Determination of immunophenotypic aberrancies provides better assessment of peripheral blood involvement by mycosis fungoides/Sézary syndrome than quantification of CD26- or CD7- CD4+ T-cells.

BACKGROUND: Blood involvement by mycosis fungoides (MF)/Sézary syndrome (SS) influences prognosis and therapeutic decisions. MF/SS blood stage is currently determined by absolute CD4 + CD26- or CD4 + CD7-cell counts, which quantification method may overestimate MF/SS by including CD26- or CD7- normal CD4+ T-cells, or underestimate disease burden when MF/SS cells show incomplete loss of CD26 and/or CD7. Recently, through the standardization effort led by the International Clinical Cytometry Society (ICCS), recommendation was made to quantify MF/SS by enumerating immunophenotypically aberrant CD4+ T-cells, rather than CD26- or CD7- in isolation. METHODS: We compared these two quantitation methods in 309 MF/SS patients who had blood samples analyzed by flow cytometry immunophenotyping (FCI) over a 1-year period. RESULTS: Using the European Organization of Research and Treatment of Cancer (EORTC)/International Society for Cutaneous Lymphomas (ISCL) criteria, 221 (71.5%) patients had a blood stage corresponding to B0, 57 (18.4%) to B1, and 31 (10%) to B2. By FCI analysis, a total of 62 patients (20.0%) were found positive for MF/SS. Among EORTC B0 patients, 11/221 (5%) were positive by FCI (false negatives), and among EORTC Stage B1 patients, 35/57 (61%) were negative by FCI (false positives). Regarding patients positive for MF/SS cells by FCI, there was an overall excellent correlation (r = .999, p < .001) between the EORTC/ISCL method and FCI method; however, four (6.5%) patients would have an altered B stage between B0 and B1. CONCLUSION: The MF/SS cell quantification method using immunophenotypic aberrancies, as recommended by the ICCS, allows to distinguish MF/SS cells from background benign T-cells and enables for more accurate staging, especially among patients currently being considered to have B0 and B1 stage diseases.

Ultra-Rapid Reporting of GENomic Targets (URGENTseq): Clinical Next-Generation Sequencing Results within 48 Hours of Sample Collection.

Next-generation sequencing (NGS)-based mutation panels profile multiple genes simultaneously, allowing the reporting of numerous genes while saving labor and resources. However, one drawback of using NGS is that the turnaround time is often longer than conventional single gene tests. This delay can be problematic if molecular results are required to guide therapy in patients with clinically aggressive diseases, such as acute myeloid leukemia. To overcome this limitation, we developed a novel custom platform designated as Ultra-rapid Reporting of GENomic Targets (URGENTseq), an integrated solution that includes workflow optimization and an innovative custom bioinformatics pipeline to provide targeted NGS results on fresh peripheral blood and bone marrow samples within an actionable time period. URGENTseq was validated for clinical use by determining mutant allelic frequency and minimum coverage in silico to achieve 100% concordance for all positive and negative calls between the URGENTseq and conventional sequencing approach. URGENTseq enables the reporting of selected genes useful for immediate diagnosis (CALR, CSF3R, JAK2, KRAS, MPL, NPM1, NRAS, SF3B1) and treatment decisions (IDH1, IDH2) in hematologic malignancies within 48 hours of specimen collection. In addition, we summarize the molecular findings of the first 272 clinical test results performed using the URGENTseq platform.

Bone marrow core biopsy in 508 consecutive patients with chronic myeloid leukemia: Assessment of potential value.

BACKGROUND: The diagnosis of chronic myeloid leukemia (CML) is based on characteristic clinical and laboratory findings and the presence of BCR/ABL1 in the blood and/or bone marrow (BM). The utility of BM core biopsy in the workup of patients with CML has been questioned. METHODS: The potential added value of BM biopsy versus aspiration in the workup of a single-institution series of 508 patients with CML at their initial presentation was systematically assessed. BM biopsy was considered essential when it was needed to establish the disease phase, often because blast counts derived from aspirate smears were misleading because the biopsy specimen was more representative of the disease. BM biopsy was considered helpful if it was needed for other nonessential reasons. RESULTS: In 127 patients (25%), BM biopsy was either essential (109 patients) or helpful (18 patients). Patients with accelerated-phase (AP) or blast-phase (BP) disease often required a biopsy related to essential reasons. High-grade myelofibrosis (MF) was more frequent in patients with AP/BP disease than patients with chronic-phase disease (P = .0005), and the identification of BP disease required a BM biopsy assessment in 75% of the patients (P = .001). A follow-up BM evaluation more often yielded inadequate aspirates in patients with inadequate BM aspirates at the time of their initial diagnosis. CONCLUSIONS: BM core biopsy remains valuable in the workup of 25% of patients with CML because it facilitates identification of the disease phase or MF. The initial grade of MF is associated with the disease stage and outcome after therapy. BM biopsy is, therefore, indicated for patients with CML who have AP/BP disease or other findings suggestive of progressive disease.

