniques and also technical skills in the management of his discipline combined with meticulous attention to details. Furthermore, he must have a clear knowledge of the basic physiopathology of surgical principles of the condition he is to manage. Experience with difficult situations and intrasurgical problems are essential for success.

Abnormal expression of the p53 tumor suppressor gene in the conjunctiva of patients with pterygium.

PURPOSE: To determine whether pterygium is a disorder of abnormal growth by examining the expression of the p53 gene in the conjunctiva of patients with pterygium. METHODS: Immunostaining for abnormal expression of p53 was performed using mouse monoclonal antibody to human p53, pAb 240, on six eyes with primary pterygium and two eyes with recurrent pterygium. RESULTS: In three of the eight eyes with pterygium, specimens were positive for abnormal expression in the epithelium of the pterygium and in the superior bulbar conjunctiva. CONCLUSION: Abnormal p53 expression in the epithelium of primary and recurrent pterygium specimens suggests that pterygium is a growth disorder rather than a degeneration.

Comparison of argon laser iridotomy and sequential argon laser and Nd:YAG laser iridotomy in dark irides.

BACKGROUND AND OBJECTIVE: A prospective study was performed to compare argon laser iridotomy and sequential argon laser and Nd:YAG laser iridotomy in dark irides. PATIENTS AND METHODS: Twenty-four eyes of 17 patients underwent laser iridotomies; 13 underwent argon laser iridotomies and 11 underwent sequential argon laser and Nd:YAG laser iridotomies. The argon laser settings were standardized at 1.2 W, 50-mum spot size, and 0.1-second duration. The Nd:YAG laser was set at 2.5 mJ and single-pulse shots were used. RESULTS: All of the iridotomies were patent at the end of one treatment session. The mean total energy used for argon laser iridotomy was 8.28 J. For sequential iridotomy, 3.12 J was used for the argon laser stage and 7.5 mJ for the Nd:YAG laser stage. CONCLUSION: The total argon laser energy used can be reduced by 2.65 times by using the sequential laser iridotomy technique.

Case report--ocular rosacea.

Rosacea is an uncommon disease of the eye and facial skin. Ocular rosacea is often undiagnosed by the ophthalmologist especially when skin manifestations are not evident yet. Early diagnosis and treatment is important to decrease morbidity of this potentially blinding disease. A case of ocular rosacea in a 14-year-old Chinese girl is reported. Our patient presented with chronic non-specific keratoconjunctivitis. Only much later did the characteristic corneal and facial skin lesions appear. She responded to guttae prednisolone, oral and guttae tetracycline. This case illustrates the difficulty of early diagnosis when ocular manifestations precede those of the skin. We believe this is the first case of ocular rosacea reported in Singapore.