Multicenter Prospective Evaluation of Diagnostic Potential of Flow Cytometric Aberrancies in Myelodysplastic Syndromes

Abstract Background: Myelodysplastic syndromes (MDS) are considered clonal diseases and are diagnosed according to WHO by cytomorphology and cytogenetics. The diagnostic potential of flow cytometric aberrancies has not yet been comprehensively evaluated. Aim: Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies predefined according to European LeukemiaNet (ELN). Methods: 1682 patients undergoing diagnostics for suspected MDS according to WHO 2016 criteria were analyzed in parallel by flow cytometry according to ELN recommendations. Results: Median age was 72 years (18-97). MDS, MPN-RS-T or CMML were confirmed by cytomophology in 1029 (61%) cases, 653 (39%) were non-MDS. IPSS-R data was available in 857 (51%). An overall flow cytometric readout was available in 1679 (99.8%). 1001 (60%) were in agreement with MDS while 678 (40%) were not. Flow cytometric readout significantly correlated with cytomorphologic diagnosis (p<0.001): 850 (51%) were positive by both methods (flow+/cyto+), 502 (30%) were flow-/cyto-, 176 (10%) were flow-/cyto+ and 151 (9%) flow+/cyto-. The rate of flow+ was higher in high-risk MDS (MDS-EB1/2, 92%) and CMML (89%) compared to low-risk MDS (76%). Accordingly, regarding IPSS-R highest frequency of flow+ was found in very high risk (96%) and lowest one in very low risk group (64%). Non-MDS cases had a fewer myeloid progenitor cells (MPC) (mean±SD, 0.8±0.9%) compared to low-risk MDS (1.7±2.3%, p<0.001), high-risk MDS (6.3±5.0%, p<0.001) and CMML (1.9±2.6%, p<0.001). In particular, MPC >3% was strongly associated with MDS/CMML (286/293, 98%, p<0.001). Antigen expression aberrancies in MPC were more frequent in cases with MDS or CMML than in non-MDS cases but differed between entities with lower frequencies in low-risk MDS cases (table 1). Neutrophil aberrancies were found more frequently in neoplastic cases than in non-MDS cases (table 1). Again, frequencies of aberrations were higher for high-risk MDS as compared to low-risk MDS while this was not the case for CMML showing frequencies rather similar to low-risk MDS. Frequencies of aberrancies in monocytes revealed a similar figure as in neutrophils with higher rates in neoplastic cases but clearly significant numbers positive in non-MDS cases. Interestingly, frequencies were not higher in high-risk MDS as compared to low-risk MDS. As anticipated, frequencies were highest in CMML (table 1). Regarding erythroid cells only an aberrant percentage of them and aberrant CD71 expression were found in a reasonable number of cases. Importantly, rates of positivity were rather high in non-MDS cases which did not differ from CMML cases (table 1). In order to identify the diagnostic value of each individual aberrancy multivariate analyses were performed in the three subgroups, low-risk MDS, high-risk MDS and CMML, as well as in the total cohort. In low-risk MDS ten aberrancies were independently related to MDS (table 2). Five of these aberrancies were found in MPCs, two each in neutrophils and monocytes and one in erythroid cells. In high-risk MDS 11 aberrancies were independently related to MDS (table 2). Eight were found in MPCs, two in neutrophils, none in monocytes and one in erythroid cells. In CMML 12 aberrancies were independently related to CMML (table 2). Four were found in MPCs, neutrophils and monocytes, respectively, and none in erythroid cells. Considering all these three groups together and all aberrancies identified significantly related to MDS/CMML in at least one group in univariate analysis, multivariate analysis identified 12 aberrancies independently related to MDS/CMML (table 2). Six were found in MPCs, two in neutrophils, three in monocytes and one in erythroid cells. Taking into consideration only aberrancies independently associated with MDS/CMML, three such aberrancies resulted in an 80% agreement with the cytomorphologic diagnosis of MDS/CMML, i.e. 20% concordantly negative and 60% concordantly positive. Importantly, this applies without need of at least two cell compartments being affected as specified in the ELN recommendations. Conclusions: This multicenter prospective evaluation confirms the diagnostic potential of flow cytometric aberrancies. A core set of 17 markers identified as independently related to a diagnosis of MDS/CMML is suggested mandatory for flow cytometric evaluation of suspected MDS. An MPC count >3% should be considered indicative of MDS/CMML. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Van de Loosdrecht: Celgene: Consultancy, Research Funding; Amgen: Consultancy; Roche: Consultancy; Novartis: Consultancy; Alexion: Consultancy.

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Hesk Ratio: A Complementary Tool for the Diagnosis of Myelodysplastic Syndromes and a Novel Method for Flow Cytometry Analysis

Blood ◽

10.1182/blood.v120.21.4946.4946 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4946-4946

Author(s):

Evgenia Verigou ◽

Georgia Kolliopoulou ◽

Nikoleta Smirni ◽

Elisavet Hala ◽

Polixeni Lampropoulou ◽

...

