The tumor microenvironment (TME) and limited immune surveillance
play important roles in lymphoma pathogenesis. Here we
aimed to characterize immunological profiles of diffuse large B-cell
lymphoma ...(DLBCL) and predict the outcome in response to
immunochemotherapy. We profiled the expression of 730 immune-related
genes in tumor tissues of 81 patients with DLBCL utilizing the Nanostring
platform, and used multiplex immunohistochemistry to characterize T-cell
phenotypes, including cytotoxic T cells (CD8, Granzyme B, OX40, Ki67),
T-cell immune checkpoint (CD3, CD4, CD8, PD1, TIM3, LAG3), as well as
regulatory T-cells and Th1 effector cells (CD3, CD4, FOXP3, TBET) in 188
patients. We observed a high degree of heterogeneity at the transcriptome
level. Correlation matrix analysis identified gene expression signatures
with highly correlating genes, the main cluster containing genes for cytolytic
factors, immune checkpoint molecules, T cells and macrophages, together
named a TME immune cell signature. Immunophenotyping of the distinct
cell subsets revealed that a high proportion of immune checkpoint positive
T cells translated to unfavorable survival. Together, our results demonstrate
that the immunological profile of DLBCL TME is heterogeneous and clinically
meaningful. This highlights the potential impact of T-cell immune
checkpoint in regulating survival and resistance to immunochemotherapy.
(Registered at clinicaltrials.gov identifiers: NCT01502982 and NCT01325194.)
Separating and labeling each nuclear instance (instance-aware segmentation) is the key challenge in nuclear image segmentation. Deep Convolutional Neural Networks have been demonstrated to solve ...nuclear image segmentation tasks across different imaging modalities, but a systematic comparison on complex immunofluorescence images has not been performed. Deep learning based segmentation requires annotated datasets for training, but annotated fluorescence nuclear image datasets are rare and of limited size and complexity. In this work, we evaluate and compare the segmentation effectiveness of multiple deep learning architectures (U-Net, U-Net ResNet, Cellpose, Mask R-CNN, KG instance segmentation) and two conventional algorithms (Iterative h-min based watershed, Attributed relational graphs) on complex fluorescence nuclear images of various types. We propose and evaluate a novel strategy to create artificial images to extend the training set. Results show that instance-aware segmentation architectures and Cellpose outperform the U-Net architectures and conventional methods on complex images in terms of F1 scores, while the U-Net architectures achieve overall higher mean Dice scores. Training with additional artificially generated images improves recall and F1 scores for complex images, thereby leading to top F1 scores for three out of five sample preparation types. Mask R-CNN trained on artificial images achieves the overall highest F1 score on complex images of similar conditions to the training set images while Cellpose achieves the overall highest F1 score on complex images of new imaging conditions. We provide quantitative results demonstrating that images annotated by under-graduates are sufficient for training instance-aware segmentation architectures to efficiently segment complex fluorescence nuclear images.
Primary chronic cold agglutinin disease is a rare hemolytic disease mediated by monoclonal IGHV4-34-encoded cold agglutinins with a predominant specificity for the blood group antigen I. Bone marrow ...from 54 patients was studied to type the underlying lymphoproliferative disorder better. Bone marrow biopsies showed circumscribed intra-parenchymatous nodules with small monotonous monoclonal B cells in 40/54 patients (median infiltration: 10% of marrow cells) with a CD20(+), IgMs(+), IgDs(+), CD27(+), CD5(-/+), CD11c(-), CD23(-), CD38(-) immunophenotype. Neither plasmacytoid cytological features nor expression of plasma cell differentiation-associated transcription factors MUM1, XBP1 and BLIMP1 were noted in these B cells. However, a limited number of mature monoclonal IgM(+), IgD(-) plasma cells were present outside the lymphoid nodules and were diffusely scattered throughout the marrow. Of interest, the MYD88 L265P mutation, typical of lymphoplasmacytic lymphoma, was not detected (17/17 cases). Somatically mutated monoclonal IGHV4-34 gene rearrangement was demonstrated in eight patients with frozen samples (mean sequence homology 95.4%). However, mutations of BCL6 intron 1 were not demonstrated, except in one patient, suggesting that the lymphoma cells had not matured in the germinal center. In conclusion, cold agglutinin-associated lymphoproliferative disease displays homogeneous histological and immunophenotypic features. The absence of plasmacytoid cells, the presence of plasma cells predominantly outside the nodular lymphoid infiltrates, IGHV4-34 restriction and absence of MYD88 L265P mutation strongly suggest that cold agglutinin-associated lymphoproliferative disease is a distinct entity that is different from lymphoplasmacytic lymphoma.
