Flow cytometric immunophenotypic differentiation patterns of bone marrow eosinophilopoiesis.

BACKGROUND: Flow cytometry has been widely used to study immunophenotypic patterns of maturation of most hematopoietic lineages in normal human bone marrow aspirates, thus allowing identification of changes in patterns in many myeloid malignancies. Eosinophils play an important role in a wide variety of disorders, including some myeloid neoplasms. However, changes in flow cytometric immunophenotypic patterns during normal and abnormal bone marrow eosinophilopoiesis have not been well studied. METHODS: Fresh bone marrow aspirates from 15 healthy donors, 19 patients with hypereosinophilic syndromes (HES), and 11 patients with systemic mastocytosis (SM) were analyzed for candidate markers that included EMR-1, Siglec-8, CCR3, CD9, CD11a, CD11b, CD11c, CD13, CD16, CD29, CD34, CD38, CD45, CD44, CD49d, CD49f, CD54, CD62L, CD69, CD117, CD125 (IL-5Rα), HLA-DR, using 10 parameter flow cytometry. Putative CD34-negative immature and mature normal eosinophil populations were first identified based on changes in expression of the above markers in healthy donors, then confirmed using fluorescence-based cell sorting and morphological evaluation of cytospin preparations. The normal immunophenotypic patterns were then compared to immunophenotypic patterns of eosinophilopoiesis in patients with HES and SM. RESULTS: The eosinophilic lineage was first verified using the human eosinophil-specific antibody EMR-1 in combination with anti-IL-5Rα antibody. Then, a combination of Siglec-8, CD9, CD11b, CCR3, CD49d, and CD49f antibodies was used to delineate normal eosinophilic maturational patterns. Early stages (eosinophilic promyelocytes/myelocytes) were identified as Siglec-8 dim/CD11b dim to moderate/CD9 dim/CCR3 dim/CD49d bright/CD49f dim, intermediate stages (eosinophilic myelocytes/metamyelocytes) as Siglec-8 moderate/CD11b moderate to bright/CD9 moderate/CCR3 moderate/CD49d moderate/CD49f moderate and mature bands/segmented eosinophils as Siglec-8 bright/CD11b bright/CD9 bright/CCR3 bright/CD49d dim/CD49f bright. Overall maturational patterns were also similar in patients with HES and SM; however, the expression levels of several surface markers were altered compared to normal eosinophils. CONCLUSION: A novel flow cytometric antibody panel was devised to detect alterations in immunophenotypic patterns of bone marrow eosinophil maturation and evaluated in normal, HES and SM samples. This approach will allow us to elucidate changes in immunophenotypic patterns of bone marrow eosinophilopoiesis in other hematological diseases.

End-to-end deep learning method for predicting hormonal treatment response in women with atypical endometrial hyperplasia or endometrial cancer.

Purpose: Endometrial cancer (EC) is the most common gynecologic malignancy in the United States, and atypical endometrial hyperplasia (AEH) is considered a high-risk precursor to EC. Hormone therapies and hysterectomy are practical treatment options for AEH and early-stage EC. Some patients prefer hormone therapies for reasons such as fertility preservation or being poor surgical candidates. However, accurate prediction of an individual patient's response to hormonal treatment would allow for personalized and potentially improved recommendations for these conditions. This study aims to explore the feasibility of using deep learning models on whole slide images (WSI) of endometrial tissue samples to predict the patient's response to hormonal treatment. Approach: We curated a clinical WSI dataset of 112 patients from two clinical sites. An expert pathologist annotated these images by outlining AEH/EC regions. We developed an end-to-end machine learning model with mixed supervision. The model is based on image patches extracted from pathologist-annotated AEH/EC regions. Either an unsupervised deep learning architecture (Autoencoder or ResNet50), or non-deep learning (radiomics feature extraction) is used to embed the images into a low-dimensional space, followed by fully connected layers for binary prediction, which was trained with binary responder/non-responder labels established by pathologists. We used stratified sampling to partition the dataset into a development set and a test set for internal validation of the performance of our models. Results: The autoencoder model yielded an AUROC of 0.80 with 95% CI [0.63, 0.95] on the independent test set for the task of predicting a patient with AEH/EC as a responder vs non-responder to hormonal treatment. Conclusions: These findings demonstrate the potential of using mixed supervised machine learning models on WSIs for predicting the response to hormonal treatment in AEH/EC patients.