Keyword(s):

Mathematical Model ◽

Bone Marrow ◽

Flow Cytometry ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Myeloid Cells ◽

Antigen Expression ◽

Low Risk ◽

Empirical Parameter ◽

Analytical Approaches

Abstract Abstract 4946 Establishing the diagnosis of Myelodysplastic Syndromes (MDS) is a challenging task for hematologists due to the heterogeneity of this clinical entity. Several attempts have been made to include findings from advanced technologies to the diagnostic criteria of MDS, but still in the majority of cases, morphology of peripheral blood and bone marrow remains the cornerstone for the diagnosis. Flow cytometry(FC) can identify abnormal antigen expression on myeloid cells. FC has been proposed as a complementary method in the diagnosis of low and intermediate risk MDS, particularly for patients not exhibiting characteristic karyotype abnormalities. On the other hand, recent literature suggests that these findings are not MDS-related, questioning the specificity of immunophenotyping for the diagnosis of MDS. The aim of the present study is to maximize the utility of FC data and simplify their interpretation for the diagnosis of MDS, by developing new analytical approaches of digital data, other than the conventional sequential biparametric analysis. The applied methodology was based on a mathematical model of scale analysis. Bone marrow(BM) samples from 50 subjects were analysed for the expression of CD45PC7, CD11bPC5, CD16FITC and CD13PE (antigens by Beckman Coulter, FC500 flow cytometer Beckman Coulter). 36 patients were diagnosed with MDS (23 low risk, 13 high risk) and 14 patients had other than an MDS diagnosis (ITP, chronic idiopathic neutropenia, systemic lupus erythematosus, LGL leukemia, age-related cytopenias, aplastic anemia, myelofibrosis etc). Additionally, 3 BM samples of patients with post-MDS acute myeloid leukemia(AML) were analysed. The data used for the development of the mathematical model were the following: two populations (neutro1, neutron2) were gated according to their CD45 and CD13/CD16 antigen expression (Figure 1i-1v).Seven subpopulations of Neutrophils were defined on CD11b/CD16 density plot N=g+h+i and O=k+j (Figure 1vi). In an attempt to identify correlations between data that cannot be routinely revealed by sequential biparametric analysis, we have developed the HeSK* ratio, which is given by: where x is the median of CD11b in gate O, y is the median of CD16 in gate O, z is the median of CD45 in gate neutro, pO is the percentage of gate O in the total CD11b/CD16 diagram gated in neutro, pN is the percentage of gate N in the total CD11b/CD16 gated in neutro and 1000 is an empirical parameter. The HeSK ratio combines fluorescence levels of CD16, CD11b and CD45 with the percentage of two distinct neutrophil populations (N and O), which differ in their maturation and differentiation stage. The ratio can quantify the abnormal differentiation profile of mature myeloid cells and thus distinguish MDS from non-MDS samples with statistical significance P<0. 0001 (Kruskal Wallis test) as indicated in graph 1. Descriptive statistics are shown in table 1. · HeSK ratio is based upon a novel FC analysis method that could change the conventional biparametric routine FC analysis and quantify patterns that are not evaluated properly. Mathematical modeling of antigen expression patterns optimizes the interpretation of single immunophenotype findings. · The present study proposes HeSK as a complementary diagnostic tool for MDS and a strong indicator for the classification of the patients according to their prognosis as well. *the name HeSK comes from the initials of the 4 main authors (H=Hala, e=Evgenia, S=Smirni, K=Kolliopoulou). Table 1 non MDS low risk MDS high risk MDS Number of values 14 23 13 Minimum 50,76 4,789 0,2850 25% Percentile 304,8 26,11 17,05 Median 2133 92,52 47,64 75% Percentile 10650 228,9 144,3 Maximum 55040 3043 671,7 Mean 10320 316,1 122,7 Std. Deviation 17860 647,9 185,1 Std. Error 4773 135,1 51,33 Figure 1 Figure 1. Disclosures: No relevant conflicts of interest to declare.

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Degree of Aberrant Antigen Expression in Myelodysplastic Syndromes Correlates with the Number of Molecular Mutations

Blood ◽

10.1182/blood.v126.23.2856.2856 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2856-2856

Author(s):

Wolfgang Kern ◽

Manja Meggendorfer ◽

Seishi Ogawa ◽

Claudia Haferlach ◽

Susanne Schnittger ◽

...