Assessment of CD37 B-cell antigen and cell of origin significantly improves risk prediction in diffuse large B-cell lymphoma.

CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its clinical and biologic significance in 773 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and 231 patients treated with CHOP. We found that CD37 loss (CD37-) in ∼60% of DLBCL patients showed significantly decreased survival after R-CHOP treatment, independent of the International Prognostic Index (IPI), germinal center B-cell-like (GCB)/activated B-cell-like (ABC) cell of origin, nodal/extranodal primary origin, and the prognostic factors associated with CD37-, including TP53 mutation, NF-κBhigh, Mychigh, phosphorylated STAT3high, survivinhigh, p63-, and BCL6 translocation. CD37 positivity predicted superior survival, abolishing the prognostic impact of high IPI and above biomarkers in GCB-DLBCL but not in ABC-DLBCL. Combining risk scores for CD37- status and ABC cell of origin with the IPI, defined as molecularly adjusted IPI for R-CHOP (M-IPI-R), or IPI plus immunohistochemistry (IHC; IPI+IHC) for CD37, Myc, and Bcl-2, significantly improved risk prediction over IPI alone. Gene expression profiling suggested that decreased CD20 and increased PD-1 levels in CD37- DLBCL, ICOSLG upregulation in CD37+ GCB-DLBCL, and CD37 functions during R-CHOP treatment underlie the pivotal role of CD37 status in clinical outcomes. In conclusion, CD37 is a critical determinant of R-CHOP outcome in DLBCL especially in GCB-DLBCL, representing its importance for optimal rituximab action and sustained immune responses. The combined molecular and clinical prognostic indices, M-IPI-R and IPI+IHC, have remarkable predictive values in R-CHOP-treated DLBCL.

Myelodysplastic syndromes following therapy with hypomethylating agents (HMAs): development of acute erythroleukemia may not influence assessment of treatment response.

This study followed 28 patients with myelodysplastic syndromes (MDS) who showed a rise of bone marrow (BM) erythroids to ≥ 50% following three cycles (1-60) of hypomethylating agent (HMA) therapy. If BM blasts were calculated as a percentage of non-erythroids, 12 (42.9%) patients met the diagnostic criteria for acute erythroleukemia, erythroid/myeloid (AEL). However, none of the patients showed clonal cytogenetic evolution or new mutations. When compared to 47 de novo AEL patients, these 12 patients were less anemic and thrombocytopenic, had less complex karyotypes (p = 0.044) and showed a longer survival, either calculated from diagnosis (p < 0.001) or from the time of AEL (p = 0.005). These findings illustrate that ≥ 50% erythroids may appear in BM post-HMA therapy, likely a combination of reduction of BM granulocytes (p < 0.001) and promotion of normal or abnormal erythroid proliferation. Enumeration of blasts as a percentage of non-erythroid cells may lead to a diagnosis of AEL and mis-interpretation as disease progression.

Single-institution experience in the treatment of primary mediastinal B cell lymphoma treated with immunochemotherapy in the setting of response assessment by 18fluorodeoxyglucose positron emission tomography.

