FISH patterns of ROS1, MET, and ALK with a correlation of ALK immunohistochemistry in lung cancer: a case for introducing ALK immunohistochemistry ‘Equivocal’ interpretation category in the Ventana anti?ALK (D5F3) CDx assay ? A tertiary cancer center experience

Background: Mutations in ROS1, ALK, and MET genes are targetable alterations in non-small cell lung cancer (NSCLC). They can be evaluated by different techniques, most commonly fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Methods: We explored the prevalence of ROS1, ALK, MET mutations, discuss clinicopathological associations and FISH signal patterns on 413 consecutive cases of EGFR negative lung carcinoma from March 2016 to April 2017 using FISH for ALK, ROS1, and MET along with ALK (D5F3) IHC. Results: ROS1 gene rearrangement, ALK positivity (IHC and/or FISH), and MET amplification were seen in 18/358 (5%) cases, 76/392 cases (19.4%), and 10/370 (2.7%) cases, respectively. ALK FISH and ALK IHC were positive in 51/300 (17%) and 58/330 cases (17.57%), respectively, while 8/330 (2.4%) cases were ALK IHC “equivocal” of which 3/8 (37.5%) were ALK FISH positive. Of ALK FISH and IHC co-tested cases, 43/238 (18.07%) cases were positive by both techniques, while 15/43 (34.88%) of ALK positive cases showed discordant ALK FISH and IHC results. All ROS1 rearranged and MET amplified cases were adenocarcinoma. Signet ring cell histology was associated with 78.57% likelihood of being either ALK or ROS1 positive. Genomic heterogeneity was seen in 30% of MET amplified cases. Conclusions: ALK/ROS1/MET gene alterations were found in 25.18% of NSCLC cases. An ALK IHC “equivocal” interpretation category should be incorporated into practice. Atypical patterns of ROS1 and genomic heterogeneity need to be evaluated further for any clinical relevance.

Evaluation of Meat Consumption Pattern and Meat Quality in North Indian Cities

A survey was conducted in north Indian cities (Ghaziabad, Noida, Gurugram, Delhi, Lucknow and Bareilly) with an aim to evaluate meat consumption pattern and meat products quality. Retailers from 15 randomly chosen shops, viz., Shop-A, B, C...O and 403 consumers visiting those shops were interviewed. Sample meat products from 10 shops were collected and quality evaluated. Among retailers, 73.33% told meat Kabab (chicken and chevon), is the most sold out product. As per 50.37% & 34.24% consumers chicken and goat meat was 1st and 2nd preference of meat species respectively, 53.46% of consumers preferred “taste” as their first criteria of meat products purchase, 62.85% consumers preferred convenience meat products as compared to traditional counterparts, highest percentage of (43.42%) consumers chosen seekh kabab (chicken and chevon) as their first preference of product. Evaluation of market products quality revealed that, maximum and minimum protein percentage in Shop-A, Chicken Kabab (16.73%) and Shop-C, Chicken Kabab (5.32%) respectively. Cholesterol content found maximum and minimum amount in Mutton Kabab from Shop-J (60.72 mg/100g) and Chicken Sausages from Shop-N (33.54 mg/100g) respectively. Calcium content found highest in Chicken Kabab from Shop-C (1.63%). Highest Total Plate Count (TPC) detected in chicken Kabab from Shop-D (5.41 log 10 cfu/g)

Introduction of a teaching module on intravenous drug administration and communication skills in pharmacology