Keyword(s):

Flow Cytometry ◽

Molecular Genetics ◽

Myelodysplastic Syndromes ◽

Antigen Expression ◽

Erythroid Cells ◽

Employment Equity ◽

Cell Compartment ◽

Flow Cytometric ◽

Equity Ownership ◽

Myeloid Progenitors

Abstract Introduction: The diagnostic approach for suspected myelodysplastic syndromes (MDS) is evolving and flow cytometry and molecular genetics are increasingly considered to be applied in addition to cytomorphology and cytogenetics. While reports on comparisons of flow cytometric findings with results of cytomorphology and cytogenetics are available, data on comparisons between results obtained by flow cytometry and molecular genetics, however, have not yet been presented in detail. Aims: 1) To assess the correlation between flow cytometric findings on MDS-specific aberrant antigen expression and the presence of molecular mutations in patients with cytomorphologically proven MDS. 2) To determine the respective impact of flow cytometric findings and of molecular mutations on survival in patients with MDS. Patients and methods: In 256 patients (male/female, 161/95; median age 72 years, range 24-90) with proven MDS (137 low-risk MDS, 119 RAEB1/2) we compared data on aberrantly expressed antigens (AEA) determined according to ELN guidelines (Westers, Leukemia 2012) to the previously published mutational status of 104 genes (Haferlach, Leukemia 2014). Results: Median numbers (ranges) of AEA were 0 (0-3) in myeloid progenitors, 2 (0-4) in granulocytes, 1 (0-5) in monocytes and 0 (0-1) in erythroid cells. Median number of mutation was 2 (0-7). The number of AEA in myeloid progenitors, granulocytes and monocytes increased with increasing number of mutations (r=0.257, p<0.001). Accordingly, in cases with ≥3 mutations the number of AEA in myeloid progenitors, granulocytes and monocytes was higher than in cases with ≤2 mutations (mean±SD, 3.9±1.9 vs. 3.0±2.0, p=0.001). This correlation was significant also when considering granulocytes as a single cell compartment (r=0.308, p<0.001) but non-significant trends only for myeloid progenitors and monocytes. No such correlation was observed for erythroid cells. Specifically, mutations in each of the genes TET2, ASXL1, SRSF2, STAG2, ZRSR2 or NF1 were associated with significantly higher numbers of AEA in ≥1 cell compartment. Cases with mutations in ≥1 of these genes (n=145), as compared to those without these 6 mutations (n=111), had higher numbers of AEA in myeloid progenitors (0.4±0.7 vs. 0.2±0.5, p=0.037), granulocytes (2.0±1.1 vs. 1.4±1.1, p<0.001) and monocytes (1.5±1.3 vs. 1.0±1.0, p=0.002). Consequently, the difference in the total of AEA was even larger (3.9±2.0 vs. 2.7±1.9, p<0.001). Regarding scoring points according to IPSS-R, there was a significant correlation with the number of AEA in granulocytes (r=0.189, p=0.004) as well as with the number of AEA in monocytes (r=0.159, p=0.017). Consequently, there was also a significant correlation between the IPSS-R scoring points and the number of AEA in myeloid progenitors, granulocytes and monocytes (r=0.227, p=0.001). Overall survival was impacted by the presence of mutations in ≥1 of the genes TP53, EZH2, ETV6, RUNX1 and ASXL1 (p<0.001, HR 2.9) published by Bejar (NEJM 2011) as well as by the presence of ≥3 AEA in myeloid progenitors, granulocytes and monocytes (p=0.015, HR 1.7) and by IPSS-R (p<0.001, HR 1.4). Multivariate analysis considering mutations and AEA revealed an independent significance for both of them (mutations, p<0.001, HR 2.9; AEA, p=0.017, HR 1.7). However, inclusion of also IPSS-R as a covariate resulted in a trend only for AEA (p=0.16, HR 1.4) and independent significance for mutations (p<0.001, HR 2.3) and IPSS-R (p<0.001, HR 1.3). Conclusions: This data demonstrates that the degree of flow cytometric findings on MDS-related aberrant antigen expression correlates with the number of molecular mutations as well as with the IPSS-R. The present result therefore further support the consideration of both flow cytometry and molecular genetics for the diagnostic work-up of MDS in an integrated approach in combination with cytomorphology and cytogenetics. Disclosures Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Comparison of the Prognoses of FIGO Stage I to Stage II Adenosquamous Carcinoma and Adenocarcinoma of the Uterine Cervix Treated With Radical Hysterectomy

International Journal of Gynecological Cancer ◽

10.1097/igc.0b013e31826b5d9b ◽

2012 ◽

Vol 22 (8) ◽

pp. 1389-1397 ◽

Cited By ~ 14

Author(s):

Seiji Mabuchi ◽

Mika Okazawa ◽

Yasuto Kinose ◽

Koji Matsuo ◽

Masateru Fujiwara ◽

...

