The shape of cancer relapse: Topological data analysis predicts recurrence in paediatric acute lymphoblastic leukaemia.

Although children and adolescents with acute lymphoblastic leukaemia (ALL) have high survival rates, approximately 15-20% of patients relapse. Risk of relapse is routinely estimated at diagnosis by biological factors, including flow cytometry data. This high-dimensional data is typically manually assessed by projecting it onto a subset of biomarkers. Cell density and "empty spaces" in 2D projections of the data, i.e. regions devoid of cells, are then used for qualitative assessment. Here, we use topological data analysis (TDA), which quantifies shapes, including empty spaces, in data, to analyse pre-treatment ALL datasets with known patient outcomes. We combine these fully unsupervised analyses with Machine Learning (ML) to identify significant shape characteristics and demonstrate that they accurately predict risk of relapse, particularly for patients previously classified as 'low risk'. We independently confirm the predictive power of CD10, CD20, CD38, and CD45 as biomarkers for ALL diagnosis. Based on our analyses, we propose three increasingly detailed prognostic pipelines for analysing flow cytometry data from ALL patients depending on technical and technological availability: 1. Visual inspection of specific biological features in biparametric projections of the data; 2. Computation of quantitative topological descriptors of such projections; 3. A combined analysis, using TDA and ML, in the four-parameter space defined by CD10, CD20, CD38 and CD45. Our analyses readily extend to other haematological malignancies.

High BMP4 expression in low/intermediate risk BCP-ALL identifies children with poor outcomes.

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) outcome has improved in the last decades, but leukemic relapses are still one of the main problems of this disease. Bone morphogenetic protein 4 (BMP4) was investigated as a new candidate biomarker with potential prognostic relevance, and its pathogenic role was assessed in the development of disease. A retrospective study was performed with 115 pediatric patients with BCP-ALL, and BMP4 expression was analyzed by quantitative reverse transcription polymerase chain reaction in leukemic blasts at the time of diagnosis. BMP4 mRNA expression levels in the third (upper) quartile were associated with a higher cumulative incidence of relapse as well as a worse 5-year event-free survival and central nervous system (CNS) involvement. Importantly, this association was also evident among children classified as having a nonhigh risk of relapse. A validation cohort of 236 patients with BCP-ALL supported these data. Furthermore, high BMP4 expression promoted engraftment and rapid disease progression in an NSG mouse xenograft model with CNS involvement. Pharmacological blockade of the canonical BMP signaling pathway significantly decreased CNS infiltration and consistently resulted in amelioration of clinical parameters, including neurological score. Mechanistically, BMP4 favored chemoresistance, enhanced adhesion and migration through brain vascular endothelial cells, and promoted a proinflammatory microenvironment and CNS angiogenesis. These data provide evidence that BMP4 expression levels in leukemic cells could be a useful biomarker to identify children with poor outcomes in the low-/intermediate-risk groups of BCP-ALL and that BMP4 could be a new therapeutic target to blockade leukemic CNS disease.

CD8+ T lymphocytes are sensitive to NKG2A/HLA-E licensing interaction: role in the survival of cancer patients.

NK and CD8+ T cells are the main cytolytic effectors involved in innate and adaptive tumor immune surveillance, respectively. Although their educational pathways differ, similarities in their development and function suggest that CD8+ T lymphocytes could be sensitive to NK cell licensing signals, which might influence their antitumor response. To demonstrate this hypothesis, we retrospectively evaluated the impact that NK cell licensing interactions have on the expression of CD226 on CD8+ T lymphocytes and on the survival of patients with different hematopoietic and solid cancers (n = 1,023). Prospectively, we analyzed by multiparametric flow cytometry the anti-CD3/CD28-induced proliferation and immune-receptor expression of purified CD8+ T lymphocytes from healthy donors (n = 17) with different combinations of NK cell licensing ligands. Results show that methionine/threonine (M/T) dimorphism at position -21 of the HLA-B leader peptide, but not other HLA class-I dimorphisms involved in the education of NK cells (HLA-C1/C2 or HLA-Bw4), is associated with greater survival and expression of CD226 in cancer patients, which was proportional to the number of methionines present in their genotype. CD8+ T lymphocytes from healthy donors with -21 M showed higher proliferation rates and lower expression of TIGIT after in vitro stimulation. Therefore, CD8+ T lymphocytes, like NK cells, appear to be sensitive to the -21 M/T dimorphism of HLA-B leader peptide, which results in the modulation of CD226 in vivo and the proliferation and expression of TIGIT after in vitro stimulation, all of which could be related to their immune-surveillance capacity and the survival of cancer patients.

