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