Keyword(s):

Risk Factors ◽

Cervical Cancer ◽

High Risk ◽

Uterine Cervix ◽

Radical Hysterectomy ◽

Adenosquamous Carcinoma ◽

Early Stage ◽

Univariate Analysis ◽

Low Risk ◽

Histological Subtype

ObjectivesTo evaluate the significance of adenosquamous carcinoma (ASC) compared with adenocarcinoma (AC) in the survival of surgically treated early-stage cervical cancer.MethodsWe retrospectively reviewed the medical records of 163 patients with International Federation of Gynecology and Obstetrics stage IA2 to stage IIB cervical cancer who had been treated with radical hysterectomy with or without adjuvant radiotherapy between January 1998 and December 2008. The patients were classified according to the following: (1) histological subtype (ASC group or AC group) and (2) pathological risk factors (low-risk or intermediate/high-risk group). Survival was evaluated using the Kaplan-Meier method and compared using the log-rank test. Multivariate analysis of progression-free survival (PFS) was performed using the Cox proportional hazards regression model to investigate the prognostic significance of histological subtype.ResultsClinicopathological characteristics were similar between the ASC and AC histology groups. Patients with the ASC histology displayed a PFS rate similar to that of the patients with the AC histology in both the low-risk and intermediate/high-risk groups. Neither the recurrence rate nor the pattern of recurrence differed between the ASC group and the AC group. Univariate analysis revealed that patients with pelvic lymph node metastasis and parametrial invasion achieved significantly shorter PFS than those without these risk factors.ConclusionsCharacteristics of the patients and the tumors as well as survival outcomes of ASC were comparable to adenocarcinoma of early-stage uterine cervix treated with radical hysterectomy. Our results in part support that the management of ASC could be the same as the one of AC of the uterine cervix.

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Risk category system to identify pituitary adenoma patients with AIP mutations

Journal of Medical Genetics ◽

10.1136/jmedgenet-2017-104957 ◽

2018 ◽

Vol 55 (4) ◽

pp. 254-260 ◽

Cited By ~ 16

Author(s):

Francisca Caimari ◽

Laura Cristina Hernández-Ramírez ◽

Mary N Dang ◽

Plamena Gabrovska ◽

Donato Iacovazzo ◽

...

Keyword(s):

Pituitary Adenoma ◽

Family History ◽

High Risk ◽

Pituitary Adenomas ◽

Univariate Analysis ◽

Low Risk ◽

Individual Risk ◽

Risk Category ◽

Moderate Risk ◽

Category System

BackgroundPredictive tools to identify patients at risk for gene mutations related to pituitary adenomas are very helpful in clinical practice. We therefore aimed to develop and validate a reliable risk category system for aryl hydrocarbon receptor-interacting protein (AIP) mutations in patients with pituitary adenomas.MethodsAn international cohort of 2227 subjects were consecutively recruited between 2007 and 2016, including patients with pituitary adenomas (familial and sporadic) and their relatives. All probands (n=1429) were screened for AIP mutations, and those diagnosed with a pituitary adenoma prospectively, as part of their clinical screening (n=24), were excluded from the analysis. Univariate analysis was performed comparing patients with and without AIP mutations. Based on a multivariate logistic regression model, six potential factors were identified for the development of a risk category system, classifying the individual risk into low-risk, moderate-risk and high-risk categories. An internal cross-validation test was used to validate the system.Results1405 patients had a pituitary tumour, of which 43% had a positive family history, 55.5% had somatotrophinomas and 81.5% presented with macroadenoma. Overall, 134 patients had an AIP mutation (9.5%). We identified four independent predictors for the presence of an AIP mutation: age of onset providing an odds ratio (OR) of 14.34 for age 0-18 years, family history (OR 10.85), growth hormone excess (OR 9.74) and large tumour size (OR 4.49). In our cohort, 71% of patients were identified as low risk (<5% risk of AIP mutation), 9.2% as moderate risk and 20% as high risk (≥20% risk). Excellent discrimination (c-statistic=0.87) and internal validation were achieved.ConclusionWe propose a user-friendly risk categorisation system that can reliably group patients into high-risk, moderate-risk and low-risk groups for the presence of AIP mutations, thus providing guidance in identifying patients at high risk of carrying an AIP mutation. This risk score is based on a cohort with high prevalence of AIP mutations and should be applied cautiously in other populations.

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Risk Stratification for Survival and Leukemic Transformation in Essential Thrombocythemia: A Single Institutional Study of 605 Patients.

Blood ◽

10.1182/blood.v108.11.3599.3599 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3599-3599

Author(s):

Naseema Gangat ◽

Alexandra Wolanskyj ◽

Rebecca F. McClure ◽

Chin Y. Li ◽

Susan M. Schwager ◽

...

