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.

Myeloid sarcoma presenting as nasopharyngeal mass: A rare clinicopathological scenario

Myeloid sarcoma (MS) is considered as an extramedullary manifestation of acute myeloid leukemia (AML) with or without concurrent AML. It can present at any age and any site, however, nasopharynx being an extremely rare site of manifestation. MS may precede AML by weeks, months or years, thereby necessitating an early diagnosis and timely intervention and treatment. We report a case of MS in a young female who presented with nasal obstruction and epistaxis for 3 months. The present case also highlights the significance of judicious use of immunohistochemistry panel while dealing with a hematolymphoid neoplasm devoid of expression of B-cell or T cell specific markers in head and neck region.

Correlation NKX2.2 IHC and EWSR1 break-apart FISH in the diagnosis of Ewing sarcoma: Can combined NKX2.2 and CD99 immunoexpression obviate or minimize the need of FISH testing? First assessment study from Indian tertiary cancer care center

Context: Ewing sarcoma (ES) are malignant small round cell tumors (MSRCT) characterized by rearrangements of EWSR1 gene. Although gold standard for diagnosis is detection of specific fusion genes by molecular testing, these ancillary tests are costly and only available in limited number of settings. There is a persuasive evidence for reliability of NKX2.2 immunohistochemistry (IHC) as a surrogate marker for EWSR1 gene rearrangement in ES. Aims: The aim of this study is to correlate the NKX2.2 immuno-expression with genetically confirmed ES cases and also to assess the reliability and accuracy of NKX2.2 along with combined positivity of NXX2.2 and CD99 in diagnosing ES and differentiating it from other relevant histological mimics. Settings and Design: The present study is a retrospective study conducted over a period of 6-year duration in a tertiary cancer care center. Methods and Material: We evaluated NKX2.2 immunoexpression in 35 genetically confirmed cases of ES and also in pertaining differential entities (n = 58) of ES including rhabdomyosarcoma (n = 20), lymphoblastic lymphoma (n = 14), Wilms tumor (n = 10), poorly differentiated synovial sarcoma (n = 4), small-cell osteosarcoma (n = 4), neuroblastoma (n = 5), and mesenchymal chondrosarcoma (n = 1). CD99 was performed in the category of MSRCTs showing NKX2.2 positivity to evaluate combined specificity for the diagnosis of ES. Results: Of the 35 genetically confirmed cases of ES, 29 cases (83%) showed NKX2.2-positive expression (83% sensitivity). Compared to ES, NKX2.2 was positive in only 05% cases (3/58 cases) of non-ES MSRCT. Only two of five cases of neuroblastomas and one case of mesenchymal chondrosarcoma showed NKX2.2 positivity. CD99 positivity was seen in 100% of ES and in the single case of mesenchymal chondrosarcoma. All five cases (100%) of neuroblastoma were negative for CD99. Conclusions: The presented study, which is the first from an Indian oncology center, showed NKX2.2 IHC is quite reliable in diagnosis of ES in the right clinicopathological context. With remarkable sensitivity and specificity of NKX2.2 IHC for diagnosis of ES, we propose that combined positivity of CD99 and NKX2.2 IHC can obviate or minimize the need of EWSR1 gene rearrangement molecular testing for diagnosis of ES.

Angiomatoid fibrous histiocytoma: Report of two cases, initially construed as sarcoma with unusual clinico-pathological features

Angiomatoid fibrous histiocytoma (AFH) is a rare soft tissue tumor of uncertain differentiation with low metastatic potential, most commonly occurring in children, adolescents, and young adults, involving extremities. Due to its rare nature and diverse presentation, both clinically and morphologically, it is often misdiagnosed. It becomes important to correctly diagnose this lesion, given its distinct therapeutic implications. Here, we present the clinical, radiologic, and pathologic findings of two rare cases of AFH. Since AFH is a rare soft tissue tumor with low malignant potential, both pathologists and clinicians should be aware of this entity, when encountered with a soft tissue mass in extremities of a child or adolescent, so as to accord appropriate treatment in such cases.