PURPOSE: Excellent outcomes obtained after infusional dose-adjusted etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone, and rituximab (R-EPOCH) alone have led some to question the role of consolidative radiation therapy (RT) in the treatment of primary mediastinal B cell lymphoma (PMBL). We reviewed the outcomes in patients treated with 1 of 3 rituximab-containing regimens (cyclophosphamide, doxorubicin, vincristine, prednisone [R-CHOP]; hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone [R-HCVAD], or R-EPOCH) with or without RT. We also evaluated the ability of positron emission tomography-computed tomography (PET-CT) to identify patients at risk of relapse. METHODS AND MATERIALS: We retrospectively identified 97 patients with diagnoses of stage I/II PMBCL treated at our institution between 2001 and 2013. The clinical characteristics, treatment outcomes, and toxicity were assessed. We analyzed whether postchemotherapy PET-CT could identify patients at risk for progressive disease according to a 5 point scale (5PS) Deauville score assigned. RESULTS: Among 97 patients (median follow-up time, 57 months), the 5-year overall survival rate was 99%. Of patients treated with R-CHOP, 99% received RT; R-HCVAD, 82%; and R-EPOCH, 36%. Of 68 patients with evaluable end-of-chemotherapy PET-CT scans, 62% had a positive scan (avidity above that of the mediastinal blood pool [Deauville 5PS = 3]), but only 9 patients experienced relapse (n=1) or progressive disease (n=8), all with a 5PS of 4 to 5. Of the 25 patients who received R-EPOCH, 4 experienced progression, all with 5PS of 4 to 5; salvage therapy (RT and autologous stem cell transplantation) was successful in all cases. CONCLUSION: Combined modality immunochemotherapy and RT is well tolerated and effective for treatment of PMBCL. A postchemotherapy 5PS of 4 to 5, rather than 3 to 5, can identify patients at high risk of progression who should be considered for therapy beyond chemotherapy alone after R-EPOCH.

Quantitative assessment of mutant allele burden in solid tumors by semiconductor-based next-generation sequencing.

OBJECTIVES: Identification of tumor-specific somatic mutations has had a significant impact on both disease diagnosis and therapy selection. The ability of next-generation sequencing (NGS) to provide a quantitative assessment of mutant allele burden, in numerous target genes in a single assay, provides a significant advantage over conventional qualitative genotyping platforms. METHODS: We assessed the quantitative capability of NGS and a primer extension-based matrix-assisted laser desorption ionization-time-of-flight (PE-MALDI) assay and directly correlated NGS mutant allele burden determination to morphologic assessment of tumor percentage in H&E-stained slides. RESULTS: Our results show a 100% concordance between NGS and PE-MALDI in mutant allele detection and a significant correlation between NGS and PE-MALDI for determining mutant allele burden when mutant allele burden is 10% or more. CONCLUSIONS: NGS-based mutation screening provides a quantitative assessment comparable to that of PE-MALDI. In addition, NGS also allows for a high degree of multiplexing and uses nanogram quantities of DNA, thereby preserving precious material for future analysis. Furthermore, this study provides evidence that H&E-based morphologic assessment of tumor burden does not correlate to actual tumor mutant allele burden frequency.

Assessment of chronic lymphocytic leukemia and small lymphocytic lymphoma by absolute lymphocyte counts in 2,126 patients: 20 years of experience at the University of Texas M.D. Anderson Cancer Center.

PURPOSE: Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are currently considered the same entity, but controversy remains over whether CLL and SLL should be treated similarly. We assessed whether characteristics of patients with CLL and SLL differ in ways other than the absolute lymphocyte count (ALC) and evaluated treatment outcomes and prognostic factors. METHODS: We searched the electronic database for patients with CLL or SLL who presented to The University of Texas M.D. Anderson Cancer Center (Houston, TX) between 1985 and 2005. We reviewed patient records to determine presenting characteristics, treatment, and clinical outcomes. Cox models using training and validation sets of patients and resampling methods were used to develop a model predicting survival. RESULTS: Among 2,126 consecutive CLL/SLL patients, 312 (15%) had ALC less than 5 x 10(9)/L. Patients with ALC less than 5 x 10(9)/L had lower rates of cytogenetic abnormalities (P = .0002) and higher rates of CD38-positive results (P = .0002) and had mutated immunoglobulin heavy-chain variable region gene status (P = .034). Rates of response, survival, and failure-free survival (FFS) were not different among ALC groups. Regimens that included rituximab and a nucleoside analog were associated with superior rates of response and FFS compared with other therapies, irrespective of ALC. Deletion 17p or 6q with or without other cytogenetic abnormalities, age at least 60 years, beta2-microglobulin at least 2 mg/L, albumin less than 3.5 g/dL, and creatinine at least 1.6 mg/dL were each found to independently predict shorter survival and formed the basis of a scoring system. CONCLUSION: Patients with CLL or SLL can be treated similarly. A new prognostic score is proposed.