Keyword(s):

Risk Factors ◽

High Risk ◽

Univariate Analysis ◽

Low Risk ◽

Prognostic Models ◽

Intermediate Risk ◽

Prognostic Variables ◽

Leukemic Transformation ◽

Male Sex

Abstract Background It is widely recognized that advanced age and prior thrombosis predict recurrent thrombosis in essential thrombocythemia (ET) and are used to risk-stratify patients. However, the paucity of large sample size and long-term follow-up has limited the development of similar prognostic models for survival and leukemic transformation (LT). Methods Data was abstracted from the medical records of a consecutive cohort of patients with WHO-defined ET seen at the Mayo Clinic. Cox proportional hazards was used to determine the impact of clinical and laboratory variables on survival and LT. Overall survival and leukemia-free survival was estimated by Kaplan-Meier plots. Results i. Patient characteristics and outcome The study cohort included 605 patients of which 399 (66%) were females (median age, 57 years; range 5–91). Median follow-up was 84 months (range; 0–424). During this period, 155 patients (26%) have died and LT was documented in 20 patients (3.3%) occurring at a median of 138 months (range; 23–422) from ET diagnosis. ii. Prognostic variables for overall survival Univariate analysis of parameters at diagnosis identified age ≥ 60 years, hemoglobin less than normal (defined as < 12 g/dL in females and < 13.5 g/dL in males), leukocyte count ≥ 15 x 109/L, tobacco use, diabetes mellitus, thrombosis, male sex, and the absence of microvascular symptoms as independent predictors of inferior survival. All of the above except the last two (i.e. male sex and the absence of microvascular symptoms) sustained their prognostic significance on multivariate analysis. Based on the first three prognostic variables: age, hemoglobin level, and leukocyte count, we constructed a prognostic model for survival: low-risk (none of the risk factors), intermediate-risk (1of 3 risk factors), and high-risk (≥ 2 risk factors). The respective median survivals were 278, 200, and 111 months (p<0.0001; Figure 1) iii. Prognostic variables for leukemic transformation On univariate analysis of parameters at ET diagnosis, LT was significantly associated with platelet count ≥ 1000 x 109/L, hemoglobin less than normal, and exposure to P-32. However, on multivariate analysis, only hemoglobin less than normal and platelet count ≥ 1000 x 109/L maintained independent prognostic value. Accordingly, we utilized these two variables, to construct a prognostic model for LT: low-risk (none of the risk factors), intermediate-risk (1 risk factor), and high-risk (both risk factors). Only 1 of the 239 patients (0.4%) in the low-risk group vs. 14 of the 289 (4.8%) in the intermediate-risk and 5 of the 77 (6.5%) in the high-risk group underwent LT (p=0.0009; Figure 2). Conclusion The current study provides clinician-friendly prognostic models for both survival and LT in ET. Figure 1 Figure 1. Figure 2 Figure 2.

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Prognostic Indices in Older DLBCL Patients Receiving R-CHOP: An Analysis of the U.S. Intergroup Study (E4494, CALGB 9793).

Blood ◽

10.1182/blood.v108.11.813.813 ◽

2006 ◽

Vol 108 (11) ◽

pp. 813-813

Author(s):

R.H. Advani ◽

H. Chen ◽

T.M. Habermann ◽

V.A. Morrison ◽

E. Weller ◽

...

Keyword(s):

Overall Survival ◽

High Risk ◽

Univariate Analysis ◽

Risk Groups ◽

Low Risk ◽

Risk Category ◽

Free Survival ◽

Prognostic Tool ◽

The Impact ◽

The U.S

Abstract Background: We reported that addition of rituximab (R) to chemotherapy significantly improves outcome in DLBCL patients (pt) >60 years (JCO24:3121–27, 2006). Although the IPI is a robust clinical prognostic tool in DLBCL, Sehn et al (ASH 2005: abstract 492) reported that a revised (R) IPI more accurately predicted outcome in pt treated with rituximab-chemotherapy. Methods: We evaluated outcomes of the Intergroup study with respect to the standard IPI, R-IPI, age-adjusted (aa) IPI for evaluable pt treated with R-CHOP alone or with maintenance rituximab. We further assessed a modified IPI (mIPI) using age ≥ 70 y as a cutoff rather than age 60 y. Results: The 267 pt in this analysis were followed for a median of 4 y. Pt characteristics were: age > 70 (48%) (median=69), male 52%, stage III/IV 75%, >1 EN site 30%, LDH elevated 60%, PS ≥2 15%. On univariate analysis all of these characteristics were significant for 3 y failure-free survival (FFS) and overall survival (OS). The IPI provided additional discrimination of risk compared to the R-IPI with significant differences in FFS and OS for 3 vs 4–5 factors. The aa-IPI defined relatively few pt as low or high risk. The impact of age was studied using a cut-off of 70 years in a modified IPI, yielding 4 risk groups as shown below. Conclusions: For pt ≥ 60 treated with rituximab-chemotherapy the distinction between 3 vs 4,5 factors in the IPI was significant.The IPI also provided additional discrimination of risk compared to the R-IPI. In this older group of pt, use of an age cutoff ≥70 y placed more patients in the low risk category. It is of interest to apply the mIPI in other datasets with DLBCL pt >60 y. Group # Factors # Pt % 3y FFS* % 3y OS* *All risk groups significantly different; logrank p < 0.001 **95 % CI: FFS (0.46,0.66), OS (0.58,0.78) ***95 % CI: FFS (0.21,0.45), OS (0.31,0.55) L: Low, LI: Low Intermediate, HI: High Intermediate, H; High IPI L 0–1 12 78 83 LI 2 28 70 80 HI 3 33 56** 68** H 4–5 37 33*** 43*** R-IPI Very Good 0 0 - - Good 1–2 40 72 81 Poor 3–5 60 46 57 aa-IPI L 0 12 78 83 LI 1 35 68 78 HI 2 44 47 59 H 3 9 31 35 mIPI (age ≥ 70) L 0–1 27 77 86 LI 2 28 62 74 HI 3 29 47 58 H 4–5 16 28 36

