Round-robin testing for LMO2 and MYC as immunohistochemical markers to screen MYC rearrangements in aggressive large B-cell lymphoma.

Aggressive large B-cell lymphomas (aLBCL) include a heterogeneous group of lymphomas with diverse biological features. One of the approaches to the diagnosis of aLBCL is based on the identification of MYC rearrangements (MYC-R), in addition to BCL2 and BCL6 rearrangements by genetic techniques, mainly fluorescent in situ hybridization (FISH). Because of the low incidence of MYC-R, the identification of useful immunohistochemistry markers to select cases for MYC FISH testing may be useful in daily practice. In a previous work, we identified a strong association between the profile CD10 positive/LMO2 negative expression and the presence of MYC-R in aLBCL and obtained good intralaboratory reproducibility. In this study, we wanted to evaluate external reproducibility. To evaluate whether LMO2 can be a reproducible marker between observers 50 aLBCL cases were circulated among 7 hematopathologists of 5 hospitals. Fleiss' kappa index for LMO2 and MYC were 0.87 and 0.70, respectively, indicating high agreement between observers. In addition, during 2021-2022, the enrolled centers included LMO2 in their diagnostic panels to evaluate prospectively the utility of the marker, and 213 cases were analyzed. Comparing LMO2 with MYC, the group of CD10 positive cases showed higher specificity (86% vs 79%), positive predictive value (66% vs 58%), likelihood positive value (5.47 vs 3.78), and accuracy (83% vs 79%), whereas the negative predictive values remained similar (90% vs 91%). These findings place LMO2 as a useful and reproducible marker to screen MYC-R in aLBCL.

MYC activation impairs cell-intrinsic IFNγ signaling and confers resistance to anti-PD1/PD-L1 therapy in lung cancer.

Elucidating the adaptive mechanisms that prevent host immune response in cancer will help predict efficacy of anti-programmed death-1 (PD1)/L1 therapies. Here, we study the cell-intrinsic response of lung cancer (LC) to interferon-γ (IFNγ), a cytokine that promotes immunoresponse and modulates programmed death-ligand 1 (PD-L1) levels. We report complete refractoriness to IFNγ in a subset of LCs as a result of JAK2 or IFNGR1 inactivation. A submaximal response affects another subset that shows constitutive low levels of IFNγ-stimulated genes (IγSGs) coupled with decreased H3K27ac (histone 3 acetylation at lysine 27) deposition and promoter hypermethylation and reduced IFN regulatory factor 1 (IRF1) recruitment to the DNA on IFNγ stimulation. Most of these are neuroendocrine small cell LCs (SCLCs) with oncogenic MYC/MYCL1/MYCN. The oncogenic activation of MYC in SCLC cells downregulates JAK2 and impairs IγSGs stimulation by IFNγ. MYC amplification tends to associate with a worse response to anti-PD1/L1 therapies. Hence alterations affecting the JAK/STAT pathway and MYC activation prevent stimulation by IFