Alloimmunization following antigen-negative red blood cell transfusion.

BACKGROUND: Red blood cell (RBC) alloimmunization can occur secondary to transfusion or pregnancy. It is observed most frequently among patients with hemoglobinopathies and myeloid neoplasms. Although previous antigen exposure is generally required for alloimmunization, some alloantibodies may develop naturally without prior exposure. Other alloantibodies may become evanescent, only to reemerge at a detectable titer following a stimulatory event. In a minute fraction of cases, 'non-naturally occurring' alloantibodies may appear without a known antigenic stimulus. METHODS AND MATERIALS: All testing (antibody detection tests and identification, antigen phenotyping, and crossmatching) was performed using the same method and reagents, but occurred at two hospitals within the Yale New Haven Hospital delivery network, and was performed by technologists utilizing different instruments and reagent lots. RESULTS: We present two cases of seemingly de novo alloimmunization (anti-E and anti-K), and one case of re-emergence of a known, previously evanescent alloantibody (anti-K) following transfusion of RBCs that were antigen-negative for the corresponding antibodies. CONCLUSION: While the exact mechanism underlying the development and/or re-emergence of RBC alloantibodies in the absence of antigenic stimulation remains unclear, these cases highlight this unusual phenomenon, underscoring the general immunogenicity, as well as the potential consequences, of RBC transfusion and reiterates the importance of concluding an alloantibody specificity, even in the absence of known transfusion of RBCs with a particular antigen.

Osteoclast-rich, proximal-type epithelioid sarcoma: clinicopathologic features of 3 unusual cases expanding the histomorphological spectrum.

Epithelioid sarcoma (ES) displays a wide clinicopathologic spectrum. On histopathology, osteoclast-like giant cells have been rarely described in these tumors. A 45-year-old gentleman presented with a perineal swelling of 6-month duration. Radiologic imaging disclosed a large, highly vascular tumor mass in his perineal region that was diagnosed elsewhere as pigmented villonodular synovitis. A 58-year-old lady presented with a recurrent tumor in her right inguinolabial region for which she underwent multiple tumor resections in the past. A 33-year-old lady presented with a right inguinal swelling of 1-month duration that was diagnosed elsewhere as a non-Hodgkin lymphoma on fine needle aspiration cytology. Histopathologic examination of tumors in all the 3 cases revealed epithelioid to "rhabdoid-like" cells arranged in a diffuse pattern interspersed with many osteoclast-like giant cells. The first tumor also revealed focal pseudoangiosarcomatous areas and heterotopic bone formation. By immunohistochemistry, tumor cells in all 3 cases were positive for AE1/AE3, epithelial membrane antigen, and CD34 and were completely negative for INI1/SMARCB1. CD68 immunostaining in 2 tumors highlighted osteoclast-like giant cells. Osteoclast-rich, proximal-type ES are unusual tumors, indicative of an expanding spectrum of ESs. Awareness of this histopathologic pattern and diagnostic confirmation with necessary immunohistochemical stains is crucial to avoid misinterpretation, as these tumors are clinically aggressive and are treated with wide local excision and optional adjuvant radiation therapy.

Isolation of the copper redox steps in the standard selective catalytic reduction on Cu-SSZ-13.

Operando X-ray absorption experiments and density functional theory (DFT) calculations are reported that elucidate the role of copper redox chemistry in the selective catalytic reduction (SCR) of NO over Cu-exchanged SSZ-13. Catalysts prepared to contain only isolated, exchanged Cu(II) ions evidence both Cu(II) and Cu(I) ions under standard SCR conditions at 473 K. Reactant cutoff experiments show that NO and NH3 together are necessary for Cu(II) reduction to Cu(I). DFT calculations show that NO-assisted NH3 dissociation is both energetically favorable and accounts for the observed Cu(II) reduction. The calculations predict in situ generation of Brønsted sites proximal to Cu(I) upon reduction, which we quantify in separate titration experiments. Both NO and O2 are necessary for oxidation of Cu(I) to Cu(II), which DFT suggests to occur by a NO2 intermediate. Reaction of Cu-bound NO2 with proximal NH4(+) completes the catalytic cycle. N2 is produced in both reduction and oxidation half-cycles.