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An Automated and Quantitative Protein Expression-Based Classification System to Identify High Risk Multiple Myeloma Patients

Blood ◽

10.1182/blood.v112.11.735.735 ◽

2008 ◽

Vol 112 (11) ◽

pp. 735-735

Author(s):

Alex Klimowicz ◽

Paola Neri ◽

Adnan Mansoor ◽

Anthony Magliocco ◽

Douglas A. Stewart ◽

...

Keyword(s):

Multivariate Analysis ◽

High Risk ◽

Protein Expression ◽

Classification System ◽

Risk Group ◽

Univariate Analysis ◽

High Risk Group ◽

Group Classification ◽

Low Risk ◽

High Expression

Abstract Background: Autologous stem cell transplantation (ASCT) has dramatically improved the survival of myeloma patients; however, this approach has significant toxicities and nearly 25% of MM patients progress within one year from their transplant. While gene expression profiling-based (GEP) molecular classification has permitted the identification of unresponsive high-risk patients, these approaches have proven too costly and complex to translate into clinical practice. Less expensive and more readily available methods are needed clinically to identify, at the time of diagnosis, MM patients who may benefit from more aggressive or experimental therapies. While protein-based tissue arrays offer such alternative, biases introduced by the “observer-dependent” scoring methods have limited their wide applicability. Methods: We have designed a simplified, fully automated and quantitative protein expression based-classification system that will allow us to accurately predict survival post ASCT in a cost effective and “observer-independent” manner. We constructed tissue microarrays using diagnostic bone marrow biopsies of 82 newly diagnosed MM patients uniformly treated with a dexamethasone based induction regimen and frontline ASCT. Using the HistoRx PM-2000 quantitative immunohistochemistry platform, coupled with the AQUA analysis software, we have examined the expression of the following proteins: FGFR3 which is associated with t(4;14), cyclin B2 and Ki-67 which are associated with cellular proliferation, TACI which is associated with maf deregulation, and phospho-Y705 STAT3 and p65NF-κB, which are associated with myeloma cell growth and survival. For FGFR3, patients were divided into FGFR3 positive and negative groups based on hierarchical clustering of their AQUA score. For all other proteins examined, based on AQUA scores, the top quartiles or quintiles of patients were classified as high expression groups. Based on the univariate analysis, patients were further classified as “High Risk” MM if they had been identified as high expressers of either TACI, p65NF-κB or FGFR3. The Kaplan-Meier method was used to estimate time to progression and overall survival. Multivariate analysis was performed using the Cox regression method. Results: 82 patients were included in this study. In univariate analysis, FGFR3 and p65NF-κB expression were associated with significantly shorter TTP (p=0.018 and p=0.009) but not OS (p=0.365 and p=0.104). TACI expression levels predicted for worse OS (p=0.039) but not TTP (p=0.384). High expression of Ki67 or phospho-Y705 STAT3 did not affect survival. Of the 82 cases, 67 were included in the multivariate analysis since they had AQUA scores available for all markers: 26 (38.8%) were considered as High Risk by their AQUA scores and had significantly shorter TTP (p=0.014) and OS (p=0.006) compared to the Low Risk group. The median TTP for the Low and High Risk groups was 2.9 years and 1.9 years, respectively. The 5-years estimates for OS were 60.6% for the High Risk group versus 83.5% for the Low Risk group. Multivariate analysis was performed using del13q and our risk group classification as variables. Both our risk group classification and del13q were independent predictors for TTP, having 2.4 and 2.3 greater risk of relapse, respectively. Our risk group classification was the only independent predictor of OS with the High Risk group having a 5.9 fold greater risk of death. Conclusions: We have found that the expression of FGFR3, TACI, and p65NF-κB, in an automated and fully quantitative tissue-based array, is a powerful predictor of survival post-ASCT in MM and eliminates the “observer-dependent” bias of scoring TMAs. A validation of this “High Risk” TMA based signature is currently underway in larger and independent cohorts. Figure Figure

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Platelet Functions and Risk Prognosis in Myelodysplastic Syndromes,

Blood ◽

10.1182/blood.v118.21.3806.3806 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3806-3806

Author(s):

Nora V. Butta ◽

Mónica Martín Salces ◽

Raquel de Paz ◽

Elena G. Arias Salgado ◽

Ihosvany Fernández Bello ◽

...

