A comparative analysis of immunohistochemistry and fluorescent in situ hybridization assay to detect anaplastic lymphoma kinase status in lung adenocarcinoma cases: A search for a testing algorithm.

INTRODUCTION: Testing for echinoderm microtubule-associated protein-like 4 (EML4) anaplastic lymphoma kinase (ALK) translocation by fluorescence in situ hybridization (FISH) is well established whereas the Food and Drug Administration (FDA) ALK immunohistochemical (IHC) test is relatively new. AIMS AND OBJECTIVE: The aim of this study is to compare FDA-approved ALK IHC test (D5F3 clone) with the standard ALK FISH test. MATERIALS AND METHODS: A validation and a test arm with 100 and 200 cases of Formalin-Fixed, Paraffin-embedded blocks of lung adenocarcinoma, respectively, comprised the material. All cases had ALK IHC test on automated Ventana Benchmark XT IHC slide stainer using anti-ALK D5F3 rabbit monoclonal primary antibody; when positive tumor cells (any percentage) showed strong granular cytoplasmic staining. For the FISH test, Vysis ALK Dual Color Break Apart Rearrangement Probe (Abbott Molecular Inc.,) was used to detect ALK gene 2p23 rearrangements; when positive the red and green signals were split two signal diameter apart and/or isolated 3'red signal were detected in more than 15% tumor cells. The ALK FISH results were available in all 100 validation cases and 64-test arm cases which formed the basis of this analysis. RESULTS: The ALK IHC test was positive in 16% cases; four discordant cases were ALK IHC positive but ALK FISH negative, but no case was ALK IHC negative and ALK FISH positive. There was 100% sensitivity, 90.5% specificity, and 93.75% accuracy. CONCLUSION: A negative ALK IHC result obviates the need for a FISH test barring those with a strong clinical profile, and a positive ALK IHC result is sufficient basis for the initiation of treatment.

RP-HPLC and UV Spectrophotometric Methods for Estimation of Pirfenidone in Pharmaceutical Formulations.

High-performance liquid chromatographic and UV spectrophotometric methods were developed and validated for the quantitative determination of pirfenidone, a novel antifibrotic agent used in idiopathic pulmonary fibrosis. Chromatography was carried out by isocratic technique on a reversed-phase C18 Zorbax Eclipse plus column with mobile phase consisting of acetonitrile:water (35:65 %v/v) at flow rate of 0.7 ml/min. The UV spectrophotometric determinations were performed at 317 nm using methanol as a solvent. The proposed methods were validated according to International Conference on Harmonization ICH Q2 (R1) guidelines. The linearity range for pirfenidone was 0.2-5.0 and 3-25 µg/ml for HPLC and UV method, respectively. Both the methods were accurate and precise with recoveries in the range of 98 and 102 % and relative standard deviation <2 %. The developed methods were successfully applied for determination of pirfenidone in tablets.

Can combination metronomic therapy overcome chemoresistance in cholangiocarcinoma? A literature review.

Cholangiocarcinoma (CCa) is relatively resistant to chemotherapy as well as radiation therapy, and complete resection is the main curative therapy for these patients. The prognosis for patients with unresectable intrahepatic CCa (iCCa) is extremely poor. A 55-year-old woman presented at our hospital with abdominal pain. After evaluation, she was diagnosed to have multifocal iCCa. She did not opt for standard chemotherapy and therefore received oral metronomic therapy with a combination of celecoxib, etoposide, and cyclophosphamide for a total of 30 months. Presently, she is 57 months post diagnosis and 27 months post cessation of all treatment and continues to be in complete radiological remission. In the present report, we review the literature and discuss whether metronomic scheduling of biologic agents and anticancer drugs will be able to overcome chemoresistance and improve the outcome in cholangiocarcinoma. References for the review were identified through searches of Pubmed for the last 10 years as well as searches of the files of the authors themselves. The final list was generated on the basis of originality and relevance to this review.

Single agent weekly paclitaxel as neoadjuvant chemotherapy in locally advanced breast cancer: a feasibility study.

