Marginal Zone Differentiation Research Articles

Low-grade follicular lymphoma with extensive marginal zone differentiation and expression of MUM1 and CD30

Low-grade follicular lymphoma with extensive marginal zone differentiation and expression of MUM1 and CD30

Pediatric-Type Indolent B-Cell Lymphomas With Overlapping Clinical, Pathologic, and Genetic Features.

Pediatric-type follicular lymphoma (PTFL) and pediatric nodal marginal zone lymphoma (PNMZL) are rare pediatric-type indolent B-cell lymphomas (PedIBCL) that differ clinicopathologically from their adult counterparts. Accurate diagnosis is important to avoid overtreatment but is often challenging. The mutational landscape of PTFL is known and may aid diagnosis, but the genetic features of PNMZL are not well understood. We analyzed 21 cases of PedIBCL according to their clinicopathologic findings and classified them into PTFL (n=11), PNMZL (n=2), and “mixed type” tumors (n=8) showing ambiguous histology. We also analyzed 2 cases of adult B-cell lymphomas showing features of PedIBCL. Targeted sequencing of 121 lymphoma-related genes was performed. The median age of PedIBCL patients was 16 years (range: 3 to 47), and all but 1 PTFL patient were male. All patients presented with limited-stage disease, and only 1 relapsed. There were no significant differences in clinical features among the 3 PedIBCL groups. The most frequently mutated genes were MAP2K1, TNFRSF14, KMT2C, IRF8, and NOTCH2. The genetic features of all groups were similar to the established mutational landscape of PTFL. The 2 adult B-cell lymphomas cases also had MAP2K1, TNFRSF14, and IRF8 mutations, but the clinical features were not typical of PedIBCL. In summary, this study demonstrated that PTFL and PNMZL are similar diseases with overlapping clinical, pathologic, and genetic features; mixed type tumors can also occur. Atypical adult cases with similar histologic features were also observed. Therefore, the disease spectrum of PedIBCL may be much broader than is currently believed.

A unifying hypothesis for PNMZL and PTFL: morphological variants with a common molecular profile

AbstractPediatric nodal marginal zone lymphoma (PNMZL) is an uncommon B-cell neoplasm affecting mainly male children and young adults. This indolent lymphoma has distinct characteristics that differ from those of conventional nodal marginal zone lymphoma (NMZL). Clinically, it exhibits overlapping features with pediatric-type follicular lymphoma (PTFL). To explore the differences between PNMZL and adult NMZL and its relationship to PTFL, a series of 45 PNMZL cases were characterized morphologically and genetically by using an integrated approach; this approach included whole-exome sequencing in a subset of cases, targeted next-generation sequencing, and copy number and DNA methylation arrays. Fourteen cases (31%) were diagnosed as PNMZL, and 31 cases (69%) showed overlapping histologic features between PNMZL and PTFL, including a minor component of residual serpiginous germinal centers reminiscent of PTFL and a dominant interfollicular B-cell component characteristic of PNMZL. All cases displayed low genomic complexity (1.2 alterations per case) with recurrent 1p36/TNFRSF14 copy number–neutral loss of heterozygosity alterations and copy number loss (11%). Similar to PTFL, the most frequently mutated genes in PNMZL were MAP2K1 (42%), TNFRSF14 (36%), and IRF8 (34%). DNA methylation analysis revealed no major differences between PTFL and PNMZL. Genetic alterations typically seen in conventional NMZL were absent in PNMZL. In summary, overlapping clinical, morphologic, and molecular findings (including low genetic complexity; recurrent alterations in MAP2K1, TNFRSF14, and IRF8; and similar methylation profiles) indicate that PNMZL and PTFL are likely part of a single disease with variation in the histologic spectrum. The term “pediatric-type follicular lymphoma with and without marginal zone differentiation” is suggested.

Retina regeneration: lessons from vertebrates.

