Immunophenotypic characterization of normal and abnormal plasma cells in bone marrow of newly diagnosed multiple myeloma patients

Background: Identification of plasma cells into abnormal (APC) and normal (NPC) compartments is of utmost importance in flow cytometric (FC) analysis of multiple myeloma (MM) and related plasma cell dyscrasias for diagnosis, prognosis, and follow-up. No single phenotypic marker is sufficient to distinguish NPC from APC. Materials and Methods: 43 newly diagnosed cases of MM and 13 controls were included in the study. Bone marrow (BM) samples from the 2nd pass were processed on the same day with antibodies against CD38, CD138, CD19, CD81, CD45, CD117, CD200, CD56, cytoKappa, and cytoLambda in a 4-color experiment with CD38 and CD138 as gating antibodies. Results: Mean APC% in cases was 96.5%. The expected Immunophenotype (IP) of APC which is CD19-/56+/45-/81-/117+/200+ was found in only 13/43 MM cases. In 30/43 cases, APC revealed deviation from expected IP either for single or a combination of markers. Sensitivity for APC detection was highest for CD19 (95.2%) followed by CD56 (90.4%) and CD81 (83.7%). Specificity was highest for CD19 (100%), CD56 (100%), and CD81 (100%) followed by CD117 (92.3%). Combination of markers with maximum sensitivity to detect APC (97.6%) was CD81- or CD19- and CD200+ or CD56+ (two markers); and for NPC (92.3%) was CD81+ and CD19+ and CD56- (three markers). Conclusion: Plasma cell IP can be highly variable with multiple minor subpopulations in both cases and normal controls. CD 19 and CD56 are highly informative markers for a 4-color experiment. Assessment of multiple markers in an 8–10 color experiment is more informative but the lack of advanced flow cytometers should not limit the use of FC in a 4-color approach. Our results emphasize that even basic equipment with limited fluorochrome can provide meaningful information if used appropriately.

Epidermal growth factor receptor mutations in adenocarcinoma lung: Comparison of techniques for mutation detection

Background: Targeted therapy using tyrosine kinase inhibitors in cases of non-small-cell lung carcinoma (NSCLC) that harbor epidermal growth factor receptor (EGFR) mutations has drastically improved the overall survival rate. The current study estimated the frequency of EGFR mutations in the Indian population by analyzing the diagnostic parameters of various techniques available for the detection of these mutations. Materials and Methods: A case series of 100 histologically diagnosed and immunohistochemically confirmed NSCLC with the adenocarcinoma phenotype comprises the study sample. EGFR mutations were detected using clone-specific immunohistochemistry (IHC), real-time polymerase chain reaction (PCR), and Sanger sequencing. Results: EGFR mutations were identified in 48% cases with 72.78% mutations involving exon 19. Clone-specific IHC had a low sensitivity of 46.43%, and the specificity was 79.17%. Sanger sequencing yielded interpretable results in 16% cases only, which were in concordance with the results of real-time PCR. Conclusion: EGFR mutations are increasingly being explored for targeted therapy and personalized medicine. Real-time PCR was found to be the best and the most accurate method for the detection of somatic EGFR mutations in adenocarcinoma primarily in the lungs.

Laboratory perspectives for Leprosy: Diagnostic, prognostic and predictive tools

The diagnosis of leprosy poses several challenges. The bacillary load, serology, and tissue response are determined by the host immune status, which make individual tests unsuitable across the spectrum. The sensitivity of tests for identifying paucibacillary cases remains limited, on the other hand, many tests lack specificity in differentiating contacts from diseased cases. Nonetheless, a plethora of laboratory tests have been added to the armamentarium of the clinicians dealing with leprosy. In the current review, we critically analyze the tests available for diagnosis, prognostication, and prediction of treatment response in leprosy. We discuss in brief the conventional tests available and detail the newer serologic and molecular tests added over the past few years with an attempt to suggest the pros and cons of each, and the tests best fit for each clinical scenario. Slit skin smears and skin or nerve biopsies are primarily performed to exclude clinical mimics, confirm a diagnosis, and immunologically subtype the case. Antibody titres of phenolic glycolipid-1 and its synthetic variants can be measured in serum and saliva and provide noninvasive means to detect leprosy with good specificity. Conventional, quantitative, real-time, and other variants of PCR can detect M. leprae DNA and have been used to effect in blood, tissue, and urine samples. T helper I and II cytokine signatures can be used to differentiate the subtypes of leprosy. Newer machine learning algorithms use combinations of these tests to predict the development of leprosy in contacts. Tests to detect treatment response, antimicrobial drug resistance, and predict the onset of reactions in leprosy can be used to advantage. We compare the characteristics of these tests and suggest an algorithm for leprosy diagnosis optimally utilizing them in various clinical settings.