Evaluation of utility of immunohistochemistry markers as a tool for objective diagnosis of low-grade myelodysplastic syndrome in routine reporting: Prospective observational study

Purpose: Diagnosis of myelodysplastic syndrome (MDS) primarily relies on the detection of morphological dysplasia in bone marrow. It is subjective and many studies have reported lack of interobserver agreement in reporting. Biopsy is preferred specimen for megakaryocyte assessment. We studied 43 bone marrow biopsies from 40 suspected MDS patient having persistent undiagnosed cytopenia. Utility of immunohistochemistry (IHC) with CD61 and p53 in detecting low-grade MDS was analyzed over routine morphology. Method and Results: Total number of megakaryocytes and number of dysplastic megakaryocytes seen on CD61 IHC was significantly higher than that on H and E stain (P value < 0.05) Out of total 43 biopsies, 13 [30.2%] cases showed dysplastic megakaryocytes that were confirmed by interobserver agreement after IHC. From 30 cases with no significant dysplasia on morphology, 21/43 [48.8%] cases showed >10% dysplastic megakaryocytes on CD61 (P value 0.0001). Nine cases showed no significant dysmegakaryopoiesis with either H and E or CD61 IHC. Fourteen cases could meet higher cut off (30%) of dysmegakaryopoiesis with CD 61 IHC. Out of total 34 cases showing significant dysplasia 7 cases (20.6%) showed positivity for p53 on IHC, which is little less than that reported in low-grade MDS. Conclusion: CD61 IHC is helpful in making correct diagnosis of MDS in cases with minimal dysplasia and should be performed before excluding possibility of MDS on morphology in a patient with undiagnosed cytopenia. IHC is cost effective tool for MDS diagnosis in developing world where access to extensive flow cytometery and molecular testing is limited.

PD-L1 expression in muscle invasive urothelial carcinoma: Comparison of SP142 and SP263 assay

Introduction: High-grade urothelial carcinoma has a different molecular pathway than superficial low grade urothelial carcinoma, and is characterized by genomic instability. The high tumor mutation burden leads to neoantigen formation, evoking an immune response. The immune response has been keenly studied in last two decades and programmed death ligand-1 (PDL-1) has emerged as acceptable immunohistochemical marker for assessment of response to therapy, prognostication and patient selection for immunotherapy. The targeting of PD-1 and PDL-1 by checkpoint inhibitors (CPIs) is an attractive strategy to unblock the inhibitor and induce cytotoxic cell death. However, the presence of complementary and companion diagnostic testing with multiple PDL-1 assays and platforms for various CPIs make a diagnostic quagmire. Thus, it is the need of hour to harmonize these assays. In this undertaken study we evaluated the concordance in PD-L1 expression between the two PD-L1 clones: SP263 and SP142, in treatment naïve muscle invasive bladder cancer (MIBC). Methods: We evaluated Ventana PD-L1 “SP263 and SP142” qualitative immunohistochemical assay using rabbit monoclonal anti-PD-L1 clones in evaluation of PDL-1 immunoexpression on Ventana autostainer platform. The study includes 30 muscle invasive urothelial carcinomas, with 10 of 30 having nodal metastasis. Results: SP263 assay was statistically more sensitive than SP142 for tumor cell (TC) scoring (P = 0.0009), whereas SP142 was more sensitive for immune cell (IC) scoring (P = 0.0067). There was no statistical significant discordance for TC or IC scoring between primary tumor and metastatic lymph node. Conclusion: PD-L1 testing status can be done on both primary tumor and metastatic site, however in metachronous metastatic setting, testing on recent metastatic site should be preferred. The harmonization of immunoexpression between 2 PD-L1 clones could not be achieved.

