Noncanonical effector functions of the T-memory-like T-PLL cell are shaped by cooperative TCL1A and TCR signaling.

T-cell prolymphocytic leukemia (T-PLL) is a poor-prognostic neoplasm. Differentiation stage and immune-effector functions of the underlying tumor cell are insufficiently characterized. Constitutive activation of the T-cell leukemia 1A (TCL1A) oncogene distinguishes the (pre)leukemic cell from regular postthymic T cells. We assessed activation-response patterns of the T-PLL lymphocyte and interrogated the modulatory impact by TCL1A. Immunophenotypic and gene expression profiles revealed a unique spectrum of memory-type differentiation of T-PLL with predominant central-memory stages and frequent noncanonical patterns. Virtually all T-PLL expressed a T-cell receptor (TCR) and/or CD28-coreceptor without overrepresentation of specific TCR clonotypes. The highly activated leukemic cells also revealed losses of negative-regulatory TCR coreceptors (eg, CTLA4). TCR stimulation of T-PLL cells evoked higher-than-normal cell-cycle transition and profiles of cytokine release that resembled those of normal memory T cells. More activated phenotypes and higher TCL1A correlated with inferior clinical outcomes. TCL1A was linked to the marked resistance of T-PLL to activation- and FAS-induced cell death. Enforced TCL1A enhanced phospho-activation of TCR kinases, second-messenger generation, and JAK/STAT or NFAT transcriptional responses. This reduced the input thresholds for IL-2 secretion in a sensitizer-like fashion. Mice of TCL1A-initiated protracted T-PLL development resembled such features. When equipped with epitope-defined TCRs or chimeric antigen receptors, these Lckpr-hTCL1Atg T cells gained a leukemogenic growth advantage in scenarios of receptor stimulation. Overall, we propose a model of T-PLL pathogenesis in which TCL1A enhances TCR signals and drives the accumulation of death-resistant memory-type cells that use amplified low-level stimulatory input, and whose loss of negative coregulators additionally maintains their activated state. Treatment rationales are provided by combined interception in TCR and survival signaling.

Capability of CI-Orbitrap for Gas-Phase Analysis in Atmospheric Chemistry: A Comparison with the CI-APi-TOF Technique.

Chemical ionization Orbitrap mass spectrometry (CI-Orbitrap) represents a promising new technique for gas-phase analysis in analytical and atmospheric chemistry mainly due to its very high mass resolving power. In this work, we performed the first side-by-side comparison between a CI-Orbitrap and the widely used atmospheric pressure interface time-of-flight mass spectrometry (CI-APi-TOF) using two different chemical ionization methods, i.e., acetate-ion-based (CH3COO-) and aminium-ion-based (n-C3H7NH3+) schemes. The capability of the CI-Orbitrap at accurately measuring low concentrations of gaseous species formed from the oxidation of α-pinene was explored. Although this study reveals a lack of linearity of the CI-Orbitrap when measuring product ions at very low concentrations (<1 × 106 molecules cm-3), very good agreement between both techniques can be achieved by applying a newly developed linearity correction. It is experimentally shown that the correction function is independent of the reagent ion used. Thus, accurate quantification of organic compounds at concentrations as low as 1 × 105 molecules cm-3 by the CI-Orbitrap can be achieved. Finally, by means of tandem mass spectrometry, the unique capability of the Orbitrap allows the direct determination of the binding energy of cluster ions between analyte and reagent ions, that is needed for the assessment of a chosen ionization scheme.

Library preparation for next generation sequencing: A review of automation strategies.

Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics. Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times. In turn, the complex and cumbersome library preparation, starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters, has been identified as a significant bottleneck. Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time. Considerable emphasis will need to be placed on standardisation to ensure robustness and reproducibility. This review presents an overview of the current state of automation of library preparation for next generation sequencing. Major challenges associated with library preparation are outlined and different automation strategies are classified according to their functional principle. Pipetting workstations allow high-throughput processing yet offer limited flexibility, whereas microfluidic solutions offer great potential due to miniaturisation and decreased investment costs. For the emerging field of single cell transcriptomics for example, microfluidics enable singularisation of tens of thousands of cells in nanolitre droplets and barcoding of the RNA to assign each nucleic acid sequence to its cell of origin. Finally, two applications, the characterisation of bacterial pathogens and the sequencing within human immunogenetics, are outlined and benefits of automation are discussed.

Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

Next-generation sequencing indicates false-positive MRD results and better predicts prognosis after SCT in patients with childhood ALL.

Minimal residual disease (MRD) monitoring via quantitative PCR (qPCR) detection of Ag receptor gene rearrangements has been the most sensitive method for predicting prognosis and making post-transplant treatment decisions for patients with ALL. Despite the broad clinical usefulness and standardization of this method, we and others have repeatedly reported the possibility of false-positive MRD results caused by massive B-lymphocyte regeneration after stem cell transplantation (SCT). Next-generation sequencing (NGS) enables precise and sensitive detection of multiple Ag receptor rearrangements, thus providing a more specific readout compared to qPCR. We investigated two cohorts of children with ALL who underwent SCT (30 patients and 228 samples). The first cohort consisted of 17 patients who remained in long-term CR after SCT despite having low MRD positivity (<0.01%) at least once during post-SCT monitoring using qPCR. Only one of 27 qPCR-positive samples was confirmed to be positive by NGS. Conversely, 10 of 15 samples with low qPCR-detected MRD positivity from 13 patients who subsequently relapsed were also confirmed to be positive by NGS (P=0.002). These data show that NGS has a better specificity in post-SCT ALL management and indicate that treatment interventions aimed at reverting impending relapse should not be based on qPCR only.

GvL effects in T-prolymphocytic leukemia: evidence from MRD kinetics and TCR repertoire analyses.

Allogeneic stem cell transplantation (alloSCT) is used for treating patients with T-prolymphocytic leukemia (T-PLL). However, direct evidence of GvL activity in T-PLL is lacking. We correlated minimal residual disease (MRD) kinetics with immune interventions and T-cell receptor (TCR) repertoire diversity alterations in patients after alloSCT for T-PLL. Longitudinal quantitative MRD monitoring was performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3) Although post-transplant immunomodulation (immunosuppression tapering or donor lymphocyte infusions) resulted in significant reduction (>1 log) of MRD levels in 7 of 10 occasions, durable MRD clearance was observed in only two patients. In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature. Novel clonotypes that could explain a clonal GvL effect did not emerge. In conclusion, TCR-based MRD quantification appears to be a suitable tool for monitoring and guiding treatment interventions in T-PLL. The MRD responses to immune modulation observed here provide first molecular evidence for GvL activity in T-PLL which, however, may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.

Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders.

In this study, we compared immunoglobulin heavy-chain-gene-based minimal residual disease (MRD) detection by real-time quantitative PCR (RQ-PCR) and next-generation sequencing (NGS) to assess whether NGS could overcome some limitations of RQ-PCR and further increase sensitivity, specificity, accuracy and reproducibility. In total, 378 samples from 55 patients with acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) or multiple myeloma (MM) were investigated for clonotype identification, clonotype identity and comparability of MRD results. Forty-five clonotypes were identified by RQ-PCR and 49 by NGS. Clonotypes identified by both tools were identical or >97% homologous in 96% of cases. Both tools were able to routinely reach a sensitivity level of 1 × E-05. A good correlation of MRD results was observed (R=0.791, P<0.001), with excellent concordance in 79.6% of cases. Few discordant cases were observed across all disease subtypes. NGS showed at least the same level of sensitivity as allele-specific oligonucleotides-PCR, without the need for patient-specific reagents. We conclude that NGS is an effective tool for MRD monitoring in ALL, MCL and MM. Prospective comparative analysis of unselected cases is required to validate the clinical impact of NGS-based MRD assessment.

EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.

PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes.

Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2-7 sequential design-evaluation-redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies.

Detection of fusion genes at the protein level in leukemia patients via the flow cytometric immunobead assay.