High-Grade Transformation and Carcinosarcoma.

CONTEXT.­: High-grade transformation, previously known as dedifferentiation, in salivary gland carcinoma and carcinosarcoma ex pleomorphic adenoma is a rare phenomenon. It is, however, clinically relevant and affects treatment and prognosis. OBJECTIVE.­: To review the existing literature, describe the histologic and immunophenotypic features, and highlight the diagnostic criteria of high-grade transformation in various salivary gland carcinomas and carcinosarcoma; to review its effect on clinical presentation and prognosis; and to review relevant molecular characteristics and recent concepts and advances. DATA SOURCES.­: Literature search in PubMed using key words such as "high-grade transformation," "dedifferentiation," and "carcinosarcoma" in salivary gland. Relevant articles were reviewed, and additional articles were curated from the references of these articles. CONCLUSIONS.­: High-grade transformation occurs rarely but has a significant impact on prognosis and management. By microscopy, the high-grade area is usually a distinct nodule and shows solid and nested architecture, cellular atypia, high mitotic count, and necrosis. The molecular features are not well established. Carcinosarcoma almost always arises in a pleomorphic adenoma and likely follows an adenoma-carcinoma-sarcoma pathway.

Synergetic impact of MgO on PMMA-ZrO2 hybrid composites: Evaluation of structural, morphological and improved mechanical behavior for dental applications.

This work aims to demonstrate the effect of ZrO2 and MgO inclusion into the Poly(methyl methacrylate) (PMMA). To fabricate novel hybrid composites via heat cure method, various composites (PZM2, PZM4 and PZM6) were synthesized in the system [(95-x) PMMA + 5 ZrO2 + x MgO] (x = 2, 4, and 6) respectively. Density of the prepared composites were determined and varying between 1.035-1.152 g/cm3. X-ray Diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) followed by EDAX and mechanical testing were performed to evaluate the fabricated composite properties. Moreover, to explore the structure of the fabricated composites the 13 C CP-MAS SSNMR and 1 H-13 C Phase-Modulated Lee Goldberg (PMLG) HETCOR Spectrum were recorded which clarify chemical shifting and motional dynamics of the composites. Mechanical tests were performed by UTM and the obtained parameters such as compressive strength, Young's modulus, fracture toughness, brittleness coefficient, flexural strength and flexural modulus are found to be in the range of 91-100 MPa, 0.48-0.51 GPa, 9.122-9.705 MPa.m1/2, 0.66-0.815, 51.03-42.78 MPa and 499-663 MPa respectively. Some more mechanical parameters such as proportional limit, elastic limit, failure strength, modulus of resilience and modulus of toughness were also calculated. Furthermore, tribological properties were also determined and the coefficient of friction (COF) was decreased by 17.4 % and 38 % for composite PZM6 at 20 N and 40 N as compared to the composite PZM2 and the lowest wear volume of 1.55 mm3 was observed for PZM2, whereas the maximum volume loss of 5.64 mm3 is observed for composite PZM6. To check out the biocompatibility, cytotoxicity and genotoxicity of the fabricated composites the Trypan-blue assay was also performed for PZM2 and PZM6 composites. Dissection on the gut of larvae was also performed on the both composites followed by DAPI and DCFH-DA staining. Therefore, these synthesized samples can be used for the fabrication of denture materials.

Metastatic paraganglioma presenting as a primary shoulder mass.

