Outcomes of acute myeloid leukemia patients who responded to venetoclax and azacitidine and stopped treatment.

Venetoclax-azacitidine is the standard of treatment for unfit acute myeloid leukemia patients. In the VIALE-A study, treatment was given until progression but there are no data on its optimal duration for responding patients who do not tolerate indefinite therapy. We retrospectively analyzed the outcome of patients who discontinued venetoclax or venetoclax-azacitidine due to poor tolerance. Sixty-two newly diagnosed (ND) AML patients and 22 patients with morphological relapse or refractory AML were included. In the ND cohort (n = 62), 28 patients stopped venetoclax and azacitidine and 34 patients continued azacitidine monotherapy. With a median follow-up of 23 months (IQR, 20-32), median overall survival and treatment-free survival were 44 (IQR, 16-NR) and 16 (IQR, 8-27) months, respectively. Patients who stopped both treatments and those who continued azacitidine monotherapy had the same outcomes. Negative minimal residual disease was associated with a 2-year treatment-free survival of 80%. In the RR cohort (n = 22), median overall survival and treatment-free survival were 19 (IQR, 17-31) and 10 (IQR, 5-NR) months, respectively. Prior number of venetoclax-azacitidine cycles and IDH mutations were associated with increased overall survival. The only factor significantly impacting treatment-free survival was the number of prior cycles. This study suggests that patients who discontinued treatment in remission have favorable outcomes supporting the rationale for prospective controlled trials.

Standardization of Flow Cytometric Immunophenotyping for Hematological Malignancies: The FranceFlow Group Experience.

Flow cytometry is broadly used for the identification, characterization, and monitoring of hematological malignancies. However, the use of clinical flow cytometry is restricted by its lack of reproducibility across multiple centers. Since 2006, the EuroFlow consortium has been developing a standardized procedure detailing the whole process from instrument settings to data analysis. The FranceFlow group was created in 2010 with the intention to educate participating centers in France about the standardized instrument setting protocol (SOP) developed by the EuroFlow consortium and to organise several rounds of quality controls (QCs) in order to evaluate the feasibility of its application and its results. Here, we report the 5 year experience of the FranceFlow group and the results of the seven QCs of 23 instruments, involving up to 19 centers, in France and in Belgium. The FranceFlow group demonstrates that both the distribution and applicability of the SOP have been successful. Intercenter reproducibility was evaluated using both normal and pathological blood samples. Coefficients of variation (CVs) across the centers were <7% for the percentages of cell subsets and <30% for the median fluorescence intensities (MFIs) of the markers tested. Intracenter reproducibility provided similar results with CVs of <3% for the percentages of the majority of cell subsets, and CVs of <20% for the MFI values for the majority of markers. Altogether, the FranceFlow group show that the 19 participating labs might be considered as one unique laboratory with 23 identical flow cytometers able to reproduce identical results. Therefore, SOP significantly improves reproducibility of clinical flow in hematology and opens new avenues by providing a robust companion diagnostic tool for clinical trials in hematology. © 2019 International Society for Advancement of Cytometry.

Ptk7-Deficient Mice Have Decreased Hematopoietic Stem Cell Pools as a Result of Deregulated Proliferation and Migration.

Hematopoietic stem cells (HSCs) located in adult bone marrow or fetal liver in mammals produce all cells from the blood system. At the top of the hierarchy are long-term HSCs endowed with lifelong self-renewal and differentiation properties. These features are controlled through key microenvironmental cues and regulatory pathways, such as Wnt signaling. We showed previously that PTK7, a tyrosine kinase receptor involved in planar cell polarity, plays a role in epithelial Wnt signaling; however, its function in hematopoiesis has remained unexplored. In this article, we show that PTK7 is expressed by hematopoietic stem and progenitor cells, with the highest level of protein expression found on HSCs. Taking advantage of a Ptk7-deficient mouse strain, we demonstrate that loss of Ptk7 leads to a diminished pool of HSCs but does not affect in vitro or in vivo hematopoietic cell differentiation. This is correlated with increased quiescence and reduced homing abilities of Ptk7-deficient hematopoietic stem and progenitor cells, unraveling novel and unexpected functions for planar cell polarity pathways in HSC fate.

