Mixed Molecular Clinical Index (MMCI) in Diffuse Large B-cell Lymphoma (DLBCL)

Study Overview
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Brief Summary
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This is a prospective observational study. The primary objective is to identify new prognostic biomarkers for DLBCL patients in terms of progression-free survival (PFS) and able to add predictive capacity to recognized important clinical factors. The secondary objectives are: to identify new biomarkers associated with overall survival (OS) and objective response rate (ORR) to characterize tissue and circulating immune microenvironment of DLBCL patients by bulk and single cell transcriptomics; to assess the correlation between the expression of immune checkpoint genes and mRNA signature; to describe the mutational status of a panel of genes relevant to DLBCL pathogenesis;. to assess the correlation between protein expression, mutational status and the messenger RNA (mRNA) signature. For each enrolled patient, immunohistochemical determinations will be performed: Cell of origin (COO) (Germinal Cell -GC- or activated B-cell - ABC- type according with Hans algorithm ), evaluation of cluster of differentiation antigen 20 (CD20), cluster of differentiation antigen 5 (CD5), cluster of differentiation antigen 10 (CD10), Bcl6, Bcl2 (cut off>50%), Multiple Myeloma 1 / Interferon Regulatory Factor 4 protein (MUM1/IRF4), c-myc (cut off>40%) and Ki67, fluorescence in situ hybridization (FISH) for c-myc and if rearranged, for Bcl2 e Bcl6 ). Moreover, paraffin embedded (FFPE) tumor specimens will be collected for RNA extraction and mRNA expression analysis and sent to Bioscience Laboratory of Istituto Scientifico Romagnolo per lo studio e la cura dei tumori (IRST-IRCCS).

Detailed Description
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Diffuse large B-cell lymphoma (DLBCL) is an heterogeneous group of cancers classified together on the basis of morphology, immunophenotype, genetic alterations and clinical behavior. The distinction of DLBCL into cell-of origin (COO) categories, based on patterns of gene expression reminiscent ( germinal center B-cell- the GC group and activated B-cell- the ABC group-), as defined and characterized by the Lymphoma & Leukemia Molecular Profiling Project (LLMPP), has profound biological, prognostic and potential therapeutic implications and in addiction, the negative prognostic effect of myelocytomatosis oncogene (MYC), B-cell lymphoma 2 (BCL2) and B-cell lymphoma-6 (BCL6) alterations in DLBCL has been showed largely dependent on COO subtypes . Furthermore, the combination of BCL2, MYC and BCL6 alterations with IPI (International Prognostic Index), identifies markedly worse prognostic groups within individual COO subtypes. The original methods used to define these entities, performed gene expression profiling (GEP) using microarrays on RNA derived from frozen tissue (FT). Subsequently, in an attempt to determine COO in standard practice using commonly available formalin-fixed paraffin-embedded tissue (FFPE) less precise but relatively inexpensive binary immunohistochemical (IHC) methods has been used . However in particular in non GC, the rate of concordance was unsatisfactory. A high degree of agreement has been demonstrated instead in COO determining, with a signature of 20 genes from formalin-fixed paraffin embedded (FFPE) tumor specimens, with Lymph2Cx kit (nCounter® Technology, NanoString Technologies), becoming the gold standard suggested in World Health Organization (WHO) classification . However recently, was demonstrated that the COO and BCL2, MYC, BCL6 status are not enough to describe the molecular risk of these patients, suggesting a genetic substructure that still to be discovered . Moreover, the tumor microenvironment and in particular the ratio of immune effectors and checkpoint molecules also have a prognostic role in DLBCL. Besides, elevated frequency of myofibroblasts, dendritic cells, and cluster of differentiation 4 (CD4) positive T cells correlated with better outcomes. In conclusion, a comprehensive genomic analysis of these patients and a deep characterization of the immune compartment and immune checkpoints (Nanostring, immunohistochemistry for BCL2, MYC, BCL6, mutation analysis, proteomic analysis etc.) joined with IPI score, will allow the creation of a mixed, molecular, clinical, index (MMCI) to identify extremely poor prognostic groups, within each COO subtype, to consider a risk-adapted treatments in future. It is a prospective observational study with a total duration of 36 months. The primary objective is the identification of new prognostic biomarkers for DLBCL patients in terms of progression-free survival (PFS) and able to add predictive capacity to recognized important clinical factors. The secondary objectives are: to identify new biomarkers associated with overall survival (OS) and objective response rate (ORR); to characterize tissue and circulating immune microenvironment of DLBCL patients by bulk and single cell transcriptomics; to assess the correlation between the expression of immune - checkpoint genes and mRNA signature; to describe the mutational status of a panel of genes relevant to DLBCL pathogenesis;. to assess the correlation between protein expression, mutational status and the mRNA signature. For each enrolled patient, immunohistochemical determinations will be performed by each Pathology Unit: COO (GC o ABC type according with Hans algorithm ), evaluation of CD20, CD5, CD10, Bcl6, Bcl2 (cut off>50%), MUM1/IRF4, c-myc (cut off>40%) and Ki67, FISH for c-myc and if rearranged, for Bcl2 e Bcl6). Moreover, paraffin embedded (FFPE) tumor specimens will be collected for RNA extraction and mRNA expression analysis and centralised at IRST-IRCCS.

Official Title
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Mixed Molecular Clinical Index (MMCI) in Diffuse Large B-cell Lymphoma (DLBCL)

Conditions
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Diffuse Large B Cell Lymphoma

Intervention / Treatment
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* Other: molecular characterization


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: New diagnosis of High grade Diffuse large B cell Lymphoma Signed written informed consent. Exclusion Criteria: Any other malignancy within 5 years (except for adequately treated carcinoma in situ of the cervix or basal or squamous cell skin cancer or surgically resected prostate cancer with normal PSA).

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| DLBCL patients<br>All patients with diagnosis of DLBCL | Other: molecular characterization<br>* immunohistochemical determinations: Cell of Origin (COO) (according with Hans algorithm), evaluation of Cluster of Differentiation (CD) (CD20, CD5, CD10), Bcl6, Bcl2 (cut off>50%), MUM1/IRF4, c-myc (cut off>40%) and Ki67, FISH for c-myc and if rearranged for Bcl2 e Bcl6. mRNA expression by Nanostring Single cell analysis Immune checkpoint expression Proteomic analysis Metabolic analysis<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| identification of new prognostic biomarkers | To identify new prognostic biomarkers for DLBCL patients that combined to clinical factors (IPI) are able to create a MMCI, predictive in terms of progression-free survival (PFS) of DLBCL patients. | 3 years | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| identification of molecular and clinical parameters | to identify molecular and clinical parameters associated with overall survival (OS) and objective response rate (ORR) | 3 years | 
| characterization of tissue and circulating immune microenvironment | to characterize tissue and circulating immune microenvironment by bulk and single cell transcriptomics | 3 years | 
| immune checkpoint genes analysis | to assess the correlation between the expression of immune checkpoint genes and mRNA signatures | 3 years | 
| mutational status | describe the mutational status of a panel of genes relevant to DLBCL pathogenesis | 3 years | 
| Correlation of protein expression, mutational status and the mRNA signatures | assess the correlation between protein expression, mutational status and the mRNA signatures. | 3 years | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Diffuse Large B cell lymphoma;, cell of origin, gene expression, immunohistochemistry