Background: Pharmacology practicals mainly focus on increasing the knowledge component but teaching on psychomotor and soft skills is largely lacking. Teaching correct method of intravenous drug administration and the communication skills about drug therapy in Pharmacology practical classes can help in minimizing the errors in drug administration and improving the patient compliance and adherence to the therapy. Objectives of this study were teaching module on intravenous drug administration and communication skills to undergraduate students in Pharmacology and to evaluate the perceptions of students and teachers towards the new teaching module.Methods: Correct methods of intravenous drug administration were demonstrated in practical classes. Role play was done to teach about communication skills regarding right method of using an inhaler and also about prescribing the correct dosage regimens. Perceptions of students and teaching staff members were collected on the teaching module.Results: Almost all of the students (>96 %) were of the opinion that learning correct drug administration methods and communication skills was relevant to the future practice and 95% students felt that after the role play sessions, they were better equipped in communicating with the patients about the medication use and were in favour of teaching these to all the medical students.Conclusions: Our study concludes that the teaching module on intravenous drug administration and communication skills was well accepted by both the teaching staff and the students and was found feasible and relevant to be introduced in the curriculum by both of them.

Fundus Flavimaculatus: Clinical Images And Review Of Literature

Stargardt disease (STGD) or fundus flavimaculatus is a progressive form of juvenile macular degeneration with considerable clinical and genetic heterogeneity. It may be considered a syndromal cone-rod dystrophy due to overlapping clinical features such as loss of color vision and photophobia in some patients. Here, we report a case of fundus flavimaculatus in a 32-year-old female and discuss the literature pertaining to it. Clinical data including medical history, findings on physical examination and local examination were suggestive of fundus flavimaculatus. Best-corrected visual acuity of the patient remained constant at 6/12 for 1 year with the patient being on 3-month follow-up. No particular or specific treatment is available for this fundus dystrophy and frequent follow-ups may help diagnose and manage associated choroidal neovascular membrane at the earliest. The only modality is available in the form of low vision aids for advanced disease

Fluconazole-induced Stevens-Jonson syndrome.

A 25-year-old girl self-administered tab fluconazole for vulvovaginal candidiasis and developed fever, myalgia and erythematous papular eruptions with itching at both upper and lower lip, mucous membrane of the mouth, vaginal region, which subsequently changed into blister and multiple ulcers. She also had difficulty in swallowing due to painful erosions of the mouth and oropharynx and severe burning pain at vulval and vaginal region. There was bilateral conjunctival hemorrhage. Investigation report revealed leukocytosis and elevated C-reactive protein, which was suggesting the diagnosis of Stevens-Johnson syndrome. She was treated mainly by corticosteroids, antihistamines and antimicrobials and improved. Time taken for resolution of the lesion was 14 days. Rechallenge with the offending drug was not done in the interest of the patient and due to ethical constraints.

The emerging therapy with probiotics in the management of inflammatory bowel disease: current status.

Inflammatory Bowel Disease (IBD) comprises Ulcerative Colitis (UC) and Crohn’s Disease (CD) with unknown aetiology. Most of the drugs used to treat IBD as standard treatment produce adverse effects during long term therapy. Evidence has suggested a role of intestinal microbiota in IBD. The use of probiotics and prebiotics is the natural approach to treat IBD. The objective of this article was to review the studies on probiotics that cover the therapeutic status in Inflammatory Bowel Disease. Appraisal of published articles from peer reviewed journals, search from PubMed and Wiley Blackwell website for English language publications using defined key words according to disease type. Studies have shown that probiotic agents play an important role in IBD and these are VSL#3, Bifido-ferminted milk, Escherichia coli Nissle 1917, Saccharomyces boulardi and “BIO-THREE for inducing remission in patients with active UC, for preventing relapses in inactive UC patients and also in UC patients with ileo-anal pouch anastomosis. Lactobacillllus rhamnosus GG and Lactobacillllus johnsonii LA1 can prevent endoscopic recurrences in patients with inactive CD. Probiotic intervention study designs in IBD patients searched were RCT vs Placebo / RCT vs standard treatment . Studies - with uncontrolled design, - with prebiotics intervention and with helminths were also searched. There is a promising role of probiotics and prebiotics in chronic mucosal inflammation that occurs in Inflammatory Bowel Disease. Sufficient evidence to support the role of probiotics in CD are not available. Well designed RCT studies based on intention -to- treat analyses are required.