AIM: To study the toxicity profile and response rates of weekly paclitaxel given as neoadjuvant chemotherapy (NACT) in patients with locally advanced breast cancer. MATERIALS AND METHODS: The study was planned as a single arm, prospective phase II study. Twenty-six patients with locally advanced breast cancer were enrolled in the study from December 2006 to October 2007. These patients underwent NACT with weekly paclitaxel at 100 mg/m(2) for 8 consecutive weeks followed by surgery. This was followed by anthracycline-based chemotherapy for three to four cycles followed by radiation. The patients received standard adjuvant hormonal therapy. The patients were carefully monitored for side-effects using common toxicity criteria. The clinical and pathological response rates were documented. The response rates were descriptively stated. RESULTS: The median age of the patients was 52 years (30-67 years) and the median tumour size was 7 cm (2.5-15 cm). Of the 208 planned weekly cycles, 207 could be given. The rates of grade 3-4 neutropenia, thrombocytopenia and neuropathy were 4, 12 and 4%, respectively. A complete clinical response was observed in 10 patients (38.5%) and a completed pathological response, defined as the absence of invasive cancer from the breast and axillary nodes, was seen in 11.5% of patients. Breast-conserving surgery was possible in 23% of patients. CONCLUSION: The regimen of weekly single agent paclitaxel is feasible in patients with locally advanced breast cancer with acceptable toxicity. It resulted in a pathological response rate that was comparable with other regimens in this group of advanced stage patients. Considering the efficacy and low toxicity of this regimen, it is worth exploring in larger studies.

A t (11; 22) (p13; q12) EWS-WT 1 positive desmoplastic small round cell tumor of the maxilla: an unusual case indicating the role of molecular diagnosis in round cell sarcomas.

A desmoplastic small round cell tumor (DSRCT) is an uncommon tumor characterized by polyphenotypic expression and a specific reciprocal translocation t (11; 22) (p13; q12). It has been rarely identified in the head and neck region. Herein, we describe a DSRCT in the maxilla of a young man, who was initially diagnosed with a primitive neuroectodermal tumor (PNET), based on histopathological appearance of a round cell tumor, with MIC2 and -FLI-1 positivity, on immunohistochemistry (IHC). Diagnosis of a DSRCT was confirmed on molecular analysis with positive -RT-PCR and sequencing results for EWS-WT1 transcript and negativity for EWS-FL1. The case is presented to highlight the value of molecular diagnosis in round cell sarcomas at uncommon sites, especially when similar IHC markers can be expressed in a PNET and a DSRCT. An exact diagnosis of a round cell sarcoma has a therapeutic relevance.

A t(X; 18) SYT-SSX2 positive synovial sarcoma in the pelvis of a young adult male: a rare case report with review of literature.

Synovial sarcoma is uncommonly documented in the pelvis. Rarely, such cases have dealt with molecular analysis. A 19-year-old boy presented with pain and swelling in his left lower limb of two months duration. He developed acute urinary retention four days prior to his hospital admission, wherein radiological examination unraveled a large soft tissue mass, displacing his pelvic muscles, along with a lytic lesion involving his right pubic bone. Biopsy showed a cellular spindle cell sarcoma, exhibiting hemangiopericytoma-like vascular pattern with focal necrosis. Immunohistochemistry (IHC) showed positivity for vimentin, BCL-2, calponin and MIC 2. Cytokeratin (CK) and epithelial membrane antigen (EMA) were negative. MIB 1 count was 70% (high). P53 was positive. Diagnosis of a poorly differentiated synovial sarcoma was offered and confirmed with a positive t(X; 18) SYT-SSX2 translocation. This case highlights the value of molecular analysis in diagnosis of a synovial sarcoma at rare sites, especially when IHC results are equivocal and the biopsy material is limited.

Chikungunya encephalomyeloradiculitis: report of 2 cases with neuroimaging and 1 case with autopsy findings.

Chikungunya, an alphavirus presenting with fever, rash, and polyarthritis, is derived from the Makonde word that means "that which bends up," in reference to the crippling manifestations of the disease. Most often it is a self-limiting febrile illness. Neurologic complications of Chikungunya infection have been reported. We are reporting the clinical and neuroimaging data in 2 patients with Chikungunya encephalomyeloradiculitis and brain autopsy findings in 1 patient.

Upfront submandibular salivary gland transfer in pharyngeal cancers.

Head and neck irradiation results in salivary dysfunction and subsequent xerostomia. Twenty two patients with squamous cancer of oropharynx or hypopharynx underwent contralateral submandibular salivary gland transfer (SMSGT) to submental triangle to shield it from subsequent radiotherapy. Resting salivary outputs of transferred and untransferred gland (control) were measured before and after SMSGT and following radiotherapy, by cannulating individual submandibular duct. They were compared by paired samples t-test. Following radiation therapy transferred gland retained 73% and untransferred gland (control) retained 27% of baseline salivary output. This significant difference in post-radiation salivary outputs suggests preservation of function of transferred salivary gland.