Unlike mammals, vertebrates such as fishes and frogs exhibit remarkable tissue regeneration including the central nervous system. Retina being part of the central nervous system has attracted the interest of several research groups to explore its regenerative ability in different vertebrate models including mice. Fishes and frogs completely restore the size, shape and tissue structure of an injured retina. Several studies have unraveled molecular mechanisms underlying retina regeneration. In teleosts, soon after injury, the Müller glial cells of the retina reprogram to form a proliferating population of Müller glia-derived progenitor cells capable of differentiating into various neural cell types and Müller glia. In amphibians, the transdifferentiation of retinal pigment epithelium and differentiation of ciliary marginal zone cells contribute to retina regeneration. In chicks and mice, supplementation with external growth factors or genetic modifications cause a partial regenerative response in the damaged retina. The initiation of retina regeneration is achieved through sequential orchestration of gene expression through controlled modulations in the genetic and epigenetic landscape of the progenitor cells. Several developmental biology pathways are turned on during the Müller glia reprogramming, retinal pigment epithelium transdifferentiation and ciliary marginal zone differentiation. Further, several tumorigenic pathways and gene expression events also contribute to the complete regeneration cascade of events. In this review, we address the various retinal injury paradigms and subsequent gene expression events governed in different vertebrate species. Further, we compared how vertebrates such as teleost fishes and amphibians can achieve excellent regenerative responses in the retina compared with their mammalian counterparts.

Transformation of low-grade follicular lymphoma with partial marginal zone differentiation: Two cases.

Two cases of low-grade follicular lymphoma, with marginal zone differentiation and/or with high proliferation rate in one of them, are reported with transformation into high grade B-cell and B-lymphoblastic lymphomas. The contribution of these features to the transforming process, although previously described, is infrequent, and has not been deciphered to date.

The complex pathology and differential diagnosis of splenic and nodal marginal zone lymphoma

: Marginal zone lymphoma is a term applied to different B-cell low-grade lymphoma disorders with some common features, originated around reactive follicles, composed by small/medium size cells with specific immunophenotype, Ig genes stereotypes and somatic mutation of genes (NOTCH2, KLF2, TNFAIP3…) involved in marginal zone differentiation and NF-kB activation. Marginal zone lymphoma differential diagnosis is based on an integration of clinical, morphological and molecular features. Here we describe the current status of splenic and nodal marginal zone lymphoma (NMZL) diagnosis, reviewing also the markers (MNDA, T-Bet, IRTA1) that facilitate their recognition and diagnosis. Splenic marginal zone lymphoma (SMZL) is a disseminated lymphoma since the initial diagnosis, with both bone marrow and peripheral blood infiltration at diagnosis in almost every case. In contrast, NMZL is frequently a tumor restricted to lymph nodes, where bone marrow and/or peripheral blood involvement is only seen ad advanced stages. Both SMZL and NMZL have a spectrum of clinical presentations ranging from subclinical monoclonal B-cell lymphocytosis at earlier stages to aggressive disorders in advanced stages where p53 mutations, increased CD30 expression or EBV presence are frequently detected.

RNA sequencing analyses reveal differentially expressed genes and pathways as Notch2 targets in B-cell lymphoma.

Splenic marginal zone lymphoma (SMZL) is a low grade, indolent B-cell neoplasm that comprises approximately 10% of all lymphoma. Notch2, a pivotal gene for marginal zone differentiation is found to be mutated in SMZL. Deregulated Notch2 signaling has been involved in tumorigenesis and also in B-cell malignancies. However the role of Notch2 and the downstream pathways that it influences for development of B-cell lymphoma remains unclear. In recent years, RNA sequencing (RNA-Seq) has become a functional and convincing technology for profiling gene expression and to discover new genes and transcripts that are involved in disease development in a single experiment. In the present study, using transcriptome sequencing approach, we have identified key genes and pathways that are probably the underlying cause in the development of B-cell lymphoma. We have identified a total of 15,083 differentially expressed genes (DEGs) and 1067 differentially expressed transcripts (DETs) between control and Notch2 knockdown B cells. Gene Ontology (GO) term enrichment and pathway analysis were applied for the identification of key genes and pathways involved in development of B-cell lymphoma. In addition, intermediate genes of top canonical pathways such as PI3K/AKT and NF-kB were found to be downregulated with Notch2 knockdown, indicating that these pathways could be the putative downstream effectors through which Notch2 mediates its oncogenic effects. Taken collectively, the identified crop of genes and pathways may be considered as targets for the treatment of B-cell lymphoma.

Discovery of Novel Recurrent Mutations and Clinically Meaningful Subgroups in Nodal Marginal Zone Lymphoma.