Activating Killer-Cell Immunoglobulin-Like Receptors Are Associated With the Severity of Coronavirus Disease 2019.

BACKGROUND: Etiopathogenesis of the clinical variability of the coronavirus disease 2019 (COVID-19) remains mostly unknown. In this study, we investigate the role of killer cell immunoglobulin-like receptor (KIR)/human leukocyte antigen class-I (HLA-I) interactions in the susceptibility and severity of COVID-19. METHODS: We performed KIR and HLA-I genotyping and natural killer cell (NKc) receptors immunophenotyping in 201 symptomatic patients and 210 noninfected controls. RESULTS: The NKcs with a distinctive immunophenotype, suggestive of recent activation (KIR2DS4low CD16low CD226low CD56high TIGIThigh NKG2Ahigh), expanded in patients with severe COVID-19. This was associated with a higher frequency of the functional A-telomeric activating KIR2DS4 in severe versus mild and/or moderate patients and controls (83.7%, 55.7% and 36.2%, P < 7.7 × 10-9). In patients with mild and/or moderate infection, HLA-B*15:01 was associated with higher frequencies of activating B-telomeric KIR3DS1 compared with patients with other HLA-B*15 subtypes and noninfected controls (90.9%, 42.9%, and 47.3%; P < .002; Pc = 0.022). This strongly suggests that HLA-B*15:01 specifically presenting severe acute respiratory syndrome coronavirus 2 peptides could form a neoligand interacting with KIR3DS1. Likewise, a putative neoligand for KIR2DS4 could arise from other HLA-I molecules presenting severe acute respiratory syndrome coronavirus 2 peptides expressed on infected an/or activated lung antigen-presenting cells. CONCLUSIONS: Our results support a crucial role of NKcs in the clinical variability of COVID-19 with specific KIR/ligand interactions associated with disease severity.

Immunological Risk Stratification of Bladder Cancer Based on Peripheral Blood Natural Killer Cell Biomarkers.

BACKGROUND: Bladder cancer (BC) is highly immunogenic. Bacillus Calmette-Guérin (BCG) immunotherapy offers the best results in non-muscle-invasive BC (NMIBC). Natural killer cells (NKcs) play decisive roles in BCG-mediated immune response and in general cancer immune-surveillance. OBJECTIVE: To analyze killer-cell immunoglobulin-like receptors (KIRs), their human leukocyte antigen class-I (HLA-I) ligands, and the expression of DNAX Accessory Molecule-1 (DNAM-1/CD226) on peripheral blood (PB) NKcs, to identify useful predictive biomarkers in BC. DESIGN, SETTING, AND PARTICIPANTS: KIR/HLA-ligand genotypes were compared between 132 BC, 201 other solid cancers, 164 plasma cell disorders, and 615 healthy Caucasoid controls. CD226 expression was evaluated by flow cytometry. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: KIR/HLA-I interactions and CD226 expression on NKcs (CD226high or CD226low) were compared across study groups, cancer stages, treatments, and progression-free and overall survival of patients, using chi-square, analysis of variance/post hoc, Kaplan-Meier/log-rank, and regression analyses. RESULTS AND LIMITATIONS: Three immunological risk groups were identified: low risk (KIR2DL1-L2+L3-/C1C1- and KIR2DL1+L2+L3+/C1C1+), intermediate risk (rest), and high risk (KIR2DL5+/HLA-C*16+ and KIR2DL1+L2+L3-), which displayed different 10-yr progression-free rates (83.3%, 48.6%, and 0%, respectively; p<0.001) and survival rates (83.3%, 54.3%, and 6.2%, respectively; p<0.001) for muscle-invasive T2/T4, and 10-yr progression-free rates (100%, 81.6%, and 50%, respectively; p<0.05) for NMIBC-T1 treated with BCG. Immunological risk stratification had an independent prognostic value to just histological staging for survival (hazard ratio=2.93, p<0.00001, Harrell C-statistic=0.779). CD226 expression on PB NKcs improved immunological stratification in intermediate-risk T1-T4 BC patients, with survival rates of 94.1% and 66.7% for CD226high and CD226low (p<0.05), respectively. CONCLUSIONS: Immunological risk stratification will complement BC histopathology to improve risk stratification and guide the selection of personalized treatments. Understanding of the molecular mechanisms of NKc tumor immune surveillance will enable the development of future NKc-based therapies. PATIENT SUMMARY: This work describes a peripheral blood test that aids in our understanding of the immune defense mechanisms against bladder cancer, is useful for classifying patient risk, and will guide personalized treatments.