Liquid biopsy in CNS tumors: Current status & future perspectives

Precise classification of central nervous system (CNS) malignancies is vital for the treatment and prognostication. Identification of noninvasive markers can be of importance to guide treatment decisions and in monitoring treatment response. CNS tumors are classified based on morphology with an essential complement of molecular changes, including mutations, amplifications, and methylation. Neuroimaging is the mainstay for initial diagnosis and monitoring tumor response with obvious limitations of imprecise tumor typing and no information on diagnostic, predictive and prognostic markers. Liquid biopsy has evolved as a diagnostic tool in body fluids and is being investigated as a surrogate for tissue biopsy in managing primary and metastatic brain tumors. Liquid biopsy refers to analyzing biological fluids such as peripheral blood, urine, pleural effusion, ascites, and cerebrospinal fluid (CSF); however, peripheral blood remains the primary source of fluid biopsy. The analytes include cell-free DNA (cfDNA) circulating tumor cells (CTCs), circulating micro RNAs (miRNAs), circulating proteins and extracellular vesicles (EVs). Analysis of these components is actively used for early cancer detection, auxiliary staging, prognosis assessment, detection of minimal residual disease (MRD), and monitoring drug resistance in various solid tumors. In recent years, liquid biopsy has been studied in CNS tumors, and analysis of CTCs and cfDNA have become relevant research topics. In the current review, we have explained the clinical potential of liquid biopsy in CNS tumors to assist in diagnosing and predicting prognosis and response to treatment.

Comparison of circulating DNA in malignant neoplasia from diverse locations: Investigating a diagnostic role

Context: Circulating free DNA (cfDNA) analysis has emerged as novel noninvasive diagnostic biomarker in several solid tumors. Raised levels have been reported in several malignancies and may correlate with clinicopathological and treatment response. The current study was designed to assess the diagnostics of cfDNA in different tumor types of malignancies correlating with tumor (T), nodes (N), and metastases (M) stage. Design: Serum samples were collected from treatment naïve cases with histologically diagnosed tumors including 23 brain tumors, 48 breasts, 50 gallbladder carcinoma (GBC), 13 lungs, 68 oral squamous cell carcinoma (OSCC), and 25 normal controls. CfDNA was quantified with real-time polymerase chain reaction (PCR), Invasive ductal carcinoma (IDC) using beta-globin gene amplification. Cut off values for diagnostics were calculated using receiver operating curve analysis. Results: Contrary to other cfDNA studies where it was postulated that cfDNA would not cross the blood–brain barrier and reach the systemic circulation, we found detectable cfDNA in glioma with median (Q1–Q3) of 349.22 ng/ml (19.87–1276.58). Median cfDNA concentration in breast, gallbladder, lung, oral and normal controls was 328.72 (128.38–624.44), 778.50 (589.88–1864.35), 348.73 (194.67–483.61), 386.27 (47.88–959.67), and 74.12 (49.66–120.00), respectively. Grades I and II glioma had significantly lower levels compared to Grades III and IV (P = 0.0001). Significant difference in median cfDNA values in IDC and GBC was observed with increasing tumor grades, stage, T stage, nodal stage and metastasis and with stage of OSCC cases. Conclusion: CfDNA levels showed good diagnostic discrimination in glioma, GBC, breast, lung carcinoma, and OSCC. Significant increase in titers was evident with increase in cancer stage from I to IV in breast, GBC and OSCC.

Comparison of laboratory-developed test & validated assay of programmed death ligand-1 immunohistochemistry in non-small-cell lung carcinoma

Background & objectives: Inhibitors of immune checkpoint regulators, programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), improve outcome in advanced non-small-cell lung carcinoma (NSCLC). Tumours expressing PD-L1 protein are more likely to benefit from this targeted therapy. Multiple concurrent clinical trials evaluating different anti-PD-1/PD-L1 therapies have validated five different immunohistochemistry (IHC) assays using varied antibody clones and staining conditions. This study was aimed at identification of a single harmonized PD-L1 assay for tumour tissue conservation and cost-effectiveness in patients with NSCLC. Methods: The performance of low-cost, manual, laboratory-developed technique (LDT) PD-L1 IHC assay using the easily available SP142 clone was compared with trial validated Ventana SP263 IHC performed on automated Ventana staining platform on tumour sections of NSCLCs. Results: Eighty cases of NSCLC were included. SP263 and SP142 stained both tumour cells and immune cells. The concordance rate of tumour cell staining was about 76 per cent, with SP263 detecting more tumour cells in 16 per cent of cases. The concordance rate of immune cell staining was only 61 per cent, with SP142 detecting more immune cells in 24 per cent of cases. The sensitivity, specificity, positive and negative predictive values of manual SP142 LDT assay against gold standard SP263 Ventana assay were 70, 94, 86 and 86 per cent, respectively, at positivity thresholds of ?1 per cent tumour cell staining. Interpretation & conclusions: The study findings suggested that LDT using SP142 clone showed only moderate concordance with SP263 Ventana assay, and the two assays were not interchangeable. More such validation studies need to be done to generate information that can complement patient therapy in cases of NSCLC.