Molecular stratification of high-grade urothelial carcinoma by immunohistochemistry with its histomorphological and clinical correlation

Introduction: Urothelial carcinoma poses a significant cause of morbidity and mortality. The recent classification of Tumors of Urinary System by World Health Organization fourth edition) has elucidated its molecular subtypes and its associated prognostic significance. Methods: We used immunohistochemistry marker expression (CK5/6, CK20, CD44, EGFR) as a surrogate marker, to stratify 150 cases of high-grade urothelial carcinoma into the intrinsic molecular subtypes. A correlation was also done with immunohistochemical markers p53, p21, E-cadherin and Ki-67. Results: On subtyping, 47.3% cases were basal, 42.7% luminal and 10% remained unclassified. We did not find GATA3 useful for molecular stratification in our study. Muscle invasion was seen in 59% of basal and 31% of luminal subtype (P = 0.016). Squamous differentiation was most commonly associated with basal subtype (P < 0.001). EGFR expression was seen in 62% of basal and 38% of luminal subtype (P = 0.014), and thus can be used as an additional marker for molecular stratification. Overexpression of p53 was seen in 64% cases of muscle invasive and 36% of non-muscle invasive high-grade carcinomas (P < 0.0001). An inverse relationship was observed between p53 and p21 immunoexpression (r = –0.494) (P < .0001). The overall survival at 1- and 2-year interval was more in the luminal subtype, suggesting an early mortality in basal group, (P = 0.827), and at 6 years both the groups had almost similar results. Conclusion: High-grade urothelial carcinoma is challenging in terms of therapeutic strategy. Increased understanding of underlying molecular basis helps identifying targetable treatment options, and newer biomarkers will enhance predictive and prognostic stratification.

Epithelioid haemangioma of bone: A series of four cases with a revision of this contentious entity

Epithelioid hemangioma of bone is a rare and locally aggressive vascular neoplasm of bone associated with a good prognosis. Because of its worrisome histomorphologic features and aggressive clinicoradiologic findings, at times with multifocal presentation, they tend to simulate malignant tumors. We report a series of four cases of epithelioid hemangioma of bone with their clinicopathologic characteristics. All had adjacent soft tissue involvement and two had multifocal bone disease. Microscopically, all cases had a tumor in lobular configuration, composed of epithelioid endothelial cells with the formation of well-formed vessels or grew in solid sheets. The tumor cells lacked significant cytologic atypia, necrosis, and increased mitosis. All cases were immunohistochemically positive for vascular markers CD34, CD31, ERG1, whereas negative for CK. Two of the cases were treated with excision, and the other two underwent curettage. None had local recurrence or metastasis on follow-up. This study highlights the importance of recognizing histomorphological and clinicoradiological features for distinguishing epithelioid hemangiomas from malignant vascular neoplasms of bone because of their distinct therapeutic implications and clinical outcomes.

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.

Diabetic retinopathy screening at primary and community health centers in Maharashtra

In order to integrate and improve eye care in noncommunicable disease (NCD) clinics, screening for diabetic retinopathy (DR) in people with diabetes mellitus (DM) was introduced in primary and secondary-level government health facilities. Initially, the project was carried out at the fixed health facilities at one district hospital (DH), two sub-district hospitals (SDH) and two community health centers (CHCs). This was combined with training of existing health care personnel, information-education-communication (IEC) campaign among patients and service providers along with the provision of essential equipment required for screening. In the revised strategy, NCD nurses were also trained for screening. Of 12,788 DM patients registered in NCD clinics, 63.8% (n = 8159) were screened for DR by trained paramedical ophthalmic assistants and the four trained NCD nurses using non-mydriatic fundus camera and teleophthalmology supported remote grading of retinopathy. DR was detected in 9.45% (n = 771) patients and sight-threatening DR (STDR) was detected in 2.35% (n = 192) in one or both eyes. Of 8,159 people screened, 55% (n = 4481) and 45% (n = 3678) were screened at CHC and mobile screening at primary health centers (PHC), respectively. DR screening in a fixed facility at CHC combined with the mobile screening at PHC level and fixed-day screening strategy provides effective coverage.