KIR+ CD8+ T Lymphocytes in Cancer Immunosurveillance and Patient Survival: Gene Expression Profiling.

Killer-cell immunoglobulin-like receptors (KIR) are expressed by natural killer (NK) and effector T cells. Although KIR+ T cells accumulate in oncologic patients, their role in cancer immune response remains elusive. This study explored the role of KIR+CD8+ T cells in cancer immunosurveillance by analyzing their frequency at diagnosis in the blood of 249 patients (80 melanomas, 80 bladder cancers, and 89 ovarian cancers), their relationship with overall survival (OS) of patients, and their gene expression profiles. KIR2DL1+ CD8+ T cells expanded in the presence of HLA-C2-ligands in patients who survived, but it did not in patients who died. In contrast, presence of HLA-C1-ligands was associated with dose-dependent expansions of KIR2DL2/S2+ CD8+ T cells and with shorter OS. KIR interactions with their specific ligands profoundly impacted CD8+ T cell expression profiles, involving multiple signaling pathways, effector functions, the secretome, and consequently, the cellular microenvironment, which could impact their cancer immunosurveillance capacities. KIR2DL1/S1+ CD8+ T cells showed a gene expression signature related to efficient tumor immunosurveillance, whereas KIR2DL2/L3/S2+CD8+ T cells showed transcriptomic profiles related to suppressive anti-tumor responses. These results could be the basis for the discovery of new therapeutic targets so that the outcome of patients with cancer can be improved.

Activating KIRs on Educated NK Cells Support Downregulation of CD226 and Inefficient Tumor Immunosurveillance.

Therapies using NK cells (NKc) expanded/activated ex vivo or stimulated in vivo with new immunostimulatory agents offer alternative opportunities for patients with recurrent/refractory tumors, but relevant biomarkers to guide the selection of patients are required for optimum results. Overall survival of 249 solid cancer patients was evaluated in relation to the genetics and/or the expression on peripheral blood NKcs of inhibitory and activating killer-cell immunoglobulin-like receptors (iKIR and aKIR, respectively), HLA class I ligands, CD226 (also known as DNAM-1), and NKG2A. Compared with patients with higher expression, patients with low expression of CD226 on total NKcs showed shorter mean overall survival (60.7 vs. 98.0 months, P < 0.001), which was further reduced in presence of telomeric aKIRs (KIR2DS1-DS5 and/or KIR3DS1, 31.6 vs. 96.8 months, P < 0.001). KIR2DL2/S2+, KIR3DL1+, KIR2DL1+, and KIR2DL3+ NKc subsets in the presence of their cognate ligands primarily contributed to shortening patients' overall survival by increasing the sensitivity to CD226 downmodulation in aKIR-rich telomeric genotypes. In patients with high tumor burden who died during the follow-up period, aKIR-rich telomeric genotypes were associated with: (i) specific downmodulation of CD226 on educated NKcs but not on CD8+ T cells or uneducated NKcs, (ii) lower expression of CD226 and higher expression of NKG2A on aKIR+ NKcs, and (iii) lower numbers of total CD56dim NKcs. The reduced expression of CD226 on NKcs with aKIR-rich genotypes may be a biomarker indicative of NKc hyporesponsiveness in patients that could benefit from new NKc immune-stimulatory therapies.

NK Cell Education in Tumor Immune Surveillance: DNAM-1/KIR Receptor Ratios as Predictive Biomarkers for Solid Tumor Outcome.

