Transcription Factors and Checkpoint Inhibitor Expression with Age: Markers of Immunosenescence?

Background: Aging is characterized by a progressive decline in immune surveillance that favors tumor development in older patients. One mechanism used by pre-malignant and/or malignant cells to escape immune surveillance is the upregulation of inhibitory immune checkpoint molecules, such as PD-1 and PD-L1. Another process associated with aging is genetic or epigenetic modifications of tumor suppressor genes (TSGs). We have reported a correlation between 6q deletion and progression into a T cell lymphoproliferative disease, identifying the BACH2 gene as a candidate TSG(1). We thus examined - on a molecular level - the expression of specific transcription factors (BACH2 and PRDM1) and checkpoint molecules (PD-1 and PD-L1) in the major lymphocytes subsets for their potential as predictive markers of immunosenescence. Methods: Lymphocyte subpopulations (CD3+/CD4+; CD3+/CD8+ and CD19+) were isolated for subsequent molecular analyses using the MACS technology (Miltenyi), with the purity of each lymphocyte subpopulation between 95%-99%. PD-1 (PDCD1), PD-L1 (CD274), IL4, IFNG, BACH2 and PRDM1 (Blimp1) mRNA transcripts were quantified in the purified subpopulations using qRT-PCR. BACH2 and PRDM1 protein expression were examined by Western blotting. Peripheral blood mononuclear cells were treated by Etoposide (VP16) to induce intracellular oxidative stress. Apoptotic function was assessed by flow cytometry (Annexin-V and Propidium Iodide). Results: Peripheral blood samples were obtained from 40 healthy donors (HDs) between the ages of 20 to 90 yrs, subdivided into <50 yrs (n=21; median: 36yo) and ³50 yrs (n=19; median: 61yo). And 40 blood samples from untreated patients with chronic lymphocytic leukemia (CLL; median: 67yo) were comparatively analyzed. BACH2-deficient mice have been shown to have increased numbers of IL4-producing CD4+ T cells after activation(2), with BACH2 known to repress PRDM1 expression in B andT cells(2,3). We, therefore, examined BACH2 gene expression in the HD groups finding it was significantly downregulated in CD4+, CD8+ T cells and CD19+ B cells from the older HD group (p=0.001; 0.004 and 0.03, respectively). BACH2 expression was further reduced in CD4+, CD8+ T cells and CD19+ B cells from CLL patients compared to HD of similar age (p=0.001; <0.001 and 0.004). In contrast, PRDM1 was significantly upregulated in CD4+ and CD8+ T cells (p=0.003; 0.001) from CLL patients but not in their leukemic B cells. As expected, BACH2 expression was inversely correlated with PRDM1 in CD4+, CD8+ T cells and CD19+ B cells (r=0.57; 0.71 and 0.62, respectively). Western blot analysis demonstrated that BACH2 and Blimp1 (PRDM1) protein expressions in the T and B cell subpopulations were significantly correlated with transcript expression. We also observed that BACH2 downregulation is correlated with increased IL-4 gene expression (r=0.61) but not IFNγ in CD4+ T cells. These observations suggest that BACH2 downregulation in CD4+ T cells could enhance expressed Th2-related genes, such as IL-4 and PRDM1. PD-1 gene expression was significantly upregulated in CD4+, CD8+ T cells (p=0.015 and 0.02) in the older HD group. PD-1 was also significantly upregulated in the same T cells subpopulations isolated from untreated CLL patients (p=0.001 and 0.002) when compared to a similar HD age group. High PD-L1 gene expression was correlated with increased age in HD B cells (p=0.04) with a further increase detected in leukemic B cells compared with the older HD group (p=0.001). We also observed an inverse correlation between BACH2 and PD-1 in CD4+, CD8+ T cells (r=0.59 and 0.68); and between BACH2 and PD-L1 in CD19+ B cells (r=0.66). We further examined VP16-induced apoptosis in isolated lymphocytes subpopulations. As recently reported in BACH2-deficient mice(4), our observation showed that BACH2 downregulation was strongly correlated with resistance to apoptosis in CD4+, CD8+ T cells and leukemic B cells (r=0.61; 0.75 and 0.69, respectively). Conclusion: These data suggest that BACH2, PRDM1, PD-1 and PD-L1 gene expression is correlated with aging and age-related immunosuppression. These effects are even more pronounced in leukemic cells from CLL patients. 1. C. Sibille, et al.,(personal data) ; 2. S. Tsukumo, et al. Proc. Natl Acad Sci USA. 2013 ; 3. H. Tanaka et al. J Biol Chem. 2016 ; 4. Muto, A., et al. J Biol Chem. 2002; Disclosures No relevant conflicts of interest to declare.

