Identification Of Plasma Cells Research Articles

A pathologist-AI collaboration framework for enhancing diagnostic accuracies and efficiencies.

In pathology, the deployment of artificial intelligence (AI) in clinical settings is constrained by limitations in data collection and in model transparency and interpretability. Here we describe a digital pathology framework, nuclei.io, that incorporates active learning and human-in-the-loop real-time feedback for the rapid creation of diverse datasets and models. We validate the effectiveness of the framework via two crossover user studies that leveraged collaboration between the AI and the pathologist, including the identification of plasma cells in endometrial biopsies and the detection of colorectal cancer metastasis in lymph nodes. In both studies, nuclei.io yielded considerable diagnostic performance improvements. Collaboration between clinicians and AI will aid digital pathology by enhancing accuracies and efficiencies.

Enhancing diagnostic accuracy of multiple myeloma through ML-driven analysis of hematological slides: new dataset and identification model to support hematologists

Multiple Myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells within the bone marrow. Diagnosing MM presents considerable challenges, involving the identification of plasma cells in cytology examinations on hematological slides. At present, this is still a time-consuming manual task and has high labor costs. These challenges have adverse implications, which rely heavily on medical professionals’ expertise and experience. To tackle these challenges, we present an investigation using Artificial Intelligence, specifically a Machine Learning analysis of hematological slides with a Deep Neural Network (DNN), to support specialists during the process of diagnosing MM. In this sense, the contribution of this study is twofold: in addition to the trained model to diagnose MM, we also make available to the community a fully-curated hematological slide dataset with thousands of images of plasma cells. Taken together, the setup we established here is a framework that researchers and hospitals with limited resources can promptly use. Our contributions provide practical results that have been directly applied in the public health system in Brazil. Given the open-source nature of the project, we anticipate it will be used and extended to diagnose other malignancies.

Performance of the new MC-80 automated digital cell morphology analyser in detection of normal and abnormal blood cells: Comparison with the CellaVision DM9600.

Mindray MC-80 is an automated system for digital imaging of white blood cells (WBCs) and their pre-classification. The objective of this work is to analyse its performance comparing it with the CellaVision® DM9600. A total of 445 samples were used, 194 normal and 251 abnormal: acute leukaemia (100), myelodysplastic syndromes/myeloproliferative neoplasms (33), lymphoid neoplasms (50), plasma cell neoplasms (14), infections (49) and thrombocytopenia (5). WBC pre-classification values with the MC-80 and DM9600 were compared with (1) the microscope, (2) Mindray BC-6800Plus differentials in only normal samples, and (3) confirmed or reclassified images (post-classification). Pearson's correlation, Lin's concordance, Passing-Bablok regression, and Bland-Altman plots were used. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values for abnormal cells using the MC-80 were calculated. The PPV and NPV were above 98% and 99%, for normal samples. For immature granulocytes (IG), NPV and PPV were 100% and 74.2%. When comparing the WBC differentials using the MC-80, the microscope and the BC-6800Plus, no differences were found except for basophils and IG. Our results showed good agreement between the pre- and post-classification of normal WBC, including IG, quantified by high correlation and concordance values (0.91-1). Sensitivity and specificity for blasts were 0.984 and 0.640. The MC-80 detected abnormal lymphocytes in 30% of the smears from patients with lymphoid neoplasm. Plasma cell identification was better using the DM9600. The sensitivity and specificity for erythroblast detection were 1 and 0.890. We found that the MC-80 shows high performance for WBC differentials for both normal samples and patients with haematological diseases.

P-368 Deep learning-based classifier for malignant plasma cell identification from single-cell RNA-seq data

P-368 Deep learning-based classifier for malignant plasma cell identification from single-cell RNA-seq data

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

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. 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. 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). 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.

IGH cytogenetic abnormalities can be detected in multiple myeloma by imaging flow cytometry

AimsCytogenetic abnormalities involving the IGH gene are seen in up to 55% of patients with multiple myeloma. Current testing is performed manually by fluorescence in situ hybridisation (FISH) on purified...

Understanding the Bioactivity and Prognostic Implication of Commonly Used Surface Antigens in Multiple Myeloma.