Paragangliomas uncommonly metastasize, including to the bones, wherein these tumors are designated as malignant paragangliomas. A 56-year-old man presented with pain and immobility in his right arm for 1 year. He had a history of controlled hypertension and diabetes mellitus for 2 years. He had also been taking anti-anxiety medications for 25 years. His shoulder imaging revealed an expansile, lytic, destructive lesion in the glenoid cavity, measuring 4.6 × 3.9 × 3.2 cm, involving the adjacent bones and soft tissues. A whole-body PET-CT scan revealed a hypermetabolic destructive mass in the right glenoid cavity and another lesion in his abdomen in the aortocaval region. Initial biopsy and subsequent scapular resection microscopically revealed a multinodular tumor with polygonal cells arranged in a nesting and diffuse pattern, in a vascularized and sclerotic stroma. Tumor cells displayed moderate to abundant, eosinophilic to clear cytoplasm, fine nuclear chromatin, focal intranuclear inclusions, and scattered mitotic figures. Immunohistochemically, tumor cells were positive for vimentin, synaptophysin, chromogranin, and CD56 and negative for AE1/AE3, CK, EMA, CD10, SMA, Melan A, HMB-45, desmin, and S100-P. Biopsy of the abdominal mass revealed foci of tumor cells resembling the scapular tumor. Diagnosis of a malignant paraganglioma was finally offered. The patient's post-operative blood pressure is controlled. Currently, his urinary vanillylmandelic acid and metanephrine levels are normal. He is asymptomatic 11 months post-surgery and is on follow-up. This unusual case is presented to increase a diagnostic index of suspicion for a malignant paraganglioma, including at unconventional musculoskeletal sites. The diagnostic challenge and therapeutic implications are discussed herewith.

Emerging Immunohistochemical Biomarkers for Myeloid Neoplasms.

CONTEXT.­: Pathologists can greatly improve patient care and advance the understanding of disease progression by adeptly employing relevant biomarkers when diagnosing myeloid neoplasms. Although the molecular era has ushered in countless molecular biomarkers in this field, the necessary techniques can be expensive and time-consuming. Novel immunohistochemical biomarkers can help to quickly and inexpensively render the correct diagnosis and predict response to targeted therapies. Hence, it is critical to continue studying and using new and promising immunohistochemical tools for myeloid neoplasms in our current era. OBJECTIVE.­: To review the emerging biomarkers in myeloid neoplasms that can be identified by immunohistochemistry and to discuss their utility, staining patterns, and pitfalls. DATA SOURCES.­: We conducted a scientific literature search of articles related to either a novel immunohistochemical marker or a new utility of an already known marker to assess myeloid neoplasms in PubMed from 2016 to September 30, 2021. We curated relevant contributing studies from the references and subsequent citations of the original articles. CONCLUSIONS.­: Immunohistochemistry is a powerful tool in analyzing biomarkers that play a significant role in the management of patients with myeloid neoplasms. We reviewed 5 immunohistochemical markers, namely, IDH1R132H, ERG, IRF8, GATA1, and NPM1. These markers, depending on the clinical scenario, can be diagnostic, predictive, and also prognostic. Immunohistochemistry also empowers us to evaluate these markers in archival samples, including pretreatment and posttreatment biopsies.

Biphenotypic Sinonasal Sarcoma With High-Grade Features: A Diagnostic Challenge.

Biphenotypic sinonasal sarcoma (BSNS) is a rare neoplasm of the sinonasal tract. These tumors show neural and myogenic differentiation and are characterized by PAX3 translocations. The immunophenotypic features reflect their dual differentiation. They are low-grade sarcomas that show monomorphic spindle cells in sheets, fascicles, and herringbone patterns and are positive for S100 and smooth muscle actin. These tumors are common in elderly female patients and have a locally aggressive course. High-grade presentation or transformation was not documented until recently. Total 3 BSNSs have now been documented in the literature and we report a fourth tumor with high-grade transformation 8 years after the initial presentation. We identify the morphologic and immunohistochemical features of the high-grade areas and we highlight the stark differences with the low-grade areas based on literature and our specimen. We also discuss the diagnostic challenges that may come up with such a presentation.

A Case of Fulminant Right Heart Failure Owing to Tumoral Pulmonary Hypertension.