High BACH2 gene Expression Is an Indicator of Poor Prognosis in Adult Diffuse Large B-Cell Lymphoma Treated with R-CHOP

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL) accounting for 30-40% of cases. It is a heterogeneous group of diseases with numerous morphological, molecular and immunophenotypical subgroups including germinal centre-like (GCB-DLBCL) and activated B-cell-like (ABC-DLBCL) subtypes. Despite advances in treatment approaches, including the introduction of Rituximab, ~40% of patients die of refractory or relapsed disease. The identification of prognostic biomarkers for R-CHOP treated DLBCL may allow differentiation of patients for whom alternate treatment approaches may be more appropriate, as well as identifying potential new targetable pathways. BACH2, a transcriptional regulator that plays a role in B-cell maturation, has been investigated as a prognostic marker in DLBCL but the results are conflicting with one study reporting that high expression correlated with good patient outcome and another reporting an association between high expression and poor outcome. Differences in treatment regimen may underlie these conflicting results as only the most recent study consisted of patients treated with a Rituximab-containing protocol. It is therefore important to investigate the prognostic significance of BACH2 in an independent cohort of DLBCL cases treated in the Rituximab-era. Furthermore, both studies used immunohistochemistry to determine BACH2 protein expression and only scored levels of cytoplasmic staining despite showing both cytoplasmic and/or nuclear expression. Evidence of the involvement of BACH2 in DLBCL lymphomagenesis has been further supported by the recent observation of BACH2 genomic deletions at high incidence (35%) in ABC-DLBCL compared to GCB-DLBCL (20%). We set-out to evaluate the significance of BACH2 expression, localisation and copy number aberrations in a cohort of adults diagnosed with DLBCL and treated with R-CHOP. 71 cases of DLBCL with sufficient material and associated clinical information were included in this study. RNA and DNA was extracted from FFPE diagnostic samples using the Allprep DNA/RNA extraction kit (Qiagen, UK) and used to evaluate BACH2 mRNA expression levels (n=71) and copy number abnormalities (n=58) by TaqMan® qRT-PCR. The patients ranged in age from 32 to 91 years (median age 65 years) with a median follow up time of 48.7 months (range 0.7-107.8). Ann Arbor stage was available for 56 patients (32 (57%) stage I-II and 24 (43%) stage II-IV). Immunophenotyping by the Hans algorithm identified 44 (65%) as GCB-DLBCL and 24 (35%) as ABC-DLBCL. Age (≥60 years) and stage (III-IV) were prognostic for poor overall survival (OS) (p=0.001 and p=0.06) and age (≥60 years), stage (III-IV) and immunophenotype (ABC-DLBCL) predicted poor progression free survival (PFS) (p=0.003, p=0.011 and p=0.043, respectively). In our cohort, high expression of BACH2 mRNA (threshold defined by ROC analysis), is an indicator of poor OS (p=0.012) and PFS (p=0.002). Within the GCB-DLBCL group, high expression of BACH2 remained an indicator of poor prognosis (OS: p=0.003, PFS: p<0.001), but this lost significance in the ABC-DLBCL group (OS and PFS: p=0.0896 and p=0.388). BACH2 was expressed at a significantly higher level in GC-like compared to ABC-DLBCL (p=0.034). BACH2 CNA data was available for 57 (80%) of patients and 11/57 (19%) demonstrated loss, 11/57 (19%) gain and 35/57 (61%) normal BACH2 status. There was no significant difference in BACH2 CNA status between GCB- and ABC-DLBCL subgroups (p >0.05). Within the groups with BACH2 gain and loss, there were 3 and 5 deaths, respectively, at the time of last follow up. There was no significant difference in OS or PFS between patients with gain, loss or normal BACH2 CNA. In conclusion, we have demonstrated that high expression of BACH2 mRNA is prognostic and is associated with shorter survival times in adult DLBCL treated with R-CHOP. Although there was no correlation between genomic loss of BACH2 locus and subtype as reported in the literature, we have identified an association between high BACH2 gene expression and GCB-DLBCL (versus ABC-DLBCL) subtype. Further work to correlate protein expression levels and BACH2 localisation with gene expression and copy number data is ongoing. Disclosures No relevant conflicts of interest to declare.