Multiple myeloma (MM) progression is dependent on its interaction with the bone marrow microenvironment and the immune system and is mediated by key surface antigens. Some antigens promote adhesion to the bone marrow matrix and stromal cells, while others are involved in intercellular interactions that result in differentiation of B-cells to plasma cells (PC). These interactions are also involved in malignant transformation of the normal PC to MM PC as well as disease progression. Here, we review selected surface antigens that are commonly used in the flow cytometry analysis of MM for identification of plasma cells (PC) and the discrimination between normal and malignant PC as well as prognostication. These include the markers: CD38, CD138, CD45, CD19, CD117, CD56, CD81, CD27, and CD28. Furthermore, we will discuss the novel marker CD24 and its involvement in MM. The bioactivity of each antigen is reviewed, as well as its expression on normal vs. malignant PC, prognostic implications, and therapeutic utility. Understanding the role of these specific surface antigens, as well as complex co-expressions of combinations of antigens, may allow for a more personalized prognostic monitoring and treatment of MM patients.

Abstract P1-08-26: Morphologic characterization of tumor-infiltrating lymphocytes and its relation with pathological response in a series of breast cancer patients treated with primary chemotherapy

Abstract Introduction: Tumor-Infiltrating Lymphocytes (TILS) is a well-known predictor of response to primary chemotherapy1,2 and a prognostic factor for improved survival in different breast cancer subtypes3. We present data on the association of the level of TILs and morphologic characteristics of such infiltrate with pCR. Material and methods: A series of 477 breast cancer patients (479 tumors) treated with primary chemotherapy at Catalan Institut of Oncolgy - H.U. Bellvitge between January 2009 and December 2016. Chemotherapy consisted on anthracyclines and taxanes (plus trastuzumab if Her-2 positive disease). Levels of percentage of TILs using hematoxylin-eosin-stained sections of diagnostic core-needle biopsy were evaluated according to international guidelines in a prospectively defined retrospective analysis. Characterization of TILS consisted on identification of plasma cells, intraepithelial infiltrate vs stromal infiltrate and homogeneous vs heterogeneous infiltrate. A sub-classification using levels of TILS and heterogeneity was done for statistical purposes. Levels of TILS and their morphological characteristics were examined for their associations with pCR adjusted for predictive clinic-pathological factors, by univariate and multivariate logistic regression, statistical significance set at 0.05. A ROC curve was performed to look for a cut-point of TILs to predict PCR. Results: The mean value of TILs was 23.79% (SD, 24%). TILs were significantly higher in ductal carcinomas (39.1% vs 0%, p=0.007), grade 3 (55.2 vs 17.7%p<0.001), ki 67 >30 (48.8% vs 24.5%, p<0.001), HER2 (56.6%) and triple negative tumors (TNBC)(56.6%) vs luminal (20%), p<0.001). The level of TILS was set in 20% to predict pCR by a ROC curve (S=62.3% E=71%). Characterization of TILs found plasma cells in 41.1% of the samples, intraepithelial infiltrate in 11.2% and homogeneous infiltrate in 60.1%. Homogeneously high infiltrate was found in 20% of the samples. In the univariate analysis pCR was higher in samples with TILs > 20% (15.2% vs 41.5%, OR: 3.96 [95%CI, 2.57-6.10]; P < 0.001); plasma cells (OR 6.61 [95%CI, 1.51-28.8]; P=0.01), intraepithelial TILS (OR: 10.34 [95%CI, 2.22-48.01]; P =0.003), homogeneous high infiltrate (OR: 13.6 [95%CI, 3.04-60.77]; P =0.001). In luminal tumors, TILs over 20% predicted pCR (OR 12.3 [95%CI, 4.0-37.7]; P < 0.001) as well as in TNBC (OR 4.32 [95%CI, 1.77-10.53]; P=0 .001) but not in those cases with HER2 positive tumors (luminalB HER2 + HER2) (OR 1.65 [95%CI, 0.88-3.07]; P=0.118). In the multivariate analyses, levels of TILs > 20% were associated with higher pCR rates (adjusted odds ratio, 2.44 [95%CI, 1.48-4.01]; P < .001). Conclusions: The presence of TILs over 20% at diagnosis is an independent, positive, predictive marker of pCR in early breast cancer treated with neoadjuvant chemotherapy. Interestingly, the predictive information added by TILs >20% was higher in luminal and triple negative tumors compared to HER2 positive cases. Careful morphological characterization of TILS may add valuable predictive information and can be done in current pathologic laboratories with a well-trained breast cancer pathologist.