Keyword(s):

Flow Cytometry ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Surface Expression ◽

Low Risk ◽

Control Group ◽

Platelet Surface ◽

High Risk Patients ◽

Fibrinogen Receptor ◽

Risk Patients

Abstract Abstract 3806 The myelodysplastic syndromes (MDS) are a heterogenous group of clonal stem cell disorders with peripheral cytopenias and increased incidence of leukemic transformation. The prognosis of MDS is determined by several factors, including the presence of specific cytogenetic abnormalities, the percentage of blastoid cells in bone marrow and peripheral blood, the number of affected cell lineages, and transfusion dependency. The most commonly used risk stratification system is the International Prognostic Scoring System (IPSS). This score divides patients into a lower risk subset (low and intermediate-1) and a higher risk subset (intermediate-2 and high). Patients with MDS may have hemorrhagic complications with serious outcomes that are among the major causes of death in this population. These bleeding episodes that are often related to thrombocytopenia also occur in MDS patients with normal platelet count. The aim of this work was to study functional characteristics of platelets in MDS patients and their relationship to risk evaluated as indicated by IPSS. Eighty diagnosed MDS patients risk-stratified according to IPSS were included: 40 with low-risk, 29 with intermediate-1-risk (I-1), 8 with intermediate-2-risk (I-2) and 3 with high-risk. Eighty healthy donors were included as control group. Platelet-related primary haemostasis was evaluated with an automated platelet function analyzer (PFA-100®, Siemens Healthcare Diagnostics). Samples of citrated blood were aspirated under a shear rate of 4,000–5,000/s through a 150-μm aperture cut into a collagen-ADP (COL-ADP) or collagen-epinephrine (COL-EPI) coated membrane. The platelet haemostatic capacity is indicated by the time required for the platelet plug to occlude the aperture (closure time, CT), which is expressed in seconds. Platelet activation was determined through FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) and FITC-P-selectin mAb binding to quiescent and 100 μM TRAP activated platelets by flow cytometry. Surface expression of fibrinogen receptor (αIIb and β3 subunits) was determined by flow cytometry with specific mAbs. Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to platelet membrane phosphatidylserine (PS) exposed in basal conditions. I-2 and high-risk patients were gathered together in a high-risk group in order to analyze experimental results. Statistical analysis was performed with one-way ANOVA and Tukey test. CTs obtained with COL-EPI and COL-ADP cartridges in controls and low risk patients were similar and significantly shorter than CTs observed in I-1-risk and high-risk MDS patients (p<0.05). Platelets from all MDS patients showed a reduced capability for being activated by 100 μM TRAP. This impairment was more evident in I-1-risk and high-risk patients: PAC-1 binding, in arbitrary units (AU), was 11368±1017 in controls; 7849±789 in low-risk MDS (p<0.05); 4161±591 in I-1-risk MDS (p<0.01 versus control and p<0.05 versus low-risk) and 492±184 in high-risk MDS (p<0.01 versus control and p<0.05 versus low-risk). The platelet surface expression of P-selectin induced by 100 μM TRAP was also reduced: 5102±340 AU in controls, 3318±400 AU in low-risk MDS (p<0.05); 1880 ±197 AU in I-1-risk MDS (p<0.05 versus control and versus low-risk), and 1211±130 AU in high-risk MDS (p<0.05 versus control and versus low-risk). Diminished responses to TRAP were not due to a reduction in surface expression of fibrinogen receptor in platelets from MDS patients. Platelets from MDS patients expressed more PS than controls under basal conditions. Mean fluorescence values for FITC-annexin binding were: 383±16 in controls; 444±21 in low-risk (p<0.05); 575±52 in I-1-risk MDS (p<0.05 versus control and versus low-risk); 611±17 in high-risk MDS (p<0.05 versus control and versus low-risk). Our results indicated that platelets from MDS patients had less ability to be activated and were more apoptotic than control ones. These dysfunctions were more pronounced when the risk of the disease was higher according to IPSS. Disclosures: No relevant conflicts of interest to declare.

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Missing the mark? Prostate cancer upgrading by systematic biopsy over fusion biopsy.

Journal of Clinical Oncology ◽

10.1200/jco.2016.34.2_suppl.62 ◽

2016 ◽

Vol 34 (2_suppl) ◽

pp. 62-62

Author(s):

Akhil Muthigi ◽

Arvin Koruthu George ◽

Amogh Iyer ◽

Michael Kongnyuy ◽

Meet Kadakia ◽

...