Identification of a Novel IGHCδ-BACH2 Fusion Transcript In B-Cell Lymphoma/Leukemia and the Expression Analysis of BACH2 Related with Other B-Cell Differentiation-Associated Genes In B-Cell Malignancies

Abstract 4450 In B-cell malignancies, the genes implicated in B-cell differentiation, germinal center formation, apoptosis, and cell cycle regulation are juxtaposed to immunoglobulin loci through chromosomal translocations. In the present study, we have identified BTB and CNC homology 2 (BACH2) as a novel translocation partner gene of the immunoglobulin heavy chain (IGH) locus, resulting in chimeric Cδ-BACH2 gene in a patient with MYC-IGH-positive highly aggressive B-cell lymphoma/leukemia carrying der(14)t(8;14) and del(6)(q15). A 71-year-old male was diagnosed as having highly aggressive B-cell lymphoma/leukemia. SKY analysis revealed the representative karyotype of tumor cells as 45,X,-Y,der(3)t(3;X)(p21.2;q24),del(6)(q15),der(14)t(8;14)(q24;q32),del(16)(q22),der(20) t(3;20)(q21;p13). FISH and long-distance PCR analyses identified a fusion of MYC with Cγ. FISH analysis also detected a small IGH signal on del(6), and a VH on del(6)(q15). Genome copy number analysis using SNP-array detected an approximately 60Mb deletion in 6q15–25 region, and its centromeric breakpoint within BACH2 gene. The cDNA bubble PCR using BACH2 primers detected multiple PCR products, and sequence analysis has revealed that one of the products contained a sequence of the first exon of IGHCδ fused to 5’ untranslated region of BACH2 exon 2. Genomic fusion point of Cδ-BACH2, was within intron 1 of Cδ and intron 1 of BACH2. Cδ-BACH2 fusion transcript consisted of exon 1 of Cδ and exons 2 to 9 of BACH2, indicating that whole coding region of BACH2 was fused to Cδ. This suggested that Cδ-BACH2 fusion cause constitutive activation of BACH2. We next analyzed the expression levels of BACH2, MYC, PRDM1, and IRF4 genes in the patient using real-time PCR and compared them with those of several hematologic malignancy cell lines, including 14 non-Hodgkin's lymphoma (NHL), 10 multiple myeloma (MM), and 3 B-lineage acute lymphoblastic leukemia, and 3 EB-virus transformed B-cell lines from normal healthy volunteers. IGH-MYC-positive MM cell lines showed increased levels of MYC expression compared with the other cell lines. The MYC expression level in our patient was lower than those in IGH-MYC-positive MM cell lines; however, it was similar to 5 IGH-MYC-positive NHL cell lines [3 Burkitt lymphoma (BL) and 2 diffuse large B-cell lymphoma (DLBCL)]. On the other hand, BACH2 expression levels were higher in NHL cell lines, especially in BL cell lines, but not correlated with the IGH-MYC translocation status. Our IGH-BACH2-positive case also showed a higher level of BACH2 expression. The expression levels of IRF4 and PRDM1 were higher in MM cell lines than in other cell lines. PRDM1 is activated by MYC through IRF4, and the MYC is negatively regulated by activated PRDM1. In MM cell lines with IGH-MYC translocation, MYC was highly expressed regardless of high expression of PRDM1, indicating that MYC activated by IGH translocation could not be inhibited by PRDM1. Unexpectedly, the expression levels of PRDM1 and IRF4 were very low in NHL including our patient, suggesting that the regulation of MYC in NHL is different from that in MM. BACH2 is a B cell-specific transcription repressor, and is specifically required for class switch recombination, somatic hypermutation, and germinal center formation. One of the target genes of BACH2 is PRDM1 at 6q21-q22.1 that is required for plasma cell differentiation. In this patient, deletion of 6q15–25 was found, indicating loss of one PRDM1 allele. It was reported that PRDM1 is inactivated by chromosomal alterations in 24% of activated B cell–like DLBCL, suggesting that PRDM1 acts as a tumor suppressor gene, and its inactivation may contribute to lymphomagenesis by blocking post–GC differentiation. The combination of BACH2 and MYC in double IGH translocations is unique and consistent with previous reports demonstrating that each partner gene found in double or multiple IGH translocations is exclusively specific to certain types of B-cell lymphoma. These results suggest that the promoter of IGHCδ and/or Eμ enhancer of IGH activate the expression of BACH2, and that BACH2 may act as oncogene in some cases with B-cell lymphoma. Although the IGH-BACH2 translocation is rare in NHL, our data suggest that the BACH2 plays a critical role in B-cell lymphomagenesis through not only IGH translocation but also activation by some other mechanisms. Disclosures: No relevant conflicts of interest to declare.