Nowadays, the presence of specific genetic aberrations is progressively used for classification and treatment stratification, because acute leukemias with the same oncogenetic aberration generally form a clinically and diagnostically homogenous disease entity with comparable prognosis. Many oncogenetic aberrations in acute leukemias result in a fusion gene, which is transcribed into fusion transcripts and translated into fusion proteins, which are assumed to play a critical role in the oncogenetic process. Fusion gene aberrations are detected by karyotyping, FISH, or RT-PCR analysis. However, these molecular genetic techniques are laborious and time consuming, which is in contrast to flow cytometric techniques. Therefore we developed a flow cytometric immunobead assay for detection of fusion proteins in lysates of leukemia cell samples by use of a bead-bound catching antibody against one side of the fusion protein and fluorochrome-conjugated detection antibody. So far, we have been able to design such fusion protein immunobead assays for BCR-ABL, PML-RARA, TEL-AML1, E2A-PBX1, MLL-AF4, AML1-ETO and CBFB-MYH11. The immunobead assay for detection of fusion proteins can be performed within 3 to 4 hours in a routine diagnostic setting, without the need of special equipment other than a flow cytometer. The novel immunobead assay will enable fast and easy classification of acute leukemia patients that express fusion proteins. Such patients can be included at an early stage in the right treatment protocols, much faster than by use of current molecular techniques. The immunobead assay can be run in parallel to routine immunophenotyping and is particularly attractive for clinical settings without direct access to molecular diagnostics.

Standardized MRD quantification in European ALL trials: proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008.

Assessment of minimal residual disease (MRD) has acquired a prominent position in European treatment protocols for patients with acute lymphoblastic leukemia (ALL), on the basis of its high prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Therefore, there is an increasing need for standardization of methodologies and harmonization of terminology. For this purpose, a panel of representatives of all major European study groups on childhood and adult ALL and of international experts on PCR- and flow cytometry-based MRD assessment was built in the context of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. The panel summarized the current state of MRD diagnostics in ALL and developed recommendations on the minimal technical requirements that should be fulfilled before implementation of MRD diagnostics into clinical trials. Finally, a common terminology for a standard description of MRD response and monitoring was established defining the terms 'complete MRD response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD terminology may allow a refined and standardized assessment of response to treatment in adult and childhood ALL, and provides a sound basis for the comparison of MRD results between different treatment protocols.

Standardized MRD flow and ASO IGH RQ-PCR for MRD quantification in CLL patients after rituximab-containing immunochemotherapy: a comparative analysis.

Rituximab-containing regimens are becoming a therapeutic standard in chronic lymphocytic leukemia (CLL), so that a validation of flow cytometric minimal residual disease (MRD) quantification (MRD flow) in the presence of this antibody is necessary. We therefore compared results obtained by real-time quantitative (RQ)-PCR to MRD flow in 530 samples from 69 patients randomized to receive chemotherapy or chemotherapy plus rituximab. Quantitative MRD levels assessed by both techniques were closely correlated irrespective of therapy (r=0.95). The sensitivity and specificity of MRD flow was not influenced by the presence of rituximab. With 58.9% positive and 26.4% negative samples by both techniques, 85.3% of assessments (452/530) were qualitatively concordant between MRD flow and RQ-PCR. Discordant samples were typically negative by MRD flow and simultaneously positive close to the detection limit of the PCR assays, indicating a higher sensitivity of PCR for very low MRD levels. However, 93.8% of all samples were concordantly classified by both methods using a threshold of 10(-4) to determine MRD positivity. MRD flow and PCR are equally effective for MRD quantification in rituximab-treated CLL patients within a sensitivity range of up to 10(-4), whereas PCR is more sensitive for detecting MRD below that level.

Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients.

BCR-ABL fusion proteins show increased signaling through their ABL tyrosine kinase domain, which can be blocked by specific inhibitors, thereby providing effective treatment. This makes detection of BCR-ABL aberrations of utmost importance for diagnosis, classification and treatment of leukemia patients. BCR-ABL aberrations are currently detected by karyotyping, fluorescence in situ hybridization (FISH) or PCR techniques, which are time consuming and require specialized facilities. We developed a simple flow cytometric immunobead assay for detection of BCR-ABL fusion proteins in cell lysates, using a bead-bound anti-BCR catching antibody and a fluorochrome-conjugated anti-ABL detection antibody. We noticed protein stability problems in lysates caused by proteases from mature myeloid cells. This problem could largely be solved by adding protease inhibitors in several steps of the immunobead assay. Testing of 145 patient samples showed fully concordant results between the BCR-ABL immunobead assay and reverse transcriptase PCR of fusion gene transcripts. Dilution experiments with BCR-ABL positive cell lines revealed sensitivities of at least 1%. We conclude that the BCR-ABL immunobead assay detects all types of BCR-ABL proteins in leukemic cells with high specificity and sensitivity. The assay does not need specialized laboratory facilities other than a flow cytometer, provides results within approximately 4 h, and can be run in parallel to routine immunophenotyping.

Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease.

Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.