Keyword(s):

Prostate Cancer ◽

High Risk ◽

Target Location ◽

Univariate Analysis ◽

High Volume ◽

Low Risk ◽

Small Subset ◽

Fusion Biopsy ◽

Low Volume ◽

Systematic Biopsy

62 Background: Multiparametric MRI (mpMRI) and fusion biopsy (FBx) detect more high risk prostate cancer (CaP) and less low risk CaP than standard systematic biopsy (SBx). However, there remains a small subset of patients where SBx captures higher grade disease than FBx. We aim to identify potential reasons for failure of FBx biopsy in detection of clinically significant (CS) CaP. Methods: A review was performed of a prospectively maintained database of patients undergoing mpMRI followed by FBx and SBx in the same session from 2007−2014. Patients upgraded to higher risk disease based on SBx results relative to FBx were identified. Independent re−review of MR imaging in this subset was conducted to identify potential proximity between MR targets and SBx region which revealed higher risk CaP. Univariate analysis was performed to determine association of patient, MRI, and pathologic characteristics with upgrading by SBx. Results: We identified 1003 total patients who underwent mpMRI and biopsy, of which 564 were found to have CaP (56.2%). Upgrading based on SBx occurred in 137/564 (24.3%) patients, of which only 55 (9.8%) were to intermediate (high volume 3+4) [N = 37, 6.5%] or high risk CaP ( ≥ 4+3) [N = 18, 3.2%]. 41 of 55 patients (75%) had a lesion identified by mpMRI with FBx in the same sextant in which SBx biopsy revealed intermediate or high risk CaP. On univariate analysis, higher prostate volume (48cc vs 42cc, p < 0.001) and lower percent core involvement (20% vs. 58%, p < 0.001) were associated with upgrading by SBx. Conclusions: MRI rarely misses CS CaP and FBx, if accurate, should reflect the true disease state. Most gleason upgrades by SBx were to low risk, low volume CaP. In patients upgraded by SBx to CS CaP, mpMRI identified a targetable lesion in proximity to the SBx sextant in a majority of patients. FBx failure may be related to suboptimal imaging or biopsy related inaccuracy including registration error, miscalculation of target location, or inadequate lesion sampling. Other possibilities include presence of tumor heterogeneity, multi−focality within the same sextant, or low volume disease. Future studies with biopsy mapping on MRI will provide insight into mechanisms of failure in patients with overlapping target and sextant sampling.

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Incidence of venous thromboembolic disease (VTE) in a cohort of patients with colorectal cancer (CRC) according to KRAS, NRAS and BRAF status.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e15136 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e15136-e15136

Author(s):

Pilar Garcia-Alfonso ◽

Laura Ortega Morán ◽

Iria Gallego Gallego ◽

Gonzalo García González ◽

Gabriela Torres Pérez-Solero ◽

...

Keyword(s):

High Risk ◽

Predictive Model ◽

Metastatic Disease ◽

Statistical Significance ◽

Univariate Analysis ◽

Low Risk ◽

Intermediate Risk ◽

Vein Thrombosis ◽

Increased Risk ◽

Cancer Associated Thrombosis

e15136 Background: A recent study has suggested that KRAS mutation could increase the risk of VTE in patients with CRC. The role of others biomarkers, such as BRAF, in this setting is unknown. The aim of this study is to analyze the incidence of cancer-associated thrombosis in a cohort of patients with CRC based on KRAS, NRAS and BRAF status. Methods: We performed a retrospective review of patients with metastatic CRC and KRAS/NRAS/BRAF status known, attended in the Medical Oncology Department of the Hospital General Universitario Gregorio Marañón (Madrid, Spain) between January 2010 and January 2018. Results: 194 patients were identified and included in the analysis. The median age was 64 years (18-86). Most were metastatic at diagnosis (58.1%). Khorana’s predictive model: low-risk 67.7%, intermediate-risk 31.0%, high-risk 2.3%. The median follow-up was 35 months (2-240). 41 patients (21.1%) experienced VTE (11 pulmonary embolism, 15 lower extremity deep-vein thrombosis, 12 visceral vein thrombosis, 2 catheter-related thrombosis, 1 unknown). Most had metastatic disease at the moment of VTE (90.2%). 40% of the events occurred at the time of diagnosis or within the first 6 months. 65% were incidental events. Khorana’s predictive model in VTE patients: low-risk 63.4%, intermediate-risk 24.5%, high-risk 7.3%. According to biomarkers, the incidence was 19.1% (13/68) in KRAS/NRAS mutated patients, 28.6% (6/21) in BRAF mutated patients and 21% (22/105) in triple-wild-type patients. 6/38 patients (15.8%) developed recurrent thrombosis. In the univariate analysis, the presence of chronic kidney disease (p = 0.022), ECOG ≥ 2 (p = 0.038) and high-risk Khorana score (p = 0.011) were significantly associated with increased risk of VTE. Metastatic disease showed a trend towards the statistical significance (p = 0.053). In the multivariate model, including this variables, age, sex and biomarkers, only ECOG ≥ 2 remained independent predictor of VTE (OR 8.73; CI 95% 1.32-57.82; p = 0.025). Conclusions: The biomarkers have not been associated with the risk of VTE. We have observed a high incidence of VTE in BRAF mutated patients that should be investigated in further studies.

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