Weakly Supervised Deep Learning for Predicting the Response to Hormonal Treatment of Women with Atypical Endometrial Hyperplasia: A Feasibility Study.

Endometrial cancer (EC) is the most common gynecologic malignancy in the US and complex atypical hyperplasia (CAH) is considered a high-risk precursor to EC. Treatment options for CAH and early-stage EC include hormone therapies and hysterectomy with the former preferred by certain patients, e.g., for fertility preservation or poor surgical candidates. Accurate prediction of response to hormonal treatment would allow for personalized and potentially improved recommendations for the treatment of these conditions. In this study, we investigate the feasibility of utilizing weakly supervised deep learning models on whole slide images of endometrial tissue samples for the prediction of patient response to hormonal treatment. We curated a clinical whole-slide-image (WSI) dataset of 112 patients from two clinical sites. We developed an end-to-end machine learning model using WSIs of endometrial specimens for the prediction of hormonal treatment response among women with CAH/EC. The model takes patches extracted from pathologist-annotated CAH/EC regions as input and utilizes an unsupervised deep learning architecture (Autoencoder or ResNet50) to embed the images into a low-dimensional space, followed by fully connected layers for binary prediction. Our autoencoder model yielded an AUC of 0.79 with 95% CI [0.61, 0.98] on a hold-out test set in the task of predicting a patient with CAH/EC as a responder vs non-responder to hormonal treatment. Our results, demonstrate the potential for using weakly supervised machine learning models on WSIs for predicting response to hormonal treatment of CAH/EC patients.

Notch signaling and efficacy of PD-1/PD-L1 blockade in relapsed small cell lung cancer.

Immune checkpoint blockade (ICB) benefits only a small subset of patients with small cell lung cancer (SCLC), yet the mechanisms driving benefit are poorly understood. To identify predictors of clinical benefit to ICB, we performed immunogenomic profiling of tumor samples from patients with relapsed SCLC. Tumors of patients who derive clinical benefit from ICB exhibit cytotoxic T-cell infiltration, high expression of antigen processing and presentation machinery (APM) genes, and low neuroendocrine (NE) differentiation. However, elevated Notch signaling, which positively correlates with low NE differentiation, most significantly predicts clinical benefit to ICB. Activation of Notch signaling in a NE human SCLC cell line induces a low NE phenotype, marked by increased expression of APM genes, demonstrating a mechanistic link between Notch activation, low NE differentiation and increased intrinsic tumor immunity. Our findings suggest Notch signaling as a determinant of response to ICB in SCLC.

Prolonged Posttreatment Virologic Control and Complete Seroreversion After Advanced Human Immunodeficiency Virus-1 Infection.

BACKGROUND: Possible human immunodeficiency virus (HIV)-1 clearance has rarely been reported. In this study, we describe a unique case of an HIV-positive, combination antiretroviral therapy (cART)-experienced woman with prior acquired immunodeficiency syndrome (AIDS) who has not experienced viral rebound for over 12 years since discontinuing cART. METHODS: Leukapheresis, colonoscopy, and lymph node excision were performed for detailed examination of virologic (including HIV reservoir) and immunologic features. Comparisons were made with chronically infected patients and healthy controls. RESULTS: No HIV-specific antibodies were detected in serum. Plasma HIV ribonucleic acid (RNA) levels were <0.2 copies/mL, and, except for low-frequency HIV deoxyribonucleic acid (DNA)+ cells in lymph node tissue (1 copy/3 × 106 cells), HIV antigen could not be detected by quantitative virus outgrowth (<0.0025 infectious units/106 CD4+ T cells) or by most measurements of HIV RNA or DNA in blood, lymph node, or gut-associated mononuclear cells. Human immunodeficiency virus-specific T-cell responses were detectable but low. Brain imaging revealed a prior biopsy site and persistent white matter disease since 1996. Human immunodeficiency virus DNA+ cells in the 1996 brain biopsy specimen confirmed her identity and initial HIV diagnosis. CONCLUSIONS: This represents the first report of complete seroreversion, prolonged posttreatment virus suppression, a profoundly small HIV reservoir, and persistent HIV-specific T cells in an adult with prior AIDS.