Chronic endometritis in in vitro fertilization failure patients

Introduction: Chronic endometritis (CE) is a common cause of infertility in asymptomatic patients and its diagnosis and treatments improved assisted reproduction technique outcome in most of the specialized centers. Diagnosis of CE in endometrial biopsy by Hematoxylin and Eosin (H&E) stain is hard to identify chronic inflammatory cells from the stroma and the use of plasma cells-specific stains is helpful. Aim of the work: Evaluation of the use of CD138 in the identification of plasma cells in endometrial biopsy of patients with previous IVF trial failure. Material and methods: Hysteroscopic and curettage endometrial biopsies from fifty-five females with previous IVF trial failure were stained with H&E and CD138 immunostaining for detection of plasma cells. Results: Plasma cells were identified in 52.7% of cases by H&E and in 6/55 by CD138 immunostaining. CD138 is more sensitive in detecting plasma cells in endometrial biopsy than H&E stain. There was a significant statistical correlation between CE and abnormal uterine bleeding, abortion and primary infertility (p > 0.5). Conclusion: Diagnosis of CE is helpful in infertility patients with IVF trial failure to improve the outcome of the maneuver. CD138 is more sensitive for plasma cells specially in endometrial biopsies than H&E.

Abstract 4271: Standardized approach for monitoring plasma cell disorders

Abstract Multi-color flow cytometry using consensus surface markers (CD138, CD38, CD19, CD45, CD27, CD56, CD81, and CD117), intracellular kappa and lambda, as well as other auxiliary markers is routinely being used for the identification and characterization of plasma cells. Here, we describe the development of a modular 10-color plasma cell panel for immunophenotyping of the plasma cell compartment. The panel was developed as an 8-color dried backbone, which can be supplemented with 2 liquid (drop in) reagents. Lyophilized or dried reagent cocktails in a unit test format are known to drive workflow efficiency in clinical settings by eliminating the need for manual cocktailing (of reagents), and frequent batch to batch verification procedures. Longer shelf life and room temperature storage are additional appealing features for lyophilized and dried products. We have developed a dried-down 8-color plasma cell panel, which is manufactured in a single test-per-tube, ready to use format, and can be supplemented with liquid (drop in) reagents. The flexibility of being able to add 2 liquid reagents to the dried backbone provides the option of using intracellular kappa and lambda to examine clonality or using CD38-multi-epitope and/or anti-p63 (VS38c) reagent(s). The single test-per-tube format is ideally suited for MRD applications, where sample size is limited and there is a need for acquiring a large number of events for data analysis. Also, the dried reagent panel is comprised of multiple high performing polymer dyes, which are brighter than conventional dyes, and afford high resolution of the relevant cell populations. Furthermore, the dried reagent tubes have a long shelf life (12 months) and significantly enhance the workflow efficiency. Comparison of the liquid and dried versions of the 10-color plasma cell panel using peripheral blood from healthy donors (as well as control cell lines) showed similar staining patterns, comparable population statistics, and fluorochrome brightness. The dried plasma cell tube(s) were also tested on bone marrow samples from healthy and diseased subjects in conjugation with intracellular staining for kappa and lambda. Our results demonstrate that the 10-color plasma cell panel can be used for identification of the plasma cell compartment (based on CD38 & CD138 expression), assessment of aberrant marker expression (CD19, CD27, CD81, CD117, CD45, CD56) for the identification of normal and abnormal plasma cells, and examining clonality in multiple patient samples. Citation Format: Ulrika Johansson, Na Li, Suraj Saksena, Natalie Golts, Brent Gaylord. Standardized approach for monitoring plasma cell disorders [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4271.