Adhesion receptors involved in HSC and early-B cell interactions with bone marrow microenvironment.

Hematopoiesis takes place in the bone marrow of adult mammals and is the process by which blood cells are replenished every day throughout life. Differentiation of hematopoietic cells occurs in a stepwise manner through intermediates of differentiation that could be phenotypically identified. This has allowed establishing hematopoietic cell classification with hematopoietic stem cells (HSCs) at the top of the hierarchy. HSCs are mostly quiescent and serve as a reservoir for maintenance of lifelong hematopoiesis. Over recent years, it has become increasingly clear that HSC quiescence is not only due to intrinsic properties, but is also mediated by cognate interactions between HSCs and surrounding cells within micro-anatomical sites called "niches". This hematopoietic/stromal crosstalk model also applies to more mature progenitors such as B cell progenitors, which are thought to reside in distinct "niches". This prompted many research teams to search for specific molecular mechanisms supporting leuko-stromal crosstalk in the bone marrow and acting at specific stage of differentiation to regulate hematopoietic homeostasis. Here, we review recent data on adhesion mechanisms involved in HSCs and B cell progenitors interactions with surrounding bone marrow stromal cells.

The PTK7 and ROR2 Protein Receptors Interact in the Vertebrate WNT/Planar Cell Polarity (PCP) Pathway.

The non-canonical WNT/planar cell polarity (WNT/PCP) pathway plays important roles in morphogenetic processes in vertebrates. Among WNT/PCP components, protein tyrosine kinase 7 (PTK7) is a tyrosine kinase receptor with poorly defined functions lacking catalytic activity. Here we show that PTK7 associates with receptor tyrosine kinase-like orphan receptor 2 (ROR2) to form a heterodimeric complex in mammalian cells. We demonstrate that PTK7 and ROR2 physically and functionally interact with the non-canonical WNT5A ligand, leading to JNK activation and cell movements. In the Xenopus embryo, Ptk7 functionally interacts with Ror2 to regulate protocadherin papc expression and morphogenesis. Furthermore, we show that Ptk7 is required for papc activation induced by Wnt5a. Interestingly, we find that Wnt5a stimulates the release of the tagged Ptk7 intracellular domain, which can translocate into the nucleus and activate papc expression. This study reveals novel molecular mechanisms of action of PTK7 in non-canonical WNT/PCP signaling that may promote cell and tissue movements.

Flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression in myelodysplastic neoplasms (MPO-MDS-Valid): protocol for a multicentre diagnostic accuracy study.

INTRODUCTION: Many patients referred for suspicion of myelodysplastic neoplasm (MDS) are subjected to unnecessary discomfort from bone marrow aspiration, due to the low disease prevalence in this population. Flow cytometric analysis of peripheral blood neutrophil myeloperoxidase expression could rule out MDS with sensitivity and negative predictive value estimates close to 100%, ultimately obviating the need for bone marrow aspiration in up to 35% of patients. However, the generalisability of these findings is uncertain due to the limited sample size, the enrolment of patients at a single study site, and the reliability issues associated with laboratory-developed tests and varying levels of operator experience. This study aims to validate the accuracy attributes of peripheral blood neutrophil myeloperoxidase expression quantified by flow cytometric analysis in an independent multicentre sample. METHODS AND ANALYSIS: The MPO-MDS-Valid project is a cross-sectional diagnostic accuracy study comparing an index test to a reference standard. Consecutive adult patients referred for suspicion of MDS are being recruited at seven university hospitals and one cancer centre in France. At each site, flow cytometric analysis of peripheral blood samples is performed by operators who are blinded to the reference diagnosis. A central adjudication committee whose members are unaware of the index test results will determine the reference diagnosis of MDS, based on cytomorphological evaluation of bone marrow performed in duplicate by experienced hematopathologists. The target sample size is 400 patients and the anticipated study recruitment completion date is 31 December 2025. ETHICS AND DISSEMINATION: An institutional review board (Comité de Protection des Personnes Nord-Ouest III, Caen, France) approved the protocol, prior to the start of the study. Participants are recruited using an opt-out approach. Efforts will be made to publish the primary results within 6 months after study completion. TRIAL REGISTRATION NUMBER: NCT05175469.