Clonal Evolution and Heterogeneity of Osimertinib Acquired Resistance Mechanisms in EGFR Mutant Lung Cancer.

Clonal evolution of osimertinib-resistance mechanisms in EGFR mutant lung adenocarcinoma is poorly understood. Using multi-region whole-exome and RNA sequencing of prospectively collected pre- and post-osimertinib-resistant tumors, including at rapid autopsies, we identify a likely mechanism driving osimertinib resistance in all patients analyzed. The majority of patients acquire two or more resistance mechanisms either concurrently or in temporal sequence. Focal copy-number amplifications occur subclonally and are spatially and temporally separated from common resistance mutations such as EGFR C797S. MET amplification occurs in 66% (n = 6/9) of first-line osimertinib-treated patients, albeit spatially heterogeneous, often co-occurs with additional acquired focal copy-number amplifications and is associated with early progression. Noteworthy osimertinib-resistance mechanisms discovered include neuroendocrine differentiation without histologic transformation, PD-L1, KRAS amplification, and ESR1-AKAP12, MKRN1-BRAF fusions. The subclonal co-occurrence of acquired genomic alterations upon osimertinib resistance will likely require targeting multiple resistance mechanisms by combination therapies.

Fatal encephalopathy with wild-type JC virus and ruxolitinib therapy.

OBJECTIVE: JC virus (JCV) infection is a lytic infection of oligodendrocytes in progressive multifocal leukoencephalopathy; less common forms of central nervous system manifestations associated with JCV infection include granule cell neuronopathy, encephalopathy, and meningitis. Presented is the first case of fatal JCV encephalopathy after immunosuppressive therapy that included ruxolitinib. METHODS: Postmortem analysis included next generation sequencing, Sanger sequencing, tissue immunohistochemistry, and formalin-fixed hemisphere 7T magnetic resonance imaging. RESULTS: JCV DNA isolated from postmortem tissue samples identified a novel 12bp insertion that altered the transcription site binding pattern in an otherwise "wild-type virus," which has long been thought to be the nonpathogenic form of JCV. Anti-VP1 staining demonstrated infection in cortical neurons, hippocampal neurons, and glial and endothelial cells. INTERPRETATION: This expands the spectrum of identified JCV diseases associated with broad-spectrum immunosuppression, including JAK-STAT inhibitors, and sheds light on an additional neurotropic virus strain of the archetype variety. ANN NEUROL 2019;86:878-884.

Durvalumab in Combination with Olaparib in Patients with Relapsed SCLC: Results from a Phase II Study.

PURPOSE: Despite high tumor mutationburden, immune checkpoint blockade has limited efficacy in SCLC. We hypothesized that poly (ADP-ribose) polymerase inhibition could render SCLC more susceptible to immune checkpoint blockade. METHODS: A single-arm, phase II trial (NCT02484404) enrolled patients with relapsed SCLC who received durvalumab, 1500 mg every 4 weeks, and olaparib, 300 mg twice a day. The primary outcome was objective response rate. Correlative studies included mandatory collection of pretreatment and during-treatment biopsy specimens, which were assessed to define SCLC immunephenotypes: desert (CD8-positive T-cell prevalence low), excluded (CD8-positive T cells in stroma immediately adjacent/within tumor), and inflamed (CD8-positive T cells in direct contact with tumor). RESULTS: A total of 20 patients were enrolled. Their median age was 64 years, and most patients (60%) had platinum-resistant/refractory disease. Of 19 evaluable patients, two were observed to have partial or complete responses (10.5%), including a patient with EGFR-transformed SCLC. Clinical benefit was observed in four patients (21.1% [95% confidence interval: 6.1%-45.6%]) with confirmed responses or prolonged stable disease (≥8 months). The most common treatment-related adverse events were anemia (80%), lymphopenia (60%), and leukopenia (50%). Nine of 14 tumors (64%) exhibited an excluded phenotype; 21% and 14% of tumors exhibited the inflamed and desert phenotypes, respectively. Tumor responses were observed in all instances in which pretreatment tumors showed an inflamed phenotype. Of the five tumors without an inflamed phenotype at baseline, no during-treatment increase in T-cell infiltration or programmed death ligand 1 expression on tumor-infiltrating immune cells was observed. CONCLUSIONS: The study combination did not meet the preset bar for efficacy. Pretreatment and during-treatment biopsy specimens suggested that tumor immune phenotypes may be relevant for SCLC responses to immune checkpoint blockade combinations. The predictive value of preexisting CD8-positive T-cell infiltrates observed in this study needs to be confirmed in larger cohorts.

