In-depth characterization of the tumor microenvironment in central nervous system lymphoma reveals implications for immune-checkpoint therapy.

Primary central nervous system lymphoma (PCNSL) is a rare type of non-Hodgkin lymphoma with an aggressive clinical course. To investigate the potential of immune-checkpoint therapy, we retrospectively studied the tumor microenvironment (TME) using high-plex immunohistochemistry in 22 PCNSL and compared to 7 secondary CNS lymphomas (SCNSL) and 7 "other" CNSL lymphomas with the presence of the Epstein-Barr virus and/or compromised immunity. The TME in PCNSL was predominantly composed of CD8+ cytotoxic T cells and CD163+ phagocytes. Despite molecular differences between PCNSL and SCNSL, the cellular composition and the functional spectrum of cytotoxic T cells were similar. But cytotoxic T cell activation was significantly influenced by pre-biopsy corticosteroids intake, tumor expression of PD-L1 and the presence of EBV. The presence of low numbers of CD8+ T cells and geographic-type necrosis each predicted inferior outcome in PCNSL. Both M1-like (CD68 + CD163low) and M2-like (CD68 + CD163high) phagocytes were identified, and an increased ratio of M1-like/M2-like phagocytes was associated with a better survival. PD-L1 was expressed in lymphoma cells in 28% of cases, while PD1 was expressed in only 0.4% of all CD8+ T cells. TIM-3, a marker for T cell exhaustion, was significantly more expressed in CD8posPD-1pos T cells compared to CD8posPD-1neg T cells, and a similar increased expression was observed in M2-like pro-tumoral phagocytes. In conclusion, the clinical impact of TME composition supports the use of immune-checkpoint therapies in PCNSL. Based on observed differences in immune-checkpoint expression, combinations that boost cytotoxic T cell activation (by blocking TIM-3 or TGFBR1) prior to the administration of PD-L1 inhibition could be of interest.

Anaplastic lymphoma kinase-positive anaplastic large cell lymphoma with the variant RNF213-, ATIC- and TPM3-ALK fusions is characterized by copy number gain of the rearranged ALK gene.

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma is characterized by 2p23/ALK aberrations, including the classic t(2;5)(p23;q35)/NPM1-ALK rearrangement present in ~80% of cases and several variant t(2p23/ALK) occurring in the remaining cases. The ALK fusion partners play a key role in the constitutive activation of the chimeric protein and its subcellular localization. Using various molecular technologies, we have characterized ALK fusions in eight recently diagnosed anaplastic large cell lymphoma cases with cytoplasmic-only ALK expression. The identified partner genes included EEF1G (one case), RNF213/ALO17 (one case), ATIC (four cases) and TPM3 (two cases). Notably, all cases showed copy number gain of the rearranged ALK gene, which is never observed in NPM1-ALK-positive lymphomas. We hypothesized that this could be due to lower expression levels and/or lower oncogenic potential of the variant ALK fusions. Indeed, all partner genes, except EEF1G, showed lower expression in normal and malignant T cells, in comparison with NPM1 In addition, we investigated the transformation potential of endogenous Npm1-Alk and Atic-Alk fusions generated by clustered regularly interspaced short palindromic repeats/Cas9 genome editing in Ba/F3 cells. We found that Npm1-Alk has a stronger transformation potential than Atic-Alk, and we observed a subclonal gain of Atic-Alk after a longer culture period, which was not observed for Npm1-Alk Taken together, our data illustrate that lymphomas driven by the variant ATIC-ALK fusion (and likely by RNF213-ALK and TPM3-ALK), but not the classic NPM1-ALK, require an increased dosage of the ALK hybrid gene to compensate for the relatively low and insufficient expression and signaling properties of the chimeric gene.

Genomic alterations of the JAK2 and PDL loci occur in a broad spectrum of lymphoid malignancies.

The recurrent 9p24.1 aberrations in lymphoid malignancies potentially involving four cancer-related and druggable genes (JAK2, CD274/PDL1, PDCD1LG2/PDL2, and KDM4C/JMJD2Cl) are incompletely characterized. To gain more insight into the anatomy of these abnormalities, at first we studied 9p24.1 alterations in 18 leukemia/lymphoma cases using cytogenetic and molecular techniques. The aberrations comprised structural (nine cases) and numerical (nine cases) alterations. The former lesions were heterogeneous but shared a common breakpoint region of 200 kb downstream of JAK2. The rearrangements predominantly targeted the PDL locus. We have identified five potential partner genes of PDL1/2: PHACTR4 (1p34), N4BP2 (4p14), EEF1A1 (6q13), JAK2 (9p24.1), and IGL (22q11). Interestingly, the cryptic JAK2-PDL1 rearrangement was generated by a microdeletion spanning the 3'JAK2-5'PDL1 region. JAK2 was additionally involved in a cytogenetically cryptic IGH-mediated t(9;14)(p24.1;q32) found in two patients. This rare but likely underestimated rearrangement highlights the essential role of JAK2 in B-cell neoplasms. Cases with amplification of 9p24.1 were diagnosed as primary mediastinal B-cell lymphoma (five cases) and T-cell lymphoma (four cases). The smallest amplified 9p24.1 region was restricted to the JAK2-PDL1/2-RANBP6 interval. In the next step, we screened 200 cases of classical Hodgkin lymphoma by interphase FISH and identified PDL1/2 rearrangement (CIITA- and IGH-negative) in four cases (2%), what is a novel finding. Forty (25%) cases revealed high level amplification of 9p24.1, including four cases with a selective amplification of PDL1/2. Altogether, the majority of 9p24.1 rearrangements occurring in lymphoid malignancies seem to target the programmed death-1 ligands, what potentiates the therapeutic activity of PD-1 blockade in these tumors. © 2016 Wiley Periodicals, Inc.