Protein tyrosine kinase 7 has a conserved role in Wnt/ß-catenin canonical signalling.

The receptor protein tyrosine kinase 7 (PTK7) was recently shown to participate in noncanonical Wnt/planar cell polarity signalling during mouse and frog embryonic development. In this study, we report that PTK7 interacts with ß-catenin in a yeast two-hybrid assay and mammalian cells. PTK7-deficient cells exhibit weakened ß-catenin/T-cell factor transcriptional activity on Wnt3a stimulation. Furthermore, Xenopus PTK7 is required for the formation of Spemann's organizer and for Siamois promoter activation, events that require ß-catenin transcriptional activity. Using epistatic assays, we demonstrate that PTK7 functions upstream from glycogen synthase kinase 3. Taken together, our data reveal a new and conserved role for PTK7 in the Wnt canonical signalling pathway.

The cell polarity PTK7 receptor acts as a modulator of the chemotherapeutic response in acute myeloid leukemia and impairs clinical outcome.

The pseudo tyrosine kinase receptor 7 (PTK7) is an orphan tyrosine kinase receptor assigned to the planar cell polarity pathway. It plays a major role during embryogenesis and epithelial tissue organization. Here we found that PTK7 is also expressed in normal myeloid progenitors and CD34(+) CD38(-) bone marrow cells in humans. We performed an immunophenotyping screen on more than 300 patients treated for hematologic malignancies. We demonstrated that PTK7 is expressed in acute myeloid leukemia (AML) and is mostly assigned to granulocytic lineage differentiation. Patients with PTK7-positive AML are more resistant to anthracycline-based frontline therapy with a significantly reduced leukemia-free survival in a multivariate analysis model. In vitro, expression of PTK7 in cultured leukemia cells promotes cell migration, cell survival, and resistance to anthracycline-induced apoptosis. The intracellular region of PTK7 is required for these effects. Furthermore, we efficiently sensitized primary AML blasts to anthracycline-mediated cell death using a recombinant soluble PTK7-Fc protein. We conclude that PTK7 is a planar cell polarity component expressed in the myeloid progenitor compartment that conveys promigratory and antiapoptotic signals into the cell and that represents an independent prognosis factor of survival in patients treated with induction chemotherapy.

Prognostic value of high-sensitivity measurable residual disease assessment after front-line chemoimmunotherapy in chronic lymphocytic leukemia.

Measurable residual disease (MRD) status is widely adopted in clinical trials in patients with chronic lymphocytic leukemia (CLL). Findings from FILO group trials (CLL2007FMP, CLL2007SA, CLL2010FMP) enabled investigation of the prognostic value of high-sensitivity (0.7 × 10-5) MRD assessment using flow cytometry, in blood (N = 401) and bone marrow (N = 339), after fludarabine, cyclophosphamide, and rituximab (FCR)-based chemoimmunotherapy in a homogeneous population with long follow-up (median 49.5 months). Addition of low-level positive MRD < 0.01% to MRD ≥ 0.01% increased the proportion of cases with positive MRD in blood by 39% and in bone marrow by 27%. Compared to low-level positive MRD < 0.01%, undetectable MRD was associated with significantly longer progression-free survival (PFS) when using blood (72.2 versus 42.7 months; hazard ratio 0.40, p = 0.0003), but not when using bone marrow. Upon further stratification, positive blood MRD at any level, compared to undetectable blood MRD, was associated with shorter PFS irrespective of clinical complete or partial remission, and a lower 5-year PFS rate irrespective of IGHV-mutated or -unmutated status (all p < 0.05). In conclusion, high-sensitivity (0.0007%) MRD assessment in blood yielded additional prognostic information beyond the current standard sensitivity (0.01%). Our approach provides a model for future determination of the optimal MRD investigative strategy for any regimen.