Standardized Approach for Monitoring Plasma Cell Disorders

Abstract Multi-color flow cytometry using consensus surface markers (CD138, CD38, CD19, CD45, CD27, CD56, CD81, and CD117), intracellular kappa and lambda, as well as other auxiliary markers is routinely used for the identification and characterization of plasma cells. Here, we describe the development of a modular 10-color plasma cell panel for immunophenotyping of the plasma cell compartment. The panel was developed as an 8-color dried backbone, manufactured in a single test-per-tube, ready to use format. The dried backbone can be supplemented with liquid (drop in) reagents, which provides the flexibility of using intracellular kappa and lambda to examine clonality or using CD38-multi-epitope and/or anti-p63 (VS38c) reagent(s). The single test-per-tube format is ideally suited for MRD applications, where sample size is limited and acquisition of millions of events is needed for data analysis. In addition, the dried reagent tubes have a long shelf life (12 months at 20–25°C vs. 24 hours for liquid cocktails with multiple BV dyes in BSB buffer at 2–8°C). Furthermore, utilization of multiple high-performing polymer dyes in the panel affords high resolution of the relevant cell populations. The dried plasma cell tube(s) were tested on bone marrow samples from healthy and diseased subjects in conjunction with intracellular staining for kappa and lambda. Our results demonstrate that the 10-color plasma cell panel can be used for identification of the plasma cell compartment (based CD38 and CD138 expression), assessment of aberrant marker expression (CD19, CD27, CD81, CD117, CD45, and CD56) for the identification of normal and abnormal plasma cells, and examining clonality in multiple patient samples (Kappa and Lambda).

Plasma cell identification based on evidential segmentation and supervised learning

Plasma cell identification based on evidential segmentation and supervised learning

Plasma cell identification based on evidential segmentation and supervised learning

Myeloma disease is among the most common type of cancer, it is characterised by proliferation of plasma cells, kind of white blood cell (WBC). Early diagnosis of the disease can improve the patient's survival rate. The manual diagnosis involves clinicians to visually examine microscopic bone marrow images for any signs of cells proliferation. This step is often laborious and can be highly subjective due to clinician's expertise. Automatic system based on WBC identification and counting provides more accurate result than manual method. This system is mainly based on three major steps: cell's segmentation, cell's characterisation and cell's classification. In the proposed system, microscopic images of bone marrow blood are segmented by the combination of watershed transform and the evidence theory, the segmented cells are characterised with shape and colour texture features, and then classified into plasma cells or not plasma cells with three supervised classifiers; support vector machines, K-nearest neighbour and decision tree. Experimental results show that the recognition of plasma cells with the K-nearest neighbour achieved 97% of correct rate with 100% of specificity.

Identification of plasma cells in peripheral blood: The role of antibody-synthesizing lymphocytes AS-LYMP

Identification of plasma cells in peripheral blood: The role of antibody-synthesizing lymphocytes AS-LYMP

Chronic Endometritis, a Common Disease Hidden Behind Endometrial Polyps in Premenopausal Women: First Evidence From a Case-Control Study

To investigate the correlation between endometrial polyps (EPs) and chronic endometritis (CE). Single-center retrospective case-control study. Academic center. A total of 480 premenopausal women with abnormal uterine bleeding (AUB) were enrolled. Group A included 240 women suffering from EPs (diagnosed by hysteroscopy and histology), and group B included 240 patients without EPs at hysteroscopy. In group A, 2 separate samples were obtained from the EPs (group A polyps) and endometrium (group A endometrium). In group B, a single sample of endometrial tissue was evaluated (group B endometrium). All tissue samples were subjected to immunohistochemistry for CD-138 for plasma cell identification. The primary study endpoint was to compare the rates of CE in group A endometrium versus group B endometrium. The secondary endpoint was to evaluate the consistency in CD-138 immunoreactivity between group A polyps and compared with group A endometrium. A higher prevalence of CE was observed in group A endometrium compared with group B endometrium (p < .0001). The total percentage of EPs showing CD-138 positivity was 76.7% (184 of 240). CE was more frequent in women with CD-138+ EPs compared to those with CD-138- EPs (p < .0001). EPs were commonly associated with CE in the premenopausal women suffering from AUB. Moreover, the majority of EPs were positive for CD-138 staining, suggesting a possible hidden association between chronic inflammation and EPs.