Nodal marginal zone lymphoma (NMZL) is a rare B-cell neoplasm, the genetic and transcriptomic landscape of which are unclear. Using high-throughput sequencing for whole-exome and transcriptome, we investigated the genetic characteristics of NMZL in a discovery cohort (n = 8) and validated their features in an extended cohort (n = 30). Novel mutations in NFKBIE and ITPR2 were found in 7.9% (3/38) and 13.9% (5/36), respectively, suggesting roles for the NF-κB pathway and B-cell-receptor-mediated calcium signaling pathway in the pathogenesis of NMZL. RNA-seq showed that NMZLs were characterized by an aberrant marginal zone differentiation, associated with an altered IRF4-NOTCH2 axis and the enrichment of various oncogenic pathways. Based on gene expression profile, two subgroups were identified. Compared with subgroup 1, subgroup 2 showed the following: the significant enrichment of cell cycle-associated and MYC-signaling pathways, a more diverse repertoire of upstream regulators, and higher Ki-67 proliferation indices. We designated two subgroups according to Ki-67 labeling, and subgroup 2 was significantly associated with a shorter progression-free survival (p = 0.014), a greater proportion of large cells (p = 0.009), and higher MYC expression (p = 0.026). We suggest that NMZL has unique features and, in this study, we provide information as to the heterogeneity of this enigmatic entity.

A rare face of follicular lymphoma: reverse variant of follicular lymphoma

BackgroundReverse Variant of Follicular Lymphoma (RVFL) is one of the rare morphological variants of FL, characterized by dark staining small centrocytes in the center and pale staining large centroblasts at the periphery of the neoplastic follicles. Only rare cases of RVFL have been described to date. The histological appearance of this little known variant of FL may be misinterpreted if pathologists are unaware of its existence. The main purpose of this study is to draw pathologists’ attention to such an uncommon growth pattern of FL so that this variant can be correctly recognized and the clinical significance further studied in the future.MethodsFour cases of FL with unusual morphologic features were evaluated for the expression pattern of CD20, CD10, BCL6, BCL2, CD21, CD23, CD3, CD5, Cyclin D1, IgD and Ki67 by immunohistochemistry. Fluorescence in situ hybridization (FISH) with break-apart probes was performed to detect BCL2 gene rearrangement.ResultsAll four cases showed distinctive morphologic pattern of RVFL; in addition, each also exemplified unique morphological features. Immunohistochemical stains confirmed the cells in both the central areas and the peripheral cuffs had the same immunophenotypic profiles, contrasting to the FL with marginal zone differentiation in which only the center of the nodules showed expression of CD10. FISH demonstrated BCL2 gene rearrangement in all cases.ConclusionThe growth pattern of this rare FL variant may mimic FL with marginal-zone differentiation and other entities including but not limited to marginal zone lymphoma (MZL), progressive transformation of germinal centers (PTGC) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). Pathologists should be familiar with this unusual morphological variant to avoid diagnostic pitfalls.

Cross-Reactive Antigen Expressed by B6 Splenocytes Drives Receptor Editing and Marginal Zone Differentiation of IgG2a-Reactive AM14 Vκ8 B Cells.

The AM14 BCR, derived from an autoimmune MRL/lpr mouse, binds autologous IgG2aa/j with low affinity, and as a result, AM14 B cells only proliferate in response to IgG2a immune complexes that incorporate DNA, RNA, or nucleic acid-binding proteins that serve as autoadjuvants. As such, AM14 B cells have served as a useful model for demonstrating the importance of BCR/TLR coengagement in the activation of autoreactive B cells. We now show that the same receptor recognizes an additional murine-encoded Ag, expressed by B6 splenocytes, with sufficient avidity to induce a TLR-independent proliferative response of BALB/c AM14 Vκ8 B cells both in vivo and in vitro. Moreover, detection of this cross-reactive Ag by B6 AM14 Vκ8 B cells promotes an anergic phenotype as reflected by suboptimal responses to BCR cross-linking and the absence of mature B cells in the bone marrow. The B6 Ag further impacts B cell development as shown by a dramatically expanded marginal zone compartment and extensive receptor editing in B6 AM14 Vκ8 mice but not BALB/c AM14 Vκ8 mice. Despite their anergic phenotypes, B6 AM14 Vκ8 B cells can respond robustly to autoantigen/autoadjuvant immune complexes and could therefore participate in both autoimmune responses and host defense.