Tumoral pulmonary hypertension is a rare cause of pulmonary hypertension. We report a patient who was thought to have idiopathic pulmonary arterial hypertension, but later developed fulminant right heart failure ultimately leading to death. Autopsy revealed substantial pulmonary tumor embolism burden originating from liver adenocarcinoma. (Level of Difficulty: Advanced.).

New Insights into Macrophage Polarization and its Prognostic Role in Patients with Colorectal Cancer Liver Metastasis.

Background: As liver metastasis is the most common cause of mortality in patients with colorectal cancer, studying colorectal cancer liver metastasis (CLM) microenvironment is essential for improved understanding of tumor biology and to identify novel therapeutic targets. Methods: We used multiplex immunofluorescence platform to study tumor associated macrophage (TAM) polarization and adaptive T cell subtypes in tumor samples from 105 CLM patients (49 without and 56 with preoperative chemotherapy). Results: CLM exhibited M2 macrophage polarization, and helper T cells were the prevalent adaptive T cell subtype. The density of total, M2 and TGFß-expressing macrophages, and regulatory T cells was lower in CLM treated with preoperative chemotherapy. CLM with right-sided primary demonstrated enrichment of TGFß-expressing macrophages, and with left-sided primary had higher densities of helper and cytotoxic T cells. In multivariate analysis, high density of M2 macrophages correlated with longer recurrence-free survival (RFS) in the entire cohort [hazard ratio (HR) 0.425, 95% CI 0.219-0.825, p=0.011) and in patients without preoperative chemotherapy (HR 0.45, 95% CI 0.221-0.932, p=0.032). High pSMAD3-expressing macrophages were associated with shorter RFS in CLM after preoperative chemotherapy. Conclusions: Our results highlight the significance of a multi-marker approach to define the macrophage subtypes and identify M2 macrophages as a predictor of favorable prognosis in CLM.

Cross Country Determinants of Investors' Sentiments Prediction in Emerging Markets Using ANN.

The paper models investor sentiments (IS) to attract investments for Health Sector and Growth in emerging markets, viz., India, Mainland China, and the UAE, by asking questions such as: What specific healthcare sector opportunities are available in the three markets? Are the USA-IS key IS predictors in the three economies? How important are macroeconomic and sociocultural factors in predicting IS in these markets? How important are economic crises and pandemic events in predicting IS in these markets? Is there contemporaneous relation in predicting IS across the three countries in terms of USA-IS, and, if yes, is the magnitude of the impact of USA-IS uniform across the three countries' IS? The artificial neural network (ANN) model is applied to weekly time-series data from January 2003 to December 2020 to capture behavioral elements in the investors' decision-making in these emerging economies. The empirical findings confirmed the superiority of the ANN framework over the traditional logistic model in capturing the cognitive behavior of investors. Health predictor-current health expenditure as a percentage of GDP, USA IS predictor-spread, and Macro-factor GDP-annual growth % are the common predictors across the 3 economies that positively impacted the emerging markets' IS behavior. USA (S&P 500) return is the only common predictor across the three economies that negatively impacted the emerging markets' IS behavior. However, the magnitude of both positive and negative impacts varies across the countries, signifying unique, diverse socioeconomic, cultural, and market features in each of the 3 economies. The results have four key implications: Firstly, US market sentiments are an essential factor influencing stock markets in these countries. Secondly, there is a need for developing a robust sentiment proxy on similar lines to the USA in the three countries. Thirdly, investment opportunities in the healthcare sector in these economies have been identified for potential investments by the investors. Fourthly, this study is the first study to investigate investors' sentiments in these three fast-emerging economies to attract investments in the Health Sector and Growth in the backdrop of UN's 2030 SDG 3 and SDG 8 targets to be achieved by these economies.

Phase II study of durvalumab (anti-PD-L1) and trametinib (MEKi) in microsatellite stable (MSS) metastatic colorectal cancer (mCRC).