BMI1, the polycomb-group gene, is recurrently targeted by genomic rearrangements in progressive B-cell leukemia/lymphoma.

BMI1, a Polycomb-group gene located at 10p12.2, is implicated in the pathogenesis of a variety of tumors. However, the genetic molecular mechanisms underlying its aberrant expression in cancer cells remain largely unknown. In this study, we show that BMI1 is recurrently targeted by chromosomal aberrations in B-cell leukemia/lymphoma. We identified a novel t(10;14)(p12;q32)/IGH-BMI1 rearrangement and its IGL variant in six cases of chronic lymphocytic leukemia (CLL) and found that these aberrations were consistently acquired at time of disease progression and high grade transformation of leukemia (Richter syndrome). The IG-BMI1 translocations were not associated with any particular molecular subtype of CLL and the leukemias were negative for common mutations of NOTCH1 and TP53, known to increase a risk of progression and transformation in CLL. In addition, using FISH and SNP array analysis, we identified a wide range of BMI1-involving 10p12 lesions in 17 cases of mantle cell lymphoma (MCL). These aberrations included various balanced and unbalanced structural abnormalities and very frequently but not exclusively, were associated with gain of the BMI1 locus and loss of the 10p terminal sequences. These findings point to genomic instability at the 10p region in MCL which likely promotes rearrangements and deregulation of BMI1. Our findings are in line with previously published observations correlating overexpression of BMI1 with tumor progression and chemoresistance. In summary, our study provides new insights into genetic molecular mechanisms underlying aberrant expression of BMI1 in lymphoma and documents its contribution in the pathogenesis of Richter syndrome and MCL.

Biallelic inactivation of NF1 in a sporadic plexiform neurofibroma.

Plexiform neurofibromas are a major cause of morbidity in individuals with neurofibromatosis type 1 (NF1). Sporadically, these tumors appear as an isolated feature without other signs of NF1. A role for the NF1 gene in solitary plexiform neurofibromas has never been described. In this study, we report a 13-year-old boy who was diagnosed with a plexiform neurofibroma, without other NF1 diagnostic criteria. The tumor was partially resected and analyzed using different techniques: karyotyping, fluorescence in situ hybridization (FISH), and microarray comparative genomic hybridization (aCGH). Tumor Schwann cell culture and subsequent karyotyping showed a rearrangement involving chromosomes 1 and 17, namely an insertion of chromosomal bands 1p36-35 at 17q11.2. FISH demonstrated that the insertion interrupted the NF1 gene. In addition, a deletion was detected affecting the other NF1 allele. Whole-genome aCGH analysis of the resected tumor confirmed the presence of an 8.28 Mb deletion including the NF1 gene locus in ∼15-20% of tumor cells. We conclude that biallelic NF1 inactivation was at the origin of the isolated plexiform neurofibroma in this patient. The insertion is most likely the "first hit" and the large deletion the "second hit."

Recurrent breakpoints in 14q32.13/TCL1A region in mature B-cell neoplasms with villous lymphocytes.

The genetic background of mature B-cell neoplasms with villous lymphocytes is poorly understood. We identified a novel breakpoint region at 14q32.13 that was rearranged together with IGH/14q32.33 in four cases of BRAF/V600E-negative leukemia/lymphoma with villous lymphocytes carrying either t(14;14)(q32.13;q32.33) (three patients) or del(14)(q32.13q32.33) (one patient). The 14q32.13 breakpoints were mapped by fluorescence in situ hybridization (FISH) in the region harboring the TCL1A/TCL1B/TCL6 genes, known to be affected by TCRA/D-mediated t(14;14)(q11;q32)/inv(14)(q11q32) occurring in T-cell leukemia/lymphoma. To identify the target of t(14;14)(q32.13; q32.33) and del(14)(q32.13q32.33), quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 25 candidate genes located centromerically and telomerically to the 14q32.13 breakpoint was performed. Any of the analyzed genes was commonly overexpressed in the presented cases. Of note, up-regulated transcription of TCL1A was observed in two cases. In summary, we provide evidence that IGH-mediated chromosomal aberrations affecting the 14q32.13/TCL1A-TCL6 region are recurrent in mature B-cell neoplasms with villous lymphocytes. Despite extensive qRT-PCR studies, molecular consequences of these novel aberrations remain elusive.