Intra-abdominal follicular dendritic cell tumor: report of two cases.

Follicular dendritic cell (FDC) tumor is an uncommon entity described mainly in the lymph nodes. We report two men with intra-abdominal FDC tumors--one arising from the colon and other presenting as a mesenteric mass. Both patients underwent successful surgical excision of the tumor.

Scrape cell-block technique for fine needle aspiration cytology smears.

An inconclusive diagnosis on fine needle aspiration cytology (FNAC) may be due to poor spreading and presence of thick tissue fragments despite aspiration of adequate material. Repeat aspiration may not be possible especially when aspirates of deep seated organs have been obtained by image guided techniques. We have resorted to a 'scrape cell-block' (SCB) technique in such cases. In this technique the cellular material on the slides which had already been fixed and stained, was carefully removed by scraping following destaining and then processed as a cell block. SCB interpretation was then compared with the smear diagnosis and histological diagnosis, wherever available. A total of 27 cases were studied. In 12 cases SCB slides added information to the FNAC smears. In 14 cases SCB did not offer any additional information. SCB was inconclusive in one case. Final histological correlation was available in eight cases and the SCB diagnosis was confirmed in six cases, whereas in two cases SCB failed to identify the tumour. Immunocytochemistry (ICC) was done in one case. SCB is a useful technique to make the best use of the available material when reaspiration is difficult.

Acutely sodded expanded polytetrafluoroethylene grafts produce only prostacyclin: a qualitative difference from saphenous veins.

Select subsets of patients require prosthetic graft material for revascularization. Although arterial prosthetic grafts of large caliber perform acceptably, grafts of <6 mm exhibit a high attrition rate. Microvessel endothelial sodding, a method resulting in the lining of prosthetic grafts with autologous endothelium, improves graft patency; however, aggressive antiplatelet therapy is still required, because terminating an antiplatelet regimen accelerates graft attrition. The present investigation was designed to address the acute production of vasoactive substances in microvessel endothelial cell sodded expanded polytetrafluoroethylene (ePTFE) grafts in an attempt to delineate a possible mechanism behind the continued requirement for antiplatelet therapy. Equal lengths of acutely sodded ePTFE grafts (canine falciform ligament source) and saphenous veins (SV) (canine source) were evaluated by superfusion bioassay. Basal secretion from ePTFE grafts relaxed the biodetector ring 1 +/- 3%, whereas SV relaxed the ring 10 +/- 3% (p < 0.05, ePTFE vs. SV). Relaxation with acetylcholine stimulation was 49 +/- 7% in grafts and 50 +/- 10% in veins (p = NS). Calcium ionophore stimulation produced relaxation of 37 +/- 9% from ePTFE grafts and 100 +/- 23% from SV (p < 0.05). Indomethacin added to perfusate reduced relaxations from sodded ePTFE grafts to 20.2 +/- 9.2% with acetylcholine stimulation and 12.5 +/- 4.3% with calcium ionophore (p < 0.05 vs. control); addition of N(G)-monomethyl-L-arginine (L-NMMA) had no effect on the release of vasoactive substances from ePTFE grafts. In contrast, relaxations of effluent from SV stimulated by acetylcholine and calcium ionophore were significantly attenuated with indomethacin and L-NMMA (p < 0.05 vs. control). Scanning electron microscopy demonstrated confluent endothelium in SV and a nonconfluent endothelial cell layer in grafts. Acutely sodded ePTFE grafts produce vasoactive substances that quantitatively and qualitatively differ from those produced by canine SV. The ePTFE grafts produce mainly prostanoids, whereas SV produce both nitric oxide and prostanoids. The endothelial cell isolation procedure and absence of immediate graft luminal confluence may contribute to the observed differences.

Hormone receptor status of breast cancer in India: a study of 798 tumours.

The objectives of this study were to document the oestrogen and progesterone receptor (ER & PR) status of breast cancer in the Indian population (as done by immunohistochemistry on paraffin blocks), and correlate the steroid receptor status of breast cancer with all relevant patient and tumour characteristics. Our current data have been compared with previously published data from other centres. In contrast to the higher rates reported in the Western literature, only 32.6% of our tumours were ER positive and 46.1% were PR positive. Tumours were separated into four categories: ER+PR+ (25%), ER+PR- (7.4%), ER-PR+ (21.1%) and ER-PR- (46.5%). ER and PR immunoreactivity increased with advancing age, and correlated with the presence of elastosis. Infiltrating lobular carcinoma, mucinous carcinoma, and mixed tumours were more frequently ER & PR positive. High-grade infiltrating duct carcinomas, pure comedo ductal carcinoma in situ, and medullary carcinoma were predominantly ER & PR negative. The presence of necrosis and lymphovascular invasion showed an inverse relationship with ER and PR reactivity.