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

Overexpression of the Promigratory and Prometastatic PTK7 Receptor Is Associated with an Adverse Clinical Outcome in Colorectal Cancer.

Biomarkers and novel therapeutic targets are urgently needed in colorectal cancer (CRC). The pseudo tyrosine kinase receptor 7 (PTK7) is involved in planar cell polarity and it is deregulated in various malignancies, including CRC. Yet, little is known about its protein expression in human CRC, or about a possible correlation of its expression with clinical endpoints. Using a clinically annotated Tissue MicroArray (TMA) produced from from 192 consecutive CRC patients treated by initial surgery, we examined PTK7 expression by immunohistochemistry in tumoral tissue and matched normal mucosae, and correlated its expression with clinico-pathological features and patient outcome. PTK7 depletion by specific shRNA in HCT116 and HCT15 CRC cell lines was found to affect cell proliferation, resistance to drugs and cell migration. Tumor growth and metastatic phenotype were investigated in vivo using a xenograft mouse model of CRC cells with modulated expression of PTK7 levels. PTK7 was significantly up-regulated in CRC tissue as compared to matched healthy mucosae, and significant overexpression was found in 34% of patients. PTK7 overexpression was significantly associated with a reduced metastasis-free survival in non-metastatic patients. In HCT116 and HCT15 cells, shRNA PTK7 reduced migration but did not affect cell proliferation and resistance to drugs. In a xenograft mouse of HCT15 cells, downregulation of PTK7 led to reduced tumor growth, whereas its overexpression in PTK7-negative cancer cells led to increased metastatic events. PTK7 expression thus represents a potential prognostic biomarker and a novel therapeutic target in CRC.

Primary cilium migration depends on G-protein signalling control of subapical cytoskeleton.

In ciliated mammalian cells, the precise migration of the primary cilium at the apical surface of the cells, also referred to as translational polarity, defines planar cell polarity (PCP) in very early stages. Recent research has revealed a co-dependence between planar polarization of some cell types and cilium positioning at the surface of cells. This important role of the primary cilium in mammalian cells is in contrast with its absence from Drosophila melanogaster PCP establishment. Here, we show that deletion of GTP-binding protein alpha-i subunit 3 (Gαi3) and mammalian Partner of inscuteable (mPins) disrupts the migration of the kinocilium at the surface of cochlear hair cells and affects hair bundle orientation and shape. Inhibition of G-protein function in vitro leads to kinocilium migration defects, PCP phenotype and abnormal hair bundle morphology. We show that Gαi3/mPins are expressed in an apical and distal asymmetrical domain, which is opposite and complementary to an aPKC/Par-3/Par-6b expression domain, and non-overlapping with the core PCP protein Vangl2. Thus G-protein-dependent signalling controls the migration of the cilium cell autonomously, whereas core PCP signalling controls long-range tissue PCP.

PTK7: a cell polarity receptor with multiple facets.

PTK7 is a tyrosine kinase receptor implicated in planar cell polarity, a process with multiple implications at the cellular and organism levels. Loss of function of PTK7 leads to profound morphogenetic defects in the mouse, such as neural tube defects, misorientation of stereocilia in the inner ear, and impaired polarized cell movements. The planar cell polarity pathway is classically assigned to a non-canonical Wnt pathway, which does not rely on b-catenin transcriptional activity. We recently revealed that PTK7 is implicated in b-catenin-dependent developmental processes in mammalian and Xenopus systems. Based on data recently obtained by our group as well as others, we discuss how PTK7 could be involved in canonical and non-canonical Wnt pathways, and which implications are expected from these data in physiology and physiopathology.