Cloning and expression analysis of multiple proteins encoding P gene of Newcastle disease virus.

Viral gene oncotherapy is emerging as a biotherapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. Newcastle disease virus (NDV) has the property to cause selective oncolysis of tumor cells sparing normal cells. NDV has a single stranded negative sense RNA genome, which is 15,186 nucleotide long and consists of six genes, which codes for eight proteins. NDV like other paramyxoviruses has the ability to generate multiple proteins from the P gene. P protein is encoded by an unedited transcript of the P gene, whereas the V and W protein are the results of RNA editing event in which one and two G residues are inserted at a conserved editing site within the P gene mRNA resulting in V and W transcripts, respectively. Although NDV is known to cause oncolysis by triggering apoptosis, the role of different viral proteins in selective oncolysis is still unclear. P gene edited products are known for its anti-apoptotic property in homologous host. In the present study, NDV P gene and its RNA edited products were amplified, cloned, sequenced and in vitro expression was done in HeLa cells. Further constructs were assayed for their apoptosis inducing ability in HeLa cells. Preliminary study suggested that P, V and W proteins are not apoptotic to HeLa cells.

In vitro expression studies of non structural 1 protein of Canine Parvo virus 2 by polyclonal antiserum raised against CPV2-NS1 protein expressed in Escherichia coli as an antigen.

The canine Parvovirus 2, non-structural 1(NS1) is a novel candidate tumor suppressor gene. To confirm the expression of the NS1 in HeLa cells after transfection there was a need to raise antiserum against CPV2- NS1. Therefore, this study was carried out to express and purify the recombinant NS1(rNS1), and characterize the polyclonal serum. CPV2-NS1, complete coding sequence (CDS) was amplified, cloned in pET32a+ and expressed in BL21 (DE3) (pLysS). SDS–PAGE analysis revealed that the expression of the recombinant protein was maximum when induced with 1.5 mM IPTG. The 6 × His tagged fusion protein was purified on Ni-NTA resin under denaturing conditions and confirmed by western blot using CPV2 specific antiserum. The rabbits were immunized with the purified rNS1 to raise anti-NS1 polyclonal antiserum. The polyclonal serum was tested for specificity and used for confirming the expression of NS1 in HeLa transfected with pcDNA.cpv2.ns1 by indirect fluorescent antibody test (IFAT), flow cytometry and western blot. The polyclonal antiserum against NS1 could be very useful to establish functional in vitro assays to explore role of NS1 in cancer therapeutics.

Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies.

In the present study recombinant VP3 (rVP3) was expressed in E.coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS–PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37ºC. The 6×His-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.

Characterization and in vitro expression of non-structural 1 protein of canine parvovirus (CPV-2) in mammalian cell line.

Parvoviruses are small, 260-Å-diameter, icosahedral, non-enveloped, single-stranded DNA viruses with a genome of approximately 5 kb. Non structural protein, (NS-1) is especially relevant, being both essential for virus replication and the main factor responsible for virus pathogenicity and cytotoxicity. This protein has also been reported to possess the property of killing of transformed cells. The present study was carried out to clone, characterize and express the NS-1 gene of canine parvovirus. NS-1 complete CDS 2020bp was amplified, cloned into eukaryotic expression vector pcDNA 3.1(+), sequenced and characterized by in vitro expression analysis. Functional activity of recombinant construct, pcDNA.cpv.NS-1, was evaluated by RT-PCR and flow cytometry for the expression of NS-1 specific mRNA and NS-1 protein, respectively, in transfected HeLa cells. This recombinant plasmid may serve as an important tool to evaluate the apoptotic potential of NS-1 protein of canine parvovirus in cultured HeLa cells.

Development and in vitro characterization of a bivalent DNA containing HN and F genes of velogenic Newcastle disease virus.