ROS-1 re-arrangements and c-MET amplifications in adenocarcinoma lung: A tertiary care center study from North India

Background: C-ros oncogene 1, receptor tyrosine kinase (ROS 1) proto-oncogene 1, receptor tyrosine kinase (ROS-1) fusions are potent oncogenic drivers and these re-arrangements promote signal transduction programs leading to uninhibited cell survival and proliferation identified in 1–2% of cases of nonsmall-cell lung cancer. Mesenchymal epithelial transition factor (MET) receptor tyrosine kinase and its ligand are predominantly involved in epithelial mesenchymal transition and tissue regeneration. The MET amplification and overexpression is oncogenic in 3–7% cases. The objectives of this study were to identify the frequency of ROS-1 and c-MET protein expression in adenocarcinoma lung and to correlate it with the clinicopathological parameters and to analyze the histomorphology of cases that harbor the characteristic mutations (c-MET and ROS-1). Materials and Methods: Study group comprised a prospective cases series of 90 cases of adenocarcinoma lung. ROS-1 protein expression was determined by immunohistochemistry using the D4D6 rabbit monoclonal antibody (Cell Signaling, Danvers, MA) and c-MET protein expressed was analyzed using the SP-44 clone (Ventana Medical Systems). Results: c-MET protein expression was identified in 33.33% cases (n = 30/90) with statistically significant thyroid transcription factor-1 (TTF-1) positivity. ROS-1 protein expression was detected in 3.33% cases (n-3/90), in biopsies from the respiratory tree with TTF-1 expression. Conclusion: This is the first study from the Indian subcontinent to identify the frequency of ROS-1 re-arrangements and MET amplification in the Indian population. The availability of targeted therapy that has a significant impact on survival makes it essential to detect these less frequent mutations.

Medical Thoracoscopy: A Useful Diagnostic Tool for Undiagnosed Pleural effusion.

Objective. We aimed to assess the role of medical thoracoscopy in patients with undiagnosed pleural effusion. Methods. Patiens presenting with pleural effusion underwent three pleural aspirations. Patients in whom pleural fluid analysis was inconclusive underwent closed pleural biopsy for diagnostic confirmation. Patients in whom closed pleural biopsy was incolcusive underwent medical thoracoscopy using a rigid thoracoscope with a viewing angle of zero degrees was done under local anaesthesia and sedation with the patient lying in lateral decubitus position with the affected side up. Biopsy specimens from parietal pleura were obtained under direct vision and were sent for histopathological examination. Results. Of the 128 patients with pleural effusion who were studied, pleural fluid examination established the diagnosis in 81 (malignancy 33, tuberculosis 33, pyogenic 14 and fungal 1); 47 patients underwent closed pleural biopsy and a diagnosis was made in 28 patients (malignancy 24, tuberculosis 4). The remaining 19 patients underwent medical thoracoscopy and pleural biopsy and the aetiological diagnosis could be confirmed in 13 of the 19 patients (69%) (adenocarcinoma 10, poorly differentiated carcinoma 2 and mesothelioma 1). Conclusion. Medical thoracoscopy is a useful tool for the diagnosis of pleural diseases. The procedure is safe with minimal complications.

Overview of intestinal parasitic prevalence in rural and urban population in Lucknow, north India.

Intestinal parasitic infestations are a common finding in the developing world, however, the patterns of parasitic distribution and rates are different everywhere. Intestinal parasitic prevalece in urban and rural areas in Lucknow, Uttar Pradesh were determined in this community-based study. Multistage random sampling was adopted to collect stool samples from urban and rural population in Lucknow district. Door to door survey was done. Stool samples were processed by standard methods for parasitological examination. One thousand and seventy one stool samples were collected from urban Alambagh, (n=648) and rural Mati, (n=343) areas. Overall one hundred and twenty three (11.5%) subjects had intestinal infection. Intestinal infestation rate was 5.4% and 20.8% in the urban and rural areas respectively. Giardia lamblia (22%) was the commonest pathogenic protozoan detected. The soil transmitted helminths detected were Ascaris (11.4%) and Hookworm (2.4%). Infection had no predilection for either sex or age group in both areas. The prevalence of parasitic infection appears to be relatively low in this region, probably due to improving access to health care. However due to the sheer numbers of affected individuals involved, intestinal parasitosis remains an important public health problem.