A pediatric-type follicular lymphoma with marginal zone and monotypic intracytoplasmic plasmacytic differentiation

Pediatric-type follicular lymphoma and pediatric nodal marginal zone lymphoma are rare indolent lymphomas that are distinctive entities with excellent prognoses. We present the histopathology and the immunophenotypic, cytogenetic, and gene rearrangement findings of a unique, heretofore, unreported case of a 15-year old boy with a right-groin mass (5.5×4.5×3.5cm) with three discrete malignant components: 1) follicular lymphoma positive for CD20, CD10, and BCL6, but negative for BCL2; by flow cytometry, 9% of the cells were kappa monotypic B-cells that expressed CD10, CD19, CD20, and CD38; 2) typical marginal zone B-cell differentiation; marginal zone B-cells were positive for CD20, CD43, BCL2, and MUM-1, but CD10 and BCL6 negative. A small subset was weakly positive for intracytoplasmic IgM and kappa consistent with early plasmacytic differentiation; 3) Plasmacytoid forms, intracytoplasmic globules and crystals strongly expressed intracytoplasmic monotypic IgM and kappa.

Primary cutaneous follicular B-cell lymphoma with marginal zone differentiation: A case discussion

Primary cutaneous follicular B-cell lymphoma with marginal zone differentiation: A case discussion

Skewed B cell differentiation affects lymphoid organogenesis but not T cell-mediated autoimmunity

B cell receptor (BCR) signalling determines B cell differentiation and may potentially alter T cell-mediated immune responses. In this study we used two transgenic strains of BCR-deficient mice expressing Epstein-Barr virus latent membrane protein (LMP)2A in B cells, where either follicular and marginal zone differentiation (D(H)LMP2A mice) or B-1 cell development (V(H)LMP2A mice) were supported, and evaluated the effects of skewed B lymphocyte differentiation on lymphoid organogenesis and T cell responses in vivo. Compared to wild-type animals, both transgenic strains displayed alterations in the composition of lymphoid organs and in the dynamics of distinct immune cell subsets following immunization with the self-antigen PLP₁₈₅₋₂₀₆. However, ex-vivo T cell proliferation to PLP₁₈₅₋₂₀₆ peptide measured in immunized D(H)LMP2A and V(H)LMP2A mice was similar to that detected in immunized control mice. Further, clinical expression of experimental autoimmune encephalitis in both LMP2A strains was identical to that of wild-type mice. In conclusion, mice with skewed B cell differentiation driven by LMP2A expression in BCR-negative B cells do not show changes in the development of a T cell mediated disease model of autoimmunity, suggesting that compensatory mechanisms support the generation of T cell responses.

Whole-exome sequencing in splenic marginal zone lymphoma reveals mutations in genes involved in marginal zone differentiation

Splenic marginal zone lymphoma (SMZL) is a B-cell neoplasm whose molecular pathogenesis remains fundamentally unexplained, requiring more precise diagnostic markers. Previous molecular studies have revealed 7q loss and mutations of nuclear factor κB (NF-κB), B-cell receptor (BCR) and Notch signalling genes. We performed whole-exome sequencing in a series of SMZL cases. Results confirmed that SMZL is an entity distinct from other low-grade B-cell lymphomas, and identified mutations in multiple genes involved in marginal zone development, and others involved in NF-κB, BCR, chromatin remodelling and the cytoskeleton.

Molecular Lesions Of Signalling Pathway Genes In Indolent B-Cell Lymphoproliferations Mimicking Splenic Marginal Zone Lymphoma