Newcastle disease (ND) is highly contagious, economically important viral disease affecting most of avian species worldwide. Newcastle disease virus (NDV) has single stranded negative sense RNA genome which encodes for six structural and two non-structural proteins. Envelope glycoproteins i.e. hemagglutinin-neuraminidase (HN) and the fusion (F), elicit protective immune response. In this study, HN and F genes of velogenic (virulent) strain were amplified and cloned at multiple cloning sites A and B, respectively into pIRES bicistronic vector for use as bivalent DNA vaccine against ND. The recombinant plasmid was characterized for its orientation by restriction enzyme digestion and PCR. Expression of HN and F genes was assessed in transfected Vero cells at RNA level using RT-PCR in total RNA as well as protein level using IFAT, IPT and western blot using NDV specific antiserum. All these experiments confirmed that HN and F genes cloned in recombinant pIRES.nd.hn.f are functionally active. The recombinant construct is being evaluated as DNA vaccine against ND.

Cloning and expression of chicken granulocyte-macrophage colony stimulating factor (GMCSF) gene.

Granulocyte-macrophage colony stimulating factor (GMCSF), a multifunctional cytokine can enhance immune responses when administered along with DNA vaccine. Aim of the present study was to clone and express the chicken GMCSF cytokine for use as ‘genetic adjuvant’. Chicken GMCSF gene 435bp was amplified using specific primers in which restriction sites of BamHI and HindIII were at forward and reverse primers respectively. The PCR product was cloned into eukaryotic expression vector pcDNA 3.1(+) and clones were confirmed by restriction digestion and nucleotide sequencing. Functional activity of recombinant GMCSF was checked by expression of GMCSF specific mRNA in transfected Vero cells by RT-PCR of total RNA isolated from transfected Vero cells. The recombinant plasmid can be used as genetic adjuvant in chicken.

Malignant peripheral nerve sheath tumors: Clinicopathological profile of 63 cases diagnosed at a tertiary cancer referral center in Mumbai, India.

Background: A malignant peripheral nerve sheath tumor (MPNST) is a rare sarcoma, characterized by an aggressive course and forms a diagnostic challenge, in view of its varied histomorphology. The present study is a comprehensive analysis, including histopathological spectrum of 63 MPNSTs that forms a substantial study from an Indian perspective. Materials and Methods: Clinicopathological features of 63 MPNSTs, diagnosed during a period from January 2002 to December 2006, at a tertiary cancer referral center in Mumbai, India, were analyzed. Statistical analysis was carried out using SPSS (version 14) and STRATA. Difference in events was noted in 50 cases with selected variables. Disease free survival (DFS) was calculated by Kaplan-Meir analysis at the end of 1 year. Results: More cases were identified in > 30 years age (36 cases, 57.14%) group; in men (46 cases, 73%), and were deep-seated (38, 60.3%). Ten cases (15.9%) showed stigmata of multiple neurofibromatosis type 1. Average tumor (T) size was 9.9 cm, with 72.9% cases having T size > 5 cm. More cases were of high grade (56, 88.8%) and high stage (22, 34.9%). Histopathologically, most cases showed hypo- and hypercellular areas (marbleized appearance) of doubly indented spindle cells. Two cases showed epithelioid differentiation. Heterologous elements in the form of osteoid, chondroid, pigmented neuroectodermal (1 case), glandular (1 case) and rhabdomyoblastic differentiation (1 case) were identified in 14 cases (22.2%). S-100 protein positivity was noted in 38/54 cases (70.3%). Maximum cases (45, 71.4%) underwent surgery, including wide excisions and amputations (R0) in 20 cases, marginal excisions (R1) in 4, and intracapsular excision (R2) in 1 case. Nineteen cases underwent adjuvant treatment. A total of 29 cases (46%) showed recurrences and 22 (34.9%) showed multifocality and/or metastasis. Four patients succumbed to the disease in 1 year. The DFS was 53.1%. Cases ≤30 years of age (P- value = 0.007), T size > 5 cm, and with high grade (P = 0.18) and stage (P = 0.00) showed more recurrences, metastasis, and death. Conclusions: A MPNST has multifaceted histomorphology. Its objective identification necessitates the incorporation of clinicopathological features and IHC with S-100 protein. Younger age, high grade and stage, and increased T size significantly relate to aggressive disease. Wide excision forms the optimal treatment with options of adjuvant CT/RT in individual cases.