Effect of health education and screening location on compliance with diabetic retinopathy screening in a rural population in Maharashtra

Purpose: To compare the acceptance of diabetic retinopathy (DR) screening by the proximity of care and health education in rural Maharashtra. Methods: Study was done in the public health facilities in four blocks (in two blocks at community health center (CHC) level and in other two blocks at primary health center (PHC) level with the provision of transport from villages to PHCs) over 3 months. Health education was not imparted in one block in each segment. Health education consisted of imparting knowledge on diabetes mellitus (DM) and DR by trained village-level workers. The screening was done using non-mydriatic fundus camera and teleophthalmology supported remote grading of DR. Results: In the study period, 1,472 people with known diabetes were screened in four blocks and 86.6% (n = 1275) gradable images were obtained from them. 9.9% (n = 126) were detected having DR and 1.9% (n = 24) having sight-threatening DR (STDR). More people accepted screening closer to their residence at the PHC than CHC (24.4% vs 11.4%; P < 0.001). Health education improved the screening uptake significantly (14.4% vs 18.7%; P < 0.01) irrespective of the place of screening—at CHC, 9.5% without health education vs 13.1% with health education; at PHC, 20.1% without health education versus 31.6% with health education. Conclusion: Conducting DR screening closer to the place of living at PHCs with the provision of transport and health education was more effective for an increase in the uptake of DR screening by people with known diabetes in rural Maharashtra.

Menopausal symptoms and quality of life in women above 40 years in an urban resettlement colony of East Delhi

Background: Awareness of menopausal symptoms and their effects on the quality of life is extremely important for better management of symptoms. Objectives: The objectives of this study was to assess the prevalence of menopausal symptoms and its effect on the quality of life in women >40 years and determine the association of sociodemographic, obstetric, and other factors with menopause. Materials and Methods: A cross-sectional study was conducted in 105 women aged 40 years and above residing in an urban resettlement colony of Delhi. An interviewer-administered questionnaire was used to obtain information about sociodemographic and menstrual history. Quality of life related to menopause and its symptoms were measured using the menopause-specific quality of life questionnaire. Statistical analysis was done using SPSS 20.0. Results: Majority of the participants were married (70%), literate (50.5%), unemployed (92.4%), and belonged to upper-lower socioeconomic class (65.7%). The most prevalent symptom was decrease in physical strength in 85.7% of women. The prevalence (89%) and mean score of botheration (3.05 ± 0.917) for physical domain of symptoms was highest and it was significantly associated with the age of subjects and attainment of menopause (P < 0.05). Psychosocial domain was also significantly affected by the age of participants. Sexual domain of symptoms was least prevalent (11%) and had the least mean score of botheration (1.38 ± 1.134). Mean age of menopause was 44.6 years. Awareness regarding menopause was poor (1.9%). Conclusions: Physical symptoms are the most prevalent. Both physical and psychosocial symptoms are associated with age of participants, while only physical symptoms are associated with menopause attainment. The awareness of menopausal symptoms was very poor in the participants.

Temporal changes in behavioural responses and serum metabolites of Cirrhinus mrigala exposed to acute hypoxia

Aim: The present study was conducted to determine the tolerance to reduced oxygen level (hypoxia) and the behavioural and biochemical responses of mrigal, Cirrhinus mrigala to environmental hypoxia. Methodology: Cirrhinus mrigala were subjected to LC50 test for 96 hr and the mortality were recorded. In a second experiment, the fishes were subjected to a stressful, but safe limit of hypoxia (0.5±0.04 mg l-1) and the behavioral responses and serum metabolites (glucose, lactate, total lipids, free amino acids and ammonia) were evaluated for 96 hrs. Results: A median lethal concentration (LC50, 96 hr) value of 0.25 mg l-1dissolved oxygen) was estimated for mrigal. The upper safe limit was 0.49 mg l-1 DO and the lower lethal limit was 0.19 mg l-1 DO. Gill ventilation frequency increased under severe hypoxic conditions and decreased with exposure time. The serum level of glucose, lactate and total lipids increased significantly (P<0.05) within 24 hr of exposure to hypoxic conditions. Free amino acids and ammonia contents were not altered by a four day exposure to hypoxia. Interpretation: The present study explains the basic metabolic and behavioural mechanism behind the hypoxia tolerance of Indian Major Carp, mrigal assisted by alterations in gill ventilation and metabolic responses.