Comparison of Minimal Residual Disease Detection in Autografts of Patients with Multiple Myeloma between 8-Color Multiparameter Flow Cytometry (EuroFlow) and Next-Generation Sequencing

Background: Autologous stem cell transplantation (ASCT) in conjunction with novel therapeutic drugs can dramatically improve response rates and the prognosis of patients with multiple myeloma (MM). However, most patients with MM are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper responses are required. Next-generation sequencing (NGS) and multiparameter flow cytometry (MFC) methods have been used to assess MRD. However, the lack of standardization of conventional MFC approaches has had a negative impact on its reproducibility. Recently, a next-generation MFC method (EuroFlow, NGF) has been developed by the EuroFlow Consortium and the International Myeloma Foundation (IMF) for a highly sensitive and standardized detection of MRD in MM.Aims: To compare the prognostic value of MRD detection in autografts in MM between NGS (Adaptive) and 8-color MFC method (EuroFlow, NGF), and also MRD levels between fresh and cryopreserved autografts.Methods: A total of 39 newly-diagnosed MM patients who underwent ASCT were enrolled in this study. Median age 60 at ASCT (range 41-69); males 22, females 17; ISS 1 (n=10), 2 (n=19), 3 (n=10). 10 patients showed high-risk chromosomal abnormalities (t(4;14) (n=9), del17p & t(4;14) (n=1)). The induction regimen was bortezomib-based chemotherapy. All patients received melphalan 200 mg/sqm as conditioning regimen before ASCT. 34 of 39 (87%) patients received maintenance therapy until progressive disease. The best response post-ASCT was as follows: 23sCR, 2CR, 12VGPR, 2PR. 39 autografts, one from each MM patient, were analyzed using NGF and NGS methods. The NGF method was based on a standardized lyse-wash-and-stain sample preparation protocol, the measurement of high numbers of cells and an optimized 8-color, 2-tubes, antibody panel, for accurate identification of plasma cells (PCs) and discrimination between phenotypically aberrant (aPC) and normal PC (nPC) (J Flores-Montero et al., Leukemia 2017). NGS-based MRD assessment was performed using Adaptive's standardized NGS-MRD Assay (Seattle, WA) (Martinez-Lopez et al., Blood 2014). To assess the correlation of MRD levels between fresh and cryopreserved autografts using NGF, 6 additional MM patients' autografts were used.Results: MRD levels in all 39 autografts were assessed using EuroFlow, while those in 32 of 39 (82%) were assessed with NGS due to limited availability of material for calibration. We identified abnormal plasma cells (aPC) in autografts based on multivariate analysis of individual cells from each patient (e.g. CD56+, CD19-, CyIgκ+, CD117+). Since there was a good correlation in MRD levels between fresh and thawed frozen autografts detected by EuroFlow (R=0.943, P=0.02), we assessed the MRD levels in thawed frozen autografts. For the MM MRD in autografts, the events from tube 1 and tube 2 were combined and a median of 7.3×106 (range: 2.2×106-37.6×106) events was acquired. The sensitivity of EuroFlow was 1×10-5-2×10-6 while that of NGS was 10-7 due to the high number of DNA derived from autografts (Takamatsu et al., Ann Oncol 2017). 21 of 39 (54%) cases were MRD positive by 8-color MFC while 22 of 32 (69%) cases were MRD positive by NGS. The correlation of MRD levels between 8-color MFC and NGS was relatively high (Fig. 1A). MRD negative by NGF (MRDMFC (-)) cases tended to show better PFS than MRDMFC (+) cases (P=0.145) (Fig. 1B) while MRD negative by NGS (MRDNGS (-)) cases showed significantly better PFS than MRDNGS (+) cases (P=0.03) (Fig. 1C). Furthermore, MRDMFC (-) MRDNGS (-) cases showed significantly better PFS than MRDMFC (-) MRDNGS (+) cases (P=0.01), but the PFS of MRDMFC (-) MRDNGS (+) cases was not different from that of MRDMFC (+) MRDNGS (+) cases (P=0.70). MRDMFC (-) and MRDNGS (-) cases showed better OS than MRDMFC (+) (P=0.14) and MRDNGS (+) (P=0.08) cases, respectively.Conclusions: Although EuroFlow is a fast and accurate method for detecting MRD of MM in autografts, in this study the NGS platform had a higher sensitivity and prognostic value than EuroFlow. The homogenous nature of the mobilized autograft relative to the focal nature of myeloma in bone marrow might provide a better sample to assess MRD. [Display omitted] DisclosuresTakamatsu:Celgene: Honoraria, Research Funding; Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria. Nakao:Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.

Cytoplasmic Immunoglobulin Light Chain Revelation and Interphase Fluorescence In Situ Hybridization in Myeloma.