Primary endobronchial synovial sarcoma confirmed by SYT-SSX1 fusion gene transcript by reverse transcriptase polymerase chain reaction.

Primary sarcomas of lung are rare compared to metastatic sarcomas. Herein, we report a rare case of primary pulmonary synovial sarcoma with polypoid endobronchial growth in a 35-year-old lady who presented with cough and dyspnea. A malignant pulmonary tumor was suspected and left pneumonectomy was performed. Grossly, a non-encapsulated polypoidal endobronchial tumor measuring 6 cm in greatest diameter, with a solid, tan-white cut surface was identified. Microscopically, tumor was characterized by a proliferation of oval to spindle-shaped cells arranged in sheets and fascicles. Focal hemangiopericytomatous pattern was noted. Immunohistochemically, tumor cells were positive for vimentin, BCL-2, MIC-2 and calponin and focally positive for pancytokeratin and epithelial membrane antigen. A subsequent molecular analysis performed using reverse transcriptase-polymerase chain reaction with RNA extracted from paraffin-embedded tissue, revealed SYT/SSX1 fusion gene which confirmed the diagnosis of synovial sarcoma. The utility of immunohistochemistry and molecular techniques in diagnosis of such a rare case is stressed and the relevant literature is discussed.

Metastatic adenocarcinoma of bilateral cavernous sinus and optic nerve with unknown primary mimicking orbital pseudotumor.

Bilateral cavernous sinus and optic nerve metastases is extremely rare. We report an extremely rare case of metastatic adenocarcinoma of bilateral cavernous sinus and optic nerve with unknown primary presenting as orbital pseudotumor. She underwent bilateral optic nerve decompression and the diagnosis was established by biopsy.

Multidrug-resistant typhoid fever.

OBJECTIVE: To study the epidemiological pattern, clinical picture, the recent trends of multidrug-resistant typhoid fever (MDRTF), and therapeutic response of ofloxacin and ceftriaxone in MDRTF. METHODS: The present prospective randomized controlled parallel study was conducted on 93 blood culture-proven Salmonella typhi children. All MDRTF cases were randomized to treatment with ofloxacin or ceftriaxone. RESULTS: Of 93 children, 62 (66.6%) were MDRTF. 24 cases were below 5 years, 26 between 5-10 years and 12 were above 10 years. Male to female ratio was 1.85: 1. Majority of cases came from lower middle socio-economic classes with poor personal hygiene. Fever was the main presenting symptom. Hepatomegaly and splenomegaly was present in 88% and 46% cases respectively. 19 (30.6%) cases developed complications. Mean defervescence time with ceftriaxone and ofloxacin was 4.258 and 4.968 days respectively. CONCLUSION: MDRTF is still emerging as serious public and therapeutic challenge. Ceftriaxone is well-tolerated and effective drug but expensive whereas ofloxacin is safe, cost-effective and therapeutic alternative in treatment of MDRTF in children with comparable efficacy to ceftriaxone.

Congenital tuberculosis.

We report a case of congenital tuberculosis diagnosed by liver biopsy in a 8-week-old infant presented with acute abdomen. Liver biopsy showed multiple lymphoepitheloid cell granulomas with Langhans' giant cells and central necrosis. The culture of caseous material from the liver showed Mycobacterium tuberculosis . Antituberculous treatment was started. Endometrial biopsy in the asymptomatic mother confirmed the source of infection.