Immunophenotyping of male breast cancer - Experience at a tertiary care centre

Background: Male breast cancers (MBCs) are uncommon and account for 1% of all breast cancers. Medical conditions that increase the estrogen to testosterone ratio are implicated as the risk factors. Morphologically similar, but MBCs have biological differences compared with female breast cancer (FBC). Purpose: The present study was aimed to examine the immunophenotype of MBC, subsequent molecular subtypes, their association with clinicopathological features, and prognosis. Materials and Methods: We analyzed clinicopathological features of 42 cases of MBC, and classified them according to molecular classification using immunohistochemistry (IHC). This is the second largest study from India. Results and Conclusion: Median age of patients was 61 years (age range: 41-87 years). Invasive duct carcinoma comprised 95.2% of cases. Tumor grade II and III was seen in 50% and 47.6% of cases, respectively, and advanced stage disease (III/IV) was seen in 45.2% cases (n = 39). Estrogen receptor (ER) was positive in 97.6% cases, progesterone receptor (PR) in 83.3%, androgen receptor (AR) in 76.2%, HER2 in 4.8%, Cyclin-D1 in 92.9%, Bcl2 in 66.7%, GCDFP-15 in 23.8%, p53 in 16.7%, and Ki67 index was low (<14%) in 66.7% cases. Molecular subtyping of these cases revealed 64.3% of luminal A, 35.7% of luminal B, and no HER2 rich/driven category or triple negative case. There was no statistical significance between luminal A and B category pertaining to overall stage of tumor (P = 0.905). Lymph node metastasis was more commonly associated with luminal B category (P = 0.089). p53 positivity showed significant association with luminal A cases (P = 0.002) and nodal metastasis (P = 0.042). GCDFP-15 positivity showed significant association with higher tumor grade (P = 0.042) and stage (P = 0.047). Stage was the most significant prognostic marker (P < 0.0001). On follow-up (n = 27), all the six cases that showed recurrence/persistent disease were high stage (III/IV) on presentation.

Safety and efficacy of dexamethasone implant along with phacoemulsification and intraocular lens implantation in children with juvenile idiopathic arthritis associated uveitis

Purpose: To assess the safety and efficacy of intraoperative intravitreal dexamethasone implant in patients of juvenile idiopathic arthritis (JIA)-associated uveitis undergoing phacoemulsification with posterior chamber intraocular lens (PCIOL) implantation. Methods: Retrospectively, data of patients with JIA-associated uveitis undergoing phacoemulsification with PCIOL implantation with intraoperative dexamethasone implant injection were analyzed. Patients with a minimum follow-up of 6 months were included. Primary outcome measures were ocular inflammation, intraocular pressure (IOP), best-corrected visual acuity (BCVA), and worsening of uveitis. Results: 8 eyes of 6 patients were included. BCVA was significantly improved at 1, 3, and 6 months postoperatively 0.20 ± 0.09, P = 0.008; 0.18 ± 0.11, P = 0.008; and 0.24 ± 0.11, P = 0.01, respectively. No statistical difference noted in mean IOP at various follow-up visits. None developed worsening of uveitis or Cystoid macular edema. Conclusion: Intraoperative intravitreal dexamethasone implant is a safe and effective in preventing and managing the postoperative inflammation in children with JIA-associated uveitic cataract.