Mediastinal Castleman's disease complicated by follicular dendritic cell tumour.

We describe a rare case of follicular dendritic cell tumour which had arisen over the background of hyaline vascular type of Castleman's disease at the mediastinal location. Constellation of histology and immunohistochemistry using CD21 antibody and non-reactivity to CD15, CD30, cytokeratin and epithelial membrane antigen helped us diagnose this case. The literature is reviewed, specially with reference to the genesis of follicular dendritic cell neoplasm at the backdrop of Castleman's disease and its clinical relevance.

Direct activation of oestrogen receptor-alpha by interleukin-6 in primary cultures of breast cancer epithelial cells.

Interleukin 6 (IL-6) is secreted by breast tumours and shows synergistic activity with 17beta-oestradiol (E2), leading to increases in reductive 17beta-hydroxysteroid dehydrogenase activity in breast cancer epithelial cells. However, the mechanisms involved are poorly understood. Using short-term epithelial cultures established from primary breast tumours, we have examined whether IL-6 could directly affect transcriptional activity of oestrogen reception alpha (ERalpha). Tumour epithelial cultures were established from 15 breast tumours, grown to 70% confluence and transiently transfected with a plasmid reporter containing the vitellogenin oestrogen response element and the luciferase coding sequence (ERE-TK-LUC). Following transfection, cells were incubated with E2, IL-6, the pure anti-oestrogen ZM 182780 or combinations of these substances for 48 h. Luciferase activity was then measured in cell lysates. E2 caused a dose-dependent increase in luciferase expression, causing a maximum threefold stimulation at 100 pM. In the presence of IL-6, transcriptional activity was increased by up to 2.5-fold in ERalpha+ cultures (11/15). In combination with E2, synergistic effects were observed with increases in luciferase activity of up to sixfold over controls. This effect could be blocked by treatment with ZM 182780. Pre-incubation of cells with an antibody directed against the signalling component of IL-6, gp130, was ineffective in blocking the E2 response. This antibody reduced, but did not completely block the effect of IL-6 either alone or in combination with E2, suggesting cross-talk between the two signalling pathways. In conclusion, these results provide evidence for direct transcriptional activation of ERalpha by IL-6.

Evidence for transcriptional activation of ERalpha by IL-1beta in breast cancer cells.

Estrogen is mitogenic in breast cancer where IL-1beta also fulfils a role. The aim of this study was to determine any relationship between IL-1beta and ERalpha in breast cancer. By RT-PCR, 26/77 tumours expressed IL-1beta, and 57/77 expressed ERalpha. Samples which were IL-1beta positive were categorised against those which expressed ERalpha. Of the 26 tumours which expressed IL-1beta, all were ERalpha positive. We next examined whether IL-1beta could directly activate ERalpha. MCF-7 cells stably transfected with a plasmid reporter (ERE-TK-LUC) were incubated with either 17beta-estradiol (E2, 10-9-10-13 M), IL-1beta (10 ng/ml), the pure antiestrogen ZM 182780 (10 nM) or combinations of these substances. Transcriptional activity was measured in cell lysates 48 h later. E2 caused a dose-dependent increase in luciferase activity. With IL-1beta, transcriptional activity was typically half of the E2 response. To determine the role of the IL-1 receptor, parallel cultures were incubated with IL-1 receptor antagonist. This reduced, but did not completely block the effect of IL-1beta, suggesting that IL-1beta was affecting transcriptional activity via another pathway. Confirmation that the effect was via ERalpha was verified using the pure antiestrogen, ZM 182370, which completely abrogated the effects of E2, when added alone or in combination with IL-1beta. These results provide compelling evidence for direct transcriptional activation of ERalpha by IL-1beta. Interactions of these factors may thus modulate hormonal activity in human breast tumours.

Tumor angiogenesis and endothelial cell modulatory factors.