Impact of a comprehensive telephone-based disease management programme on quality-of-life in patients with heart failure.

BACKGROUND: Disease management programmes for patients with heart failure have improving the quality-of-life (QOL) of patients with heart failure. METHODS: Patients attending the heart failure clinic were randomized into 2 groups of 25 patients each. The control group was managed in the heart failure clinic and the intervention group underwent the following additional interventions: (i) interactive sessions with the patient and spouse informing them about the disease, drugs, and self-management of fluid intake and diuretic dose; (ii) a telephonic helpline was established and regular telephone calls made to reinforce the information and modify drug dosages. The QOL was assessed using the Kansas City Cardiomyopathy questionnaire. Functional capacity was assessed by the 6-minute walk test. Continuous variables were compared with the Student t-test (paired or unpaired). RESULTS: There was significant improvement in the QOL and functional capacity of patients in the intervention group compared with controls over a 6-month period. The mean (SD) QOL scores in the intervention group improved from 60.0 (23.6) to 76.3 (17.3) but did not change significantly in the control group (62.2 [22.6] to 63.4 [21.9]). There was a similar improvement in the functional capacity measured by the 6-minute walk test in the intervention group (from 202.2 [81.5] to 238.1 [100.9] metres, p < 0.05) but not in the control group (193.8 [81.5] to 179.7 [112.0] metres). In the intervention group, the use of beta-blockers and angiotensin-converting enzyme inhibitors was similar but in the intervention group patients were placed on higher doses. There was no significant difference in the number of emergency room visits or admissions in either group. For every 20 patients in the intervention group, 14 patients improved by 1 functional class while in the control group this was observed in only 3 patients for every 20 treated. CONCLUSION: This study demonstrates that in the setting of a developing country, improvement in QOL by intensive management of heart failure patients through a heart failure programme with telephonic reinforcement and a helpline is greater than that usually achieved with drug therapy in a routine heart failure clinic.

Evaluation of cysticercus fasciolaris antigen for the immunodiagnosis of neurocysticercosis.

Cysticercus cellulosae antigen has been frequently used to detect antibodies for immunodiagnosis of neurocysticercosis. We have, for the first time, used membrane extract of cysticercus fasciolaris, the larval stage of Taenia taeniaeformis, in ELISA, with successful results. IgM and IgG antibodies against cysticercus were measured in serum from cases of neurocysticercosis (217), normal and diseased controls (89). 203 sera from cases of neurocysticercosis were positive for either or both IgG and IgM antibodies while 157/217 cases showed IgM and 158/217 showed IgG antibodies. Ten controls showed false postivity in IgG ELISA. Eight of these cases also had IgM antibodies. The test had an overall sensitivity of 93.54% and a specificity of 84.2% with a positive predictive value of 93.54% and a negative predictive value of 84.2%. Cysticercus fasciolaris can be conveniently produced in the experimental laboratory host, Rattus rattus, and would be of practical value in the immunodiagnosis of cysticercosis in humans.

AgNOR expression in CNS neoplasms.

Silver colloid staining of nucleolar organiser regions (AgNORs) is used for assessing the proliferative potential of tumours. The present study aimed at evaluating the AgNOR indices in normal and reactive CNS tissue, benign and malignant CNS neoplasms. The study group comprised of tissue from 22 controls and 100 cases (53 benign & 47 malignant neoplasms). The mean AgNOR index of controls was 0.95, benign neoplasms 1.25 and malignant neoplasms 2.12. A statistically significant difference was observed in controls and cases (p < 0.001) and between benign and malignant tumours (p = 0.002). Mean indices for low and high grade astrocytoma also significantly differed (p < 0.001). Using ROC curves cut off values were obtained for differentiation of neoplastic from non neoplastic (AgNOR index 1.10), benign from malignant (AgNOR index 1.75) and low grade (I & II) from anaplastic (Gr III & IV) Astrocytomas (AgNOR index 1.62). A spectrum of gradually increasing AgNOR indices from normal, reactive, benign to low and high grade malignancy indicates the usefulness of this simple technique as a proliferative marker.