Neuroblastoma (NBL), one of the main death-causing cancers in children, is known for its remarkable genetic heterogeneity and varied patient outcome spanning from spontaneous regression to widespread ...disease. Specific copy number variations and single gene rearrangements have been proven to be associated with biological behavior and prognosis; however, there is still an unmet need to enlarge the existing armamentarium of prognostic and therapeutic targets. We performed whole exome sequencing (WES) of samples from 18 primary tumors and six relapse samples originating from 18 NBL patients. Our cohort consists of 16 high-risk, one intermediate, and one very low risk patient. The obtained results confirmed known mutational hotspots in ALK and revealed other non-synonymous variants of NBL-related genes (TP53, DMD, ROS, LMO3, PRUNE2, ERBB3, and PHOX2B) and of genes cardinal for other cancers (KRAS, PIK3CA, and FLT3). Beyond, GOSeq analysis determined genes involved in biological adhesion, neurological cell-cell adhesion, JNK cascade, and immune response of cell surface signaling pathways. We were able to identify novel coding variants present in more than one patient in nine biologically relevant genes for NBL, including TMEM14B, TTN, FLG, RHBG, SHROOM3, UTRN, HLA-DRB1, OR6C68, and XIRP2. Our results may provide novel information about genes and signaling pathways relevant for the pathogenesis and clinical course in high-risk NBL.
Neuroblastoma (NBL), one of the main death-causing cancers in children, is known for its remarkable genetic heterogeneity and varied patient outcome spanning from spontaneous regression to widespread ...disease. Specific copy number variations and single gene rearrangements have been proven to be associated with biological behavior and prognosis; however, there is still an unmet need to enlarge the existing armamentarium of prognostic and therapeutic targets. We performed whole exome sequencing (WES) of samples from 18 primary tumors and six relapse samples originating from 18 NBL patients. Our cohort consists of 16 high-risk, one intermediate, and one very low risk patient. The obtained results confirmed known mutational hotspots in ALK and revealed other non-synonymous variants of NBL-related genes (TP53, DMD, ROS, LMO3, PRUNE2, ERBB3, and PHOX2B) and of genes cardinal for other cancers (KRAS, PIK3CA, and FLT3). Beyond, GOSeq analysis determined genes involved in biological adhesion, neurological cell-cell adhesion, JNK cascade, and immune response of cell surface signaling pathways. We were able to identify novel coding variants present in more than one patient in nine biologically relevant genes for NBL, including TMEM14B, TTN, FLG, RHBG, SHROOM3, UTRN, HLA-DRB1, OR6C68, and XIRP2. Our results may provide novel information about genes and signaling pathways relevant for the pathogenesis and clinical course in high-risk NBL.
Background
International standardized criteria for eligibility, evaluable disease sites, and disease response assessment in patients with refractory, progressive, or relapsed high‐risk neuroblastoma ...enrolled in early‐phase clinical trials are lacking.
Methods
A National Cancer Institute–sponsored Clinical Trials Planning Meeting was convened to develop an international consensus to refine the tumor site eligibility criteria and evaluation of disease response for early‐phase clinical trials in children with high‐risk neuroblastoma.