BACKGROUND: Monotherapy with immune checkpoint blockade is ineffective for patients (pts) with microsatellite stable (MSS) metastatic colorectal cancer (mCRC). This study investigates whether the combination of trametinib (T) with durvalumab (D) can alter the immune tumor microenvironment (TME) by successfully priming and activating T-cells. METHODS: Open-label, single-center, phase II trial with primary endpoint of immune-related response rate for combination of T+D in refractory MSS mCRC pts (NCT03428126). T is 2 mg/day orally starting 1 week prior to D, which is given 1500 mg intravenously every 4 weeks. Simon 2-stage design used to enroll 29 pts into first stage, requiring a response in two or more pts to proceed to stage 2. Tumor biopsies were collected at baseline (BL) and early on-treatment (OT) at week 4. RESULTS: Twenty nine treated pts include 48% females, median age 48 years (range 28-75), and median prior therapies 2 (range 1-5). No grade (G) 4 or 5 treatment-related adverse events (TRAE). The most common TRAE of any grade was acneiform rash, 17% being G3. One of 29 pts had confirmed partial response (PR) lasting 9.3 months (mo) for an overall response rate of 3.4%. Seven pts had stable disease (SD) and five pts (1 PR, 4 SD) demonstrated decrease in total carcinoembryonic antigen ng/mL (best percentage reduction: 94%, 95%, 42%, 34%, and 22%, respectively). Median progression-free survival was 3.2 mo (range 1.1-9.3 months). Three pts with both liver and lung metastases demonstrated discrepant responses in which clinical benefit was present in the lung metastases but not liver metastases. Comparison of BL and 4-week OT tumor tissue flow cytometry demonstrated no changes in T-cell infiltration but upregulation expression of PD-1 and Tim3 on CD8 T cells. However, expression of PD-1 and Tim3 as single markers and as coexpressed markers was observed to increase OT relative to BL (p=0.03, p=0.06 and p=0.06, respectively). CONCLUSIONS: T+D demonstrated acceptable tolerability in pts with refractory MSS mCRC. The response rate in the first stage of the study did not meet efficacy criteria to proceed to the second stage. Specific site of metastatic disease may impact outcomes in novel immunotherapy combination trials. TRIAL REGISTRATION NUMBER: NCT03428126.

Assessment of Clinical Response Following Atezolizumab and Bevacizumab Treatment in Patients With Neuroendocrine Tumors: A Nonrandomized Clinical Trial.

Importance: Therapies for patients with advanced well-differentiated neuroendocrine tumors (NETs) have expanded but remain inadequate, with patients dying of disease despite recent advances in NET therapy. While patients with other cancers have seen long-term disease control and tumor regression with the application of immunotherapies, initial prospective studies of single-agent programmed cell death 1 inhibitors in NET have been disappointing. Objective: To evaluate the response rate following treatment with the combination of the vascular endothelial growth factor inhibitor bevacizumab with the programmed cell death 1 ligand 1 inhibitor atezolizumab in patients with advanced NETs. Design, Setting, and Participants: This single-arm, open-label nonrandomized clinical study in patients with rare cancers included 40 patients with advanced, progressive grade 1 to 2 NETs (20 with pancreatic NETs [pNETs] and 20 with extrapancreatic NETs [epNETs]) treated at a tertiary care referral cancer center between March 31, 2017, and February 19, 2019. Data were analyzed from June to September 2021. Interventions: Patients received intravenous bevacizumab and atezolizumab at standard doses every 3 weeks until progression, death, or withdrawal. Main Outcomes and Measures: The primary end point was objective radiographic response using Response Evaluation Criteria in Solid Tumors, version 1.1, with progression-free survival (PFS) as a key secondary end point. Results: Following treatment of the 40 study patients with bevacizumab and atezolizumab, objective response was observed in 4 patients with pNETs (20%; 95% CI, 5.7%-43.7%) and 3 patients with epNETs (15%; 95% CI, 3.2%-37.9%). The PFS was 14.9 (95% CI, 4.4-32.0) months and 14.2 (95% CI, 10.2-19.6) months in these cohorts, respectively. Conclusions and Relevance: In this nonrandomized clinical trial, findings suggest that clinical responses in patients with NET may follow treatment with the combination of bevacizumab and atezolizumab, with a PFS consistent with effective therapies. Trial Registration: ClinicalTrials.gov Identifier: NCT03074513.