Angiogenesis is the ability of preexisting vasculature to send out capillary sprouts leading to the formation of new vasculature. It is now a well-accepted idea that progression of solid tumors is intrinsically dependent on angiogenesis for growth of the primary tumor and metastatic lesions. Investigations into tumor angiogenesis have focused on inhibition of tumor neovasculature as yet another possible mechanism for impairing tumor progression. Numerous studies have characterized cellular and molecular factors important to vascular formation and development and have led to the identification and understanding of requisite interactions between endothelium, angiogenic cytokines, and the supporting matrix. These studies have also led to the identification of cytokines involved in the proteolytic disruption of the basement membrane, the migration of endothelial cells, and the proliferation and formation of neoendothelium into functional vasculature. As therapies based on antiangiogenic strategies continue to evolve and clinical trials are conducted, these agents may become an important part of the arsenal against tumor proliferation, especially given their favorable toxicity profile. This review discusses the angiogenic cytokines which have been most intensely studied and the receptors they act upon. Additionally, we discuss select proteases and their importance in the development of neovasculature. A better understanding of these components will help in the development of novel therapeutic strategies.

Gastric emptying of solids in long-term NSAID users: correlation with endoscopic findings and Helicobacter pylori status.

OBJECTIVE: Prostaglandins regulate gastric motor function. Inhibition of prostaglandins by nonsteroidal antiinflammatory drugs (NSAIDs) may alter gastric emptying. To study gastric emptying of solids and its relation to endoscopic findings and Helicobacter pylori in patients receiving long-term NSAIDs, we undertook this study. METHODS: Ninety-five patients with arthritis, 65 taking long-term NSAIDs (Group I) and 30 not taking NSAIDs (Group II) were studied. Presence of dyspeptic symptoms was determined using a questionnaire. Mucosal damage was determined by endoscopy. H. pylori was detected by antral biopsies for rapid urease test and histology. Gastric emptying for solids was evaluated using a scintigraphic method. Thirty healthy volunteers were used as controls for gastric emptying (Group III). Patients with peptic ulcer were excluded from the analysis of gastric emptying. Logistic regression analysis was performed to identify predictive factors for gastric emptying. RESULTS: Nineteen patients from Group I with peptic ulcers were excluded. Dyspeptic symptoms were seen in 24 (52%) Group I and seven (23%) Group II patients. Gastroduodenal erosions were seen in 10 (21.7%) Group I patients and four (13.3%) Group II patients. H. pylori was detected in 17 patients in Group I (36.9%) and Group II (56.6%). Gastric emptying was delayed in 24 (52%) Group I patients, six (20%) Group II patients (p < 0.001), and in none of the Group III controls. The mean gastric emptying times were 99.5 (15.6) min and 89 (17.7) min for Groups I and II, respectively (p < 0.05). Endoscopic damage was found with similar frequency in Group I patients with delayed or normal gastric emptying. H. pylori infection was present in 37.5% Group I patients with delayed gastric emptying and in 36.3% with normal gastric emptying (p = ns). Logistic regression analysis identified NSAID therapy as the single factor most predictive of delayed gastric emptying. CONCLUSION: Delayed gastric emptying was seen in 52% of patients on long-term NSAID therapy.

Short-term primary culture of epithelial cells derived from human breast tumours.

As experimental models for breast cancer, most studies rely on established human breast cancer cell lines. However, many of these lines were established over 20 years ago, many from pleural effusions rather than the primary tumour, so the validity of using them as representative models is questionable. This paper describes our experiences, over a 3-year period, in establishing short-term epithelial-cell-enriched preparations from primary breast tumours based on differential centrifugation followed by culture in selective media. Epithelial cells were successfully cultured from 55% of samples, but culture success did not appear to be correlated with tumour histology, stage, grade or node status. Epithelial cell-enriched cultures were immunopositive for broad-spectrum cytokeratin and epithelial membrane antigen (EMA). Positivity for keratin 19 confirmed that the cultures contained tumour-derived cells, which additionally showed significantly higher activity of the reductive pathway of the steroid-converting enzyme 17beta-hydroxysteroid dehydrogenase type I. That the cultures contained tumour and not normal epithelial cells was further substantiated by the complete absence of the calmodulin-like gene NB-1 in tumour-derived cultures; this is only associated with normal breast epithelia. Eighty-five per cent of cultures established from oestrogen receptor (ER)-positive tumours expressed ER in vitro; this was functional in 66% of cultures, although ER-positive phenotype was gradually lost over time. In conclusion, epithelial cells can be isolated and maintained as short-term cultures from primary breast tumours irrespective of histopathological or clinical details, providing a model system with a greater biological and clinical relevance than breast cancer cell lines.