Results
Standardized data collection of patient and disease characteristics (including specified genomic data), eligibility criteria, a definition of evaluable disease, and response evaluations for primary and metastatic sites of disease were developed. Eligibility included two distinct patient groups: progressive disease and refractory disease. The refractory disease group was subdivided into responding persistent disease and stable persistent disease to better capture the clinical heterogeneity of refractory neuroblastoma. Requirements for defining disease evaluable for a response assessment were provided; they included requirements for biopsy to confirm viable neuroblastoma and/or ganglioneuroblastoma in those patients with soft tissue or bone disease not avid for iodine‐123 meta‐iodobenzylguanidine. Standardized evaluations for response components and time intervals for response evaluations were established.
Conclusions
The use of international consensus eligibility, evaluability, and response criteria for early‐phase clinical studies will facilitate the collection of comparable data across international trials and promote more rapid identification of effective treatment regimens for high‐risk neuroblastoma.
This international consensus statement from a National Cancer Institute‐sponsored Clinical Trials Planning Meeting refines the tumor site eligibility criteria and evaluation of disease response for early‐phase clinical trials in children with high‐risk neuroblastoma.
Mantle cell lymphoma (MCL) is clinically and biologically heterogeneous. While various prognostic features have been proposed, none currently impact therapy selection, particularly in older patients, ...for whom treatment is primarily dictated by age and comorbidities. Herein, we undertook a comprehensive comparison of clinicopathological features in a cohort of patients 60 years and older, uniformly treated with bendamustine and rituximab, with a median survival of >8 years. The strongest prognostic indicators in this cohort were a high-risk call by a simplified MCL international prognostic index (s-MIPI) (HR: 3.32, 95% CI: 1.65-6.68 compared to low risk), a high-risk call by MCL35 (HR: 10.34, 95% CI: 2.37-45.20 compared to low risk) and blastoid cytology (HR: 4.21, 95% CR: 1.92-9.22 compared to classic). Patients called high risk by both the s-MIPI and MCL35 had the most dismal prognosis (HR: 11.58, 95% CI: 4.10-32.72), while those with high risk by either had a moderate but clinically relevant prognosis (HR: 2.95, 95% CI: 1.49-5.82). A robust assay to assess proliferation, such as MCL35, along with stringent guidelines for cytological evaluation of MCL, in combination with MIPI, may be a strong path to risk-stratify older MCL patients in future clinical trials.
Post-transplant lymphoproliferative disorders (PTLDs) are iatrogenic immune deficiencyassociated lymphoid/plasmacytic proliferations developing due to immunosuppression in solid organ or ...hematopoietic stem cell allograft patients. PTLDs are characterized by abnormal proliferation of lymphoid cells and have a heterogeneous clinical behavior. We profiled expression of >700 tumor microenvironment (TME)-related genes in 75 post-transplant aggressive B-cell lymphomas (PT-ABCLs). EBV-positive PT-ABCLs clustered together and were enriched for type I interferon pathway and antiviral response genes. Additionally, a cytotoxicity gene signature associated with EBV-positivity and favorable overall survival (OS; HR 0.61, P=0.019). In silico immunophenotyping revealed two subgroups with distinct immune cell compositions. The inflamed subgroup with higher proportions of immune cells had better outcome compared to non-inflamed subgroup (median OS >200.0 vs. 15.2 months, P=0.006). In multivariable analysis with EBV status, International Prognostic Index, and rituximab-containing treatment, the inflamed TME remained as an independent predictor for favorable outcome. We also compared the TME between posttransplant and immunocompetent host diffuse large B-cell lymphomas (DLBCLs) (n=75) and discovered that the proportions of T cells were lower in PT-DLBCL. In conclusion, we provide a comprehensive phenotypic characterization of PT-ABCLs, highlighting the importance of immune cell composition of TME in determining the clinical behavior and prognosis of PT-ABCL.
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