The cytogenetic analysis of plasma cell myeloma (PCM) allows stratification of patients so that prognosis may be determined and appropriate therapeutic options can be discussed. Owing to the patchy nature of the disease in the bone marrow (BM), the low proliferative activity of plasma cells and the cryptic nature of some PCM-associated cytogenetic changes, karyotypic analysis in this disease should be augmented with targeted interphase fluorescence in situ hybridization (FISH). Immunofluorescent revelation of cytoplasmic immunoglobulin light chains, together with interphase FISH (cIg-FISH), allows the identification of plasma cells within a sample so that they may be scored preferentially. This is particularly useful in situations where there are only a small percentage of plasma cells in a sample. Where an underlying myeloid disease is suspected the cIg-FISH-negative cells can be scored separately. Two methods are provided in this chapter: the technique for cIg-FISH in fresh PCM BM samples and a procedure for use in fixed cytogenetics preparations.

A Comparison of Clinical FISH and Sequencing Based FISH Estimates in Multiple Myeloma: An Mmrf Commpass Analysis

Fluorescent in situ hybridization (FISH) is commonly used in the multiple myeloma field to subtype and risk-stratify patients. There are many benefits to FISH based assays, which are widely used around the world and represent true single cell assays. However, there are significant discrepancies in the specific assays, utilization of reflex testing strategies, and enumeration requirements between clinical centers. By comparison next-generation sequencing tests can be designed to simultaneously detect the copy number abnormalities and translocations detected by clinical FISH along with gene mutations that cannot be detected by FISH. As part of the MMRF CoMMpass Study we have compared the results attained using clinical FISH assays compared to sequencing based FISH (Seq-FISH) results.Clinical FISH reports from a random subset of 339 CoMMpass patients were extraction by a single individual based on the ISCN result lines of each report. To validate the accuracy of the central data extraction, two independent cross validations of 10% of the cohort were performed, after which our data entry error rate is expected to be less than 0.348%. The Seq-FISH results were extracted from the whole genome sequencing data available from each patient using a rapid and fully automated informatics process and the results were cross-validated using the matching exome sequencing data for copy number abnormalities and by RNA sequencing data for dysregulated immunoglobulin translocation target genes.There were 230 patients with clinical FISH and Seq-FISH results. In this cohort, 151 translocations were identified by Seq-FISH. This includes translocations to MYC, CCND2, MAFA, and those involving IgK and IgL, which are not tested by clinical FISH. After filtering non-tested translocations there are 118 translocations identified by Seq-FISH. Only 97 of these translocations had a clinical FISH assay performed with 89 (91.75%) of these being detected by clinical FISH, yet spiked target gene expression was observed in all 89 cases by RNA sequencing. Conversely, 93 translocations were called by clinical FISH, of these 89 were called by Seq-FISH(95.7%). Of the 4 translocations only called by clinical FISH, 3 were t(4;14) and 1 was a t(11;14). In two of these t(4;14) cases we did observe spiked target gene expression by RNA sequencing, suggesting these are false negatives by Seq-FISH. However, the remaining two events appear to be false positive clinical FISH results. The t(4;14) event was only observed in 1/200 cells and a co-occuring t(11;14) was also called, which was confirmed by Seq-FISH and spiked gene expression. Similarly, the one t(11;14) was observed in 3/56 cells but a del13q14 was seen in 47/50 cells, unfortunately RNA sequencing data is not available to cross-validate in this case.Plasma cell enrichment or identification is commonly used to prepare myeloma samples for FISH because even in myeloma, the total plasma cell percentage can be low (median 8.3% in the MMRF CoMMpass Baseline Cohort). Therefore, performing FISH on a sample without performing purification or plasma cell identification will indiscriminately assay non-plasma cells and limit the efficacy of the assay. We looked at the two most common translocations in myeloma, t(4;14) and t(11;14), to test the effect of enrichment on sensitivity. Sensitivity was higher for both sets of translocations in the enriched cohort. There was 1 false negative in the enriched population, yielding sensitivities of 100% (32/32) and 95%(19/20) for CCND1 and WHSC1 respectively. For those reports that did not indicate enrichment was performed the observed sensitivities were 86.36% (19/22) and 92.86% (13/14).Seq-FISH identified almost all of the translocations called by clinical FISH and simultaneously; it identified 30 translocations missed by clinical FISH. The translocations that were not reported by clinical FISH can be attributed to a mixture of the correct assay not being performed and the translocation being missed even though the assay was performed. We believe that Seq-FISH is a viable alternative to clinical FISH, with similar specificity and greater sensitivity. It is important to note that a single Seq-FISH assay is sufficient to investigate all translocations, while each translocation must be investigated separately with clinical FISH. As such, Seq-FISH obviates the concern that a translocation would be missed because the correct assay was not performed. DisclosuresMcBride:Instat: Employment.