An analysis of research biopsy core variability from over 5000 prospectively collected core samples.

Factors correlated with biopsy tissue adequacy and the prevalence of within-biopsy variability were evaluated. Totally, 1149 research biopsies were performed on 686 patients from which 5090 cores were assessed. Biopsy cores were reviewed for malignant percentage (estimated percentage of cells in the core that were malignant) and malignant area (estimated area occupied by malignant cells). Linear mixed models and generalized linear mixed models were used for the analysis. A total of 641 (55.8%) biopsies contained a core with <10% malignant percentage (inadequate core). The chance of an inadequate core was not influenced by core order, though the malignant area decreased with each consecutive core (p < 0.001). Younger age, bone biopsy location, appendiceal tumor pathology, and responding/stable disease prior to biopsy increased the odds of a biopsy containing zero adequate cores. Within-biopsy variability in core adequacy is prevalent and suggests the need for histological tumor quality assessment of each core in order to optimize translational analyses.

ARID1A Mutation May Define an Immunologically Active Subgroup in Patients with Microsatellite Stable Colorectal Cancer.

PURPOSE: AT-rich interactive domain 1A (ARID1A) is commonly mutated in colorectal cancer, frequently resulting in truncation and loss of protein expression. ARID1A recruits MSH2 for mismatch repair during DNA replication. ARID1A deficiency promotes hypermutability and immune activation in preclinical models, but its role in patients with colorectal cancer is being explored. EXPERIMENTAL DESIGN: The DNA sequencing and gene expression profiling of patients with colorectal cancer were extracted from The Cancer Genome Atlas and MD Anderson Cancer Center databases, with validation utilizing external databases, and correlation between ARID1A and immunologic features. IHC for T-cell markers was performed on a separate cohort of patients. RESULTS: Twenty-eight of 417 patients with microsatellite stable (MSS) colorectal cancer (6.7%) had ARID1A mutation. Among 58 genes most commonly mutated in colorectal cancer, ARID1A mutation had the highest increase with frameshift mutation rates in MSS cases (8-fold, P < 0.001). In MSS, ARID1A mutation was enriched in immune subtype (CMS1) and had a strong correlation with IFNγ expression (Δz score +1.91, P < 0.001). Compared with ARID1A wild-type, statistically significant higher expression for key checkpoint genes (e.g., PD-L1, CTLA4, and PDCD1) and gene sets (e.g., antigen presentation, cytotoxic T-cell function, and immune checkpoints) was observed in mutant cases. This was validated by unsupervised differential expression of genes related to immune response and further confirmed by higher infiltration of T cells in IHC of tumors with ARID1A mutation (P = 0.01). CONCLUSIONS: The immunogenicity of ARID1A-mutant cases is likely due to an increased level of neoantigens resulting from increased tumor mutational burden and frameshift mutations. Tumors with ARID1A mutation may be more susceptible to immune therapy-based treatment strategies and should be recognized as a unique molecular subgroup in future immune therapy trials.

Efficacy, Safety, and Biomarker Analysis of Combined PD-L1 (Atezolizumab) and VEGF (Bevacizumab) Blockade in Advanced Malignant Peritoneal Mesothelioma.