Natural killer cell (NKc)-based therapies offer promising outcomes in patients with tumors, but they could improve with appropriate selection of donors and optimization of methods to expand NKcs in vitro Education through licensing interactions of inhibitory killer cell immunoglobulin-like receptors (iKIR) and NKG2A with their cognate HLA class-I ligands optimizes NKc functional competence. This work has evaluated the role of licensing interactions in NKc differentiation and the survival of cancer patients. We have analyzed KIR and KIR-ligand genes, and the expression of activating (CD16 and DNAM-1/CD226) and inhibitory (NKG2A and iKIRs) receptors on peripheral blood NKcs in 621 healthy controls and 249 solid cancer patients (80 melanoma, 80 bladder, and 89 ovarian). Licensing interactions upregulated the expression of activating CD226, reduced that of iKIR receptors, and shifted the CD226/iKIR receptor ratio on NKc membranes to activating receptors. A high tumor burden decreased CD226 expression, reduced the ratio of CD226/iKIR, and negatively affected patient survival. The progression-free survival (38.1 vs. 67.0 months, P < 0.002) and overall survival (56.3 vs. 99.6 months, P < 0.00001) were significantly shorter in patients with lower expression of CD226 on NKcs. Hence, transformed cells can downmodulate these licensing-driven receptor rearrangements as a specific mechanism to escape NKc immune surveillance. Our results suggest the importance of the CD226/iKIR receptor ratio of NKcs induced by licensing interactions as critical determinants for solid cancer immune surveillance, and may provide predictive biomarkers for patient survival that may also improve the selection of donors for NKc immunotherapy.

Overexpression of KIR inhibitory ligands (HLA-I) determines that immunosurveillance of myeloma depends on diverse and strong NK cell licensing.

Missing self recognition makes cancer sensitive to natural killer cell (NKc) reactivity. However, this model disregards the NKc licensing effect, which highly increases NKc reactivity through interactions of inhibitory killer cell immunoglobulin-like receptors (iKIR) with their cognate HLA-I ligands. The influence of iKIR/HLA-ligand (HLA-C1/C2) licensing interactions on the susceptibility to and progression of plasma cell (PC) dyscrasias was evaluated in 164 Caucasian patients and 286 controls. Compared to controls, myeloma accumulates KIR2DL1-L2+L3- genotypes (2.8% vs. 13.2%, p < 0.01, OR = 5.29) and less diverse peripheral repertoires of NKc clones. Less diverse and weaker-affinity repertoires of iKIR2D/HLA-C licensing interactions increased myeloma susceptibility. Thus, the complete absence of conventional iKIR2D/HLA-C licensing interactions (KIR2DL1-L2+L3-/C2C2, 2.56% vs. 0.35%; p < 0.05; OR = 15.014), single-KIR2DL3+/C1+ (20.51% vs. 10.84%; p < 0.05; OR = 2.795) and single-KIR2DL2+/C1+ (12.82% vs. 4.9%; p < 0.01; OR = 5.18) interactions were over-represented in myeloma, compared to controls. Additionally, KIR2DL1-L2+L3- (20% vs. 83%, p < 0.00001) as well as KIR3DL1- (23% vs. 82%, p < 0.00001) genotypes had a dramatic negative impact on the 3-y progression-free survival (PFS), particularly in patients with low-tumor burden. Remarkably, myeloma-PCs, compared to K562 and other hematological cancers, showed substantial over-expression of HLA-I ("increasing-self" instead of missing-self), including HLA-C, and mild expression of ligands for NKc activating receptors (aRec) CD112, CD155, ULBP-1 and MICA/B, which apparently renders myeloma-PCs susceptible to lysis mainly by licensed NKc. KIR2DL1-L2+L3-/C2C2 patients (with no conventional iKIR2D/HLA-C licensing interactions) lyse K562 but barely lyse myeloma-PCs (4% vs. 15%; p < 0.05, compared to controls). These results support a model where immunosurveillance of no-missing-self cancers, e.g., myeloma, mainly depends on NKc licensing.