BackgroundAlmost 60% cases of splenic marginal zone lymphoma (SMZL) cases harbor molecular lesions affecting signalling pathways involved in normal marginal zone (MZ) differentiation, including the NOTCH pathway, the NF-kB pathway, the toll-like receptor (TLR) pathway and the B-cell receptor (BCR) pathway. However, little is known with regard to these lesions in other indolent B-cell lymphoproliferative disorders mimicking SMZL. MethodsCandidate gene mutations (NOTCH2, NOTCH1, BIRC3, TNFAIP3, TRAF3, IKBKB, MYD88, CD79A, CD79B, CARD11) were investigated in 60 indolent B-cell lymphoproliferative disorders, including nodal marginal zone lymphoma (NMZL=33), monoclonal B-cell lymphocytosis showing a MZL-like phenotype (MZL-like MBL=16), and variant hairy cell leukemia (vHCL=11). In all cases, tumor representation was >50% in order to allow the detection of clonal lesions. All cases lacked the BRAF V600E mutation as assessed by AS-PCR. ResultsOverall, the genetics of NMZL and MZL-like MBL was consistent with that of SMZL, suggesting the involvement of a common oncogenic pathway in these disorders. Among NMZL, 51% (17/33) of cases were characterized by mutually exclusive genetic lesions affecting MZ differentiation genes, including NOTCH2 stabilizing mutations in 24% (8/33) of cases, TNFAIP3 disrupting mutations in 12% (4/33), MYD88 activating mutations in 12% (4/33), and NOTCH1, TRAF3 and BIRC3 mutations in 3% (1/33) of cases each. Among MZL-like MBL, 37% (6/16) of cases harbored mutually exclusive lesions of MZ genes, including MYD88 mutations in 25% (4/16) of cases, NOTCH2 mutations in 12% (2/16), and TNFAIP3 and CD79B mutations in 6% (1/16) of cases each. On the contrary, all cases (n=11) of vHCL lacked mutations of NOTCH, NF-kB, TLR or BCR genes, suggesting that none of these signaling pathways plays a relevant role in this disease. Among NMZL, MYD88 mutations were enriched among cases harboring an IgM type monoclonal component (3/4, 75% vs 1/18, 5%, p=.010) and showing cytological clues of plasma cell differentiation of lymph node tumor cells (3/6, 50% vs 0/9; p=.040). NOTCH2 mutations did not correlate with any pathologic feature (i.e. lymph node pattern of infiltration, presence of monocytoid B cells, immunoglobulin gene usage or mutation status). Among MZL-like MBL, neither MYD88 mutations nor NOTCH2 mutations correlated with specific clinico-pathologic features (i.e. presence of an IgM type monoclonal component, intrasinusoidal bone marrow infiltration, plasma cell differentiation). ConclusionsThese data suggest that: i) SMZL, NMZL and MZL-like MBL share a similar genotype and are all promoted by the same molecular deregulation of MZ differentiation genes; and ii) vHCL stands as a genetically different entity among indolent B-cell lymphoproliferations. These data might help to refine the differential diagnosis of indolent B-cell lymphoproliferations mimicking SMZL. Disclosures:No relevant conflicts of interest to declare.

Prognostic implications of histologic grade and intensity of Bcl-2 expression in follicular lymphomas undergoing rituximab-containing therapy

This study aimed to determine the correlations of 7 histopathologic prognostic indicators of follicular lymphoma (follicular lymphoma grade, CD10 expression, Bcl-2 expression, IGH/BCL2 fusion, diffuse area, fibrosis, and marginal zone differentiation) with progression-free survival, overall survival, and follicular lymphoma histologic grade in 255 follicular lymphoma patients who were treated with rituximab-containing therapy. The complete response, overall response, 6-year progression-free survival, and 6-year overall survival rates were 83%, 96%, 56%, and 97%, respectively. Patients with follicular lymphoma grades 3a and 3b showed 100% 6-year and 10-year overall survival, and progression-free survival did not significantly differ between patients with follicular lymphoma grade 3 and those with follicular lymphoma grades 1 and 2. The absence or presence of Bcl-2 expression and intensity of Bcl-2 expression were not significant prognostic indicators of progression-free survival and overall survival. Likewise, the presence of IGH/BCL2 fusion, diffuse area, fibrosis, and marginal zone differentiation were not significantly correlated with progression-free survival and overall survival. Follicular lymphoma grade 3 was correlated with nodal disease and negative or lower intensity of Bcl-2 expression, but not with age, stage, or IGH/BCL2 status. In the prerituximab era, grade 3 disease was reported to be associated with a poor prognosis; however, the opposite was true for patients treated with rituximab-containing therapy, regardless of their age or disease stage. Bcl-2 expression and marginal zone differentiation were not prognostic indicators in follicular lymphoma patients treated with rituximab-containing therapy.