Interleukin-15 but not interleukin-7 abrogates vaccine-induced decrease in virus level in simian immunodeficiency virus mac251-infected macaques.

The loss of CD4(+) T cells and the impairment of CD8(+) T cell function in HIV infection suggest that pharmacological treatment with IL-7 and IL-15, cytokines that increase the homeostatic proliferation of T cells and improve effector function, may be beneficial. However, these cytokines could also have a detrimental effect in HIV-1-infected individuals, because both cytokines increase HIV replication in vitro. We assessed the impact of IL-7 and IL-15 treatment on viral replication and the immunogenicity of live poxvirus vaccines in SIV(mac251)-infected macaques (Macaca mulatta). Neither cytokine augmented the frequency of vaccine-expanded CD4(+) or CD8(+) memory T cells, clonal recruitment to the SIV-specific CD8(+) T cell pool, or CD8(+) T cell function. Vaccination alone transiently decreased the viral set point following antiretroviral therapy suspension. IL-15 induced massive proliferation of CD4(+) effector T cells and abrogated the ability of vaccination to decrease set point viremia. In contrast, IL-7 neither augmented nor decreased the vaccine effect and was associated with a decrease in TGF-beta expression. These results underscore the importance of testing immunomodulatory approaches in vivo to assess potential risks and benefits for HIV-1-infected individuals.

Subunit recombinant vaccine protects against monkeypox.

The smallpox vaccine Dryvax, a live vaccinia virus (VACV), protects against smallpox and monkeypox, but is contraindicated in immunocompromised individuals. Because Abs to VACV mediate protection, a live virus vaccine could be substituted by a safe subunit protein-based vaccine able to induce a protective Ab response. We immunized rhesus macaques with plasmid DNA encoding the monkeypox orthologs of the VACV L1R, A27L, A33R, and B5R proteins by the intradermal and i.m. routes, either alone or in combination with the equivalent recombinant proteins produced in Escherichia coli. Animals that received only DNA failed to produce high titer Abs, developed innumerable skin lesions after challenge, and died in a manner similar to placebo controls. By contrast, the animals vaccinated with proteins developed moderate to severe disease (20-155 skin lesions) but survived. Importantly, those immunized with DNA and boosted with proteins had mild disease with 15 or fewer lesions that resolved within days. DNA/protein immunization elicited Th responses and binding Ab titers to all four proteins that correlated negatively with the total lesion number. The sera of the immunized macaques recognized a limited number of linear B cell epitopes that are highly conserved among orthopoxviruses. Their identification may guide future efforts to develop simpler, safer, and more effective vaccines for monkeypox and smallpox.

Vaccine-induced CD8+ central memory T cells in protection from simian AIDS.

Critical to the development of an effective HIV vaccine is the identification of adaptive immune responses that prevent infection or disease. In this study we demonstrate in a relevant nonhuman primate model of AIDS that the magnitude of vaccine-induced virus-specific CD8(+) central memory T cells (T(CM)), but not that of CD8(+) effector memory T cells, inversely correlates with the level of SIVmac251 replication, suggesting their pivotal role in the control of viral replication. We propose that effective preventive or therapeutic T cell vaccines for HIV-1 should induce long-term protective central memory T cells.