Malignant peritoneal mesothelioma (MPeM) is a rare but aggressive malignancy with limited treatment options. VEGF inhibition enhances efficacy of immune-checkpoint inhibitors by reworking the immunosuppressive tumor milieu. Efficacy and safety of combined PD-L1 (atezolizumab) and VEGF (bevacizumab) blockade (AtezoBev) was assessed in 20 patients with advanced and unresectable MPeM with progression or intolerance to prior platinum-pemetrexed chemotherapy. The primary endpoint of confirmed objective response rate per RECISTv1.1 by independent radiology review was 40% [8/20; 95% confidence interval (CI), 19.1-64.0] with median response duration of 12.8 months. Six (75%) responses lasted for >10 months. Progression-free and overall survival at one year were 61% (95% CI, 35-80) and 85% (95% CI, 60-95), respectively. Responses occurred notwithstanding low tumor mutation burden and PD-L1 expression status. Baseline epithelial-mesenchymal transition gene expression correlated with therapeutic resistance/response (r = 0.80; P = 0.0010). AtezoBev showed promising and durable efficacy in patients with advanced MPeM with an acceptable safety profile, and these results address a grave unmet need for this orphan disease. SIGNIFICANCE: Efficacy of atezolizumab and bevacizumab vis-à-vis response rates and survival in advanced peritoneal mesothelioma previously treated with chemotherapy surpassed outcomes expected with conventional therapies. Biomarker analyses uncovered epithelial-mesenchymal transition phenotype as an important resistance mechanism and showcase the value and feasibility of performing translationally driven clinical trials in rare tumors.See related commentary by Aldea et al., p. 2674.This article is highlighted in the In This Issue feature, p. 2659.

Symptomatic intracranial Rosai-Dorfman disease in the suprasellar region treated with conformal radiotherapy - A report of two cases and literature review.

Intracranial Rosai-Dorfman Destombes (RDD) disease is a rare entity. Lesions can lead to cranial nerve palsies and visual loss, especially in suprasellar location. Resection is considered to be definitive treatment; however, complete excision is difficult to achieve in view of the close proximity of critical structures. Radiotherapy (RT) is sometimes used for refractory or progressive disease for local tumor control and amelioration of symptoms. We report two patients with suprasellar RDD's with progressive symptoms treated with conformal RT after subtotal excision. These patients were treated with high precision conformal techniques to a dose of 45 Gy with significant and durable improvement in vision.

Identification of Luminal Subtypes of Breast Carcinoma Using Surrogate Immunohistochemical Markers and Ascertaining Their Prognostic Relevance.

BACKGROUND: Therapeutic decisions in breast carcinoma are being made on the basis of tumor cell proliferation using exorbitant genomic tests. The 2013 St Gallen meeting advocated surrogate definitions for classifying tumors into luminal subtypes on the basis of immunohistochemical (IHC) markers. We studied the classification of estrogen receptor (ER)-positive tumors using these definitions as well as different methods for Ki-67 labeling index (LI) estimation. PATIENTS AND METHODS: A total of 541 ER+ invasive breast carcinoma cases from January 2012 to December 2012 were evaluated for Ki-67 LI by the average and hot spot methods. The IHC results of ER, PR, and human epidermal growth factor receptor 2 (HER2) were noted. HER2 IHC equivocal (2+) samples were subjected to HER2 fluorescence in-situ hybridization testing. Luminal subgroups created on the basis of the 2013 St Gallen meeting guidelines were correlated with clinicopathologic variables and disease-free survival. RESULTS: The distribution of luminal subtypes was as follows: luminal A-like, 13.3%; luminal B-like (HER2-), 57.9%; and luminal B-like (HER2+), 28.8%. Approximately 6% of cases were recategorized into different subgroups when the average method was used instead of the hot spot method for Ki-67 LI assessment. Younger patients (≤ 50 years), grade 3 tumors, positive axillary nodes, recurrence, and distant metastasis had a positive statistical correlation with luminal B-like (HER2-) subtype. Patients with luminal B-like (HER2-) tumors had a shorter disease-free survival compared to patients with luminal A-like tumors. CONCLUSION: Ki-67 LI, irrespective of the method of assessment, along with PR, can be efficiently used to divide ER+ tumors into prognostic subgroups in Indian patients.

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies.

In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.