Are Eosinophils a Specific Marker of Chronic Endometritis?

Abstract Objective: Chronic endometritis can be associated with abnormal uterine bleeding and infertility. The “gold standard” of diagnosing chronic endometritis is the identification of plasma cells, but this can be difficult. Immunohistochemical staining using CD 138 is helpful for identifying plasma cells but adds time and expense to the diagnostic process. It has been suggested that the more easily identified eosinophils comprise a surrogate marker for plasma cells. The current authors hypothesized that eosinophils are not a specific marker of chronic endometritis. Materials and Methods: The pathology database of the University Hospital, Newark NJ, was searched for cases of chronic endometritis from January 1, 1991 to August 1, 2015. Controls were endometrial biopsies that showed proliferative endometrium or disordered proliferative endometrium. Atrophy, pregnancy, secretory endometrium, and malignancy were exclusion criteria. Slides were evaluated to confirm the prior diagnosis and to determine the p...

Abstract A42: Quantification of MYC expression and mTORC signaling as biomarkers of BET inhibition in multiple myeloma.

Abstract Dysregulation of the MYC oncogene has been implicated in the pathogenesis of myeloma. Novel small molecule inhibitors of bromodomain extraterminal (BET) transcriptional readers inhibit MYC expression in pre-clinical settings and are currently being evaluated in phase 1 trials for myeloma. However, biomarkers of response to BET inhibitors have not been developed or validated. Here we report a strategy for flow cytometric measurements of Myc protein and phosphorylation of ribosomal S6 kinase (pS6), a marker of cell growth activation, as potential biomarkers of response to BET inhibition. We evaluated our methodology in the P493 cell line, a model of lymphoplasmacytic malignancies carrying two trans-genes that enable experimental manipulation of MYC expression through regulation of the endogeneous promoter or an ectopic promoter. As predicted, exposure to BET inhibitors: JQ1 and CPI-0610 (both 1µM for 24h) significantly decreased Myc protein levels in P493 cells expressing endogenous MYC activated by the EBNA2-ER fusion protein but had no effect on ectopic MYC under the control of the tetracycline-responsive promoter. Interestingly, in P493 cells expressing endogenous MYC, the decrease in Myc expression highly correlated with a decrease in pS6. We confirmed our results in 4 human myeloma cell lines known to express different levels of MYC and previously shown to have varying responses to BET inhibition (Delamore J.E; Cell 2011). The results from these cell lines represent various states of responses that we expect to see in patient samples and provide a rationale for Myc and pS6 measurements as biomarkers for BET inhibition. Finally, to demonstrate clinical applicability of our assay, we incorporated measurement of MYC and pS6 into a multiparameter flow cytometry panel. This panel allows identification of plasma cells based on CD45, CD38 and CD138 expression, and specific identification of malignant plasma cells based on expression of CD56, CD19, and intracellular immunoglobulin light chain restriction. Using this panel, we successfully identified malignant plasma cells and detected changes in Myc and pS6 in a fresh patient bone marrow aspirate treated ex vivo with CPI-0610. Our experimental results show that Myc protein levels can be reliably quantitated by flow cytometry as a marker of the efficacy of BET inhibitors in suppressing MYC expression and that flow cytometric quantification of pS6 may be a marker of suppression of cell growth signaling due to MYC suppression. Our strategy can be applied to fresh whole bone marrow aspirates with minimal ex vivo processing and can identify changes in MYC and pS6 levels in malignant plasma cells, with resolution at the single cell level. Our data thus provide a solid foundation for reliable and clinically applicable measurements of Myc and pS6 in myeloma cells as readout for BET inhibitor efficacy. Citation Format: Anna Kalota, Alfred Garfall, Martin P. Carroll, Edward A. Stadtmauer, Chi Dang, Dan T. Vogl. Quantification of MYC expression and mTORC signaling as biomarkers of BET inhibition in multiple myeloma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A42.