Hemophagocytic lymphohistiocytosis: Overview and diagnostic procedure. A case induced by anexpansion of monoclonal EBV-negative NK cells

Las causas más comunes de linfohistiocitosis hemofagocítica (HLH) sonexpansiones clonales de células NK y T, inducidas por EBV, así como las alteraciones genéticas que comprometen la actividad asesina de las NKs. Generalmente, HLH se desencadena por una disfunción inmune en la que se desarrolla hipercitoquinemia. En este trabajo se resumen las causas más comunes de HLH y se presenta un caso en el que una expansión monoclonal decélulas NK, EBV-negativas, se asocia a HLH en una paciente aquejada de Síndrome de Griscelli tipo-2 (GS2). Se trata de una niña de 17 meses con unamutación de nueva descripción en RAB27A, con albinismo parcial, fiebre persistente, hepatoesplenomegalia, adenopatías y citopenias al diagnóstico. Nose detectaron evidencias de infecciones virales activas, incluida EBV. Se detectó una expansión de células NKs (5300/µl) CD2+CD7+CD8+CD16+CD56+CD94+CD158a/h+CD158b/j–Perforin+Granzyme-B+. Tras el tratamiento (Protocolo HLH-2004: Cyclosporina, Etoposido y Dexametasona), la cifra de células NK se redujo a 850/µl y que aumentaron progresivamente hasta alcanzar niveles similares al diagnóstico. El ensayo de inactivación del cromosoma X demostró monoclonalidad de células NK. Dichas células manteníanintacta su actividad asesina y secretaban grandes cantidades de IFN-γ. Aldiagnóstico los niveles séricos de sIL-2R (36.8 ng/ml) e IFN-γ (400 pg/ml)estaban elevados. En conclusión, se describe un caso de una expansión monoclonal de células NK, EBV-negativas, que secretan grandes cantidades deIFN-γ como la causa más probable del episodio de HLH en una paciente conGS2. Tras el trasplante de médula ósea de su hermana HLA-idéntica, las cifrasy el fenotipo de las células NK recobraron la normalidad (AU)

Clonal natural killer (NK) and T cell expansions induced by EpsteinBarr virus (EBV) and genetic alterations compromising NK cell killing arethe most common causes of hemophagocytic lymphohistocytosis (HLH).Generally, HLH is induced by an immune dysfunction where hypercytokinemia develops into reactive hemophagocytosis. In this work wereview the causes of HLH and describe a case of a monoclonal expansionof EBV-negative NK cells associated to HLH in a seventeen-month-oldgirl suffering of Griscelli syndrome type-2 with novel RAB27A mutation and showing partial albinism, persistent fever, hepatosplenomegaly,adenopaties and cytopenias. At diagnosis, no evidence of active viral infections, including EBV, was found. Expansion of NK cells (5300/µl in peripheral blood) CD2+CD7+CD8+CD16+CD56+CD94+CD158a/h+CD158b/j–Perforin+Granzyme B+ was found. After treatment (HLH-2004 protocol:Cyclosporin, Etoposide and Dexamethasone), NK cell count fell to 850/µland progressively increased to pre-therapy levels by week 28. X-chromosome inactivation assay demonstrated NK cell monoclonality. NK cellssustained a strong killing and secreted high amounts of IFN-γ. At diagnosis, serum levels of sIL-2R (36,8 ng/ml) and IFN-γ (400 pg/ml) wereelevated. In conclusion, we describe a monoclonal expansion of EBV-negative NK cells highly secretory of IFN-γ as the most probable cause of HLHepisode in a patient with Griscelli syndrome type-2. NK cell count recovered normal levels and phenotype after bone marrow transplantationfrom her HLA identical sister (AU)

Association of monoclonal expansion of Epstein-Barr virus-negative CD158a+ NK cells secreting large amounts of gamma interferon with hemophagocytic lymphohistiocytosis.

We report the first case of hemophagocytic lymphohistiocytosis (HLH) induced by the monoclonal expansion of Epstein-Barr virus (EBV)-negative NK cells. Consanguinity of the patient's parents made it necessary to discard familial HLH in the patient and her sister with identical HLA markers and demonstrate that no cause other than the expansion of NK cells, which secrete high levels of gamma interferon, was inducing HLH in this patient.