Epidermotropic marginal zone lymphoma simulating mycosis fungoides†

Cutaneous lymphomas can usually be distinguished by architectural features, where most atypical lichenoid infiltrates implicate cutaneous T-cell lymphoma (CTCL). We report a case of an 80-year-old man who presented with asymptomatic golden brown patches and diffuse pink papules on his trunk, buttocks and hips. Biopsies revealed a lichenoid infiltrate and areas of epidermotropism. Although the overall architectural pattern was compatible with mycosis fungoides, the lymphocytes had a more monocytoid and plasmacytoid appearance, and there were interspersed mature plasma cells. Immunohistological studies revealed that the pleomorphic lymphocytes were predominantly B cells (CD20\+ and CD79a\+) with a subpopulation of smaller bland T cells (CD3\+). Moreover, the B-cell immunophenotype was compatible with marginal zone differentiation (bcl-2+, bcl-6-, CD10- and CD5-) and showed a lambda light chain restriction, confirming monoclonality. These findings were diagnostic for cutaneous marginal zone B-cell lymphoma (MZL) with epidermotropism an entity which has only been reported twice in the literature, once in the setting of primary cutaneous disease, and once as cutaneous involvement of systemic disease. This case illustrates a rare pattern of cutaneous MZL, and underscores the importance of immunophenotypic characterization of cutaneous lymphomas in order to prevent the misdiagnosis of a CTCL.

IRTA1 is selectively expressed in nodal and extranodal marginal zone lymphomas

The aim of this study was to search for a molecule selectively expressed by marginal zone (MZ) lymphomas (MZLs), whose diagnosis is currently based on morphological criteria and negativity for markers detectable in other B-cell lymphomas. Two thousand one hundred and four peripheral lymphomas of various types were immunostained with a monoclonal antibody against immunoglobulin superfamily receptor translocation-associated 1 (IRTA1), which recognizes the equivalents of MZ in human lymphoid tissues other than spleen. IRTA1 expression was restricted to extranodal (93%) and nodal MZLs (73%) and to lymphomas with MZ differentiation. Extranodal MZL cells with the strongest IRTA1 expression were usually located adjacent to epithelia, mimicking the IRTA1 expression pattern of normal and acquired mucosa-associated lymphoid tissue (MALT). The cytological features, growth pattern and IRTA1 positivity in nodal MZLs suggest they may derive from IRTA1(+) perifollicular B cells or monocytoid B cells detectable in reactive lymph nodes. Double immunostaining for IRTA1/bcl-6 tracked the colonization of B-cell follicles by MZL cells, and showed modulation of their phenotype (e.g. acquisition of bcl-6) during recirculation through germinal centres. MZL cells differentiating into plasma cells usually lost IRTA1. These results further expand our knowledge of the biology of MZLs, and highlight IRTA1 as the first positive marker for MZLs, enabling more accurate diagnosis of these neoplasms.

An unusual finding at screening colonoscopy: polypoid follicular lymphoma with marginal zone differentiation

An unusual finding at screening colonoscopy: polypoid follicular lymphoma with marginal zone differentiation

A duodenal follicular lymphoma associated with the lesion mimicking MALT lymphoma in terminal ileum and Bauhin valve

This is a case report of a 66-year-old woman who consulted us with a 1-week history of postprandial epigastric discomfort and dyspepsia. Upper and lower gastrointestinal endoscopy and double-balloon enteroscopy revealed lesions in three parts: a swelling with a shallow depression in the ampulla of Vater, flat and rough nodules in the jejunum, and a mixture of lymphoid polyposis and rough surface of follicular lymphoma of the terminal ileum and Bauhin valve. The histological, immunophenotypic, and molecular findings of the duodenal lesion confirmed the diagnosis of follicular lymphoma. We initially diagnosed the ileal lesion as MALT lymphoma immunohistochemically. However, Southern blot hybridization analysis for immunoglobulin heavy chain gene rearrangement showed identical monoclonal bands in both the duodenal and ileal lesions. The molecular cytogenetic studies were also positive for the 14;18 translocation in both lesions. Therefore, the true diagnosis of this ileal lesion should be a follicular lymphoma with marginal zone differentiation. Primary follicular lymphomas of gastrointestinal tract were suggested to have intermediate features between nodal follicular lymphoma and MALT lymphoma. This case is an important clue to prove the similarity of follicular lymphoma of gastrointestinal tract to MALT lymphoma and will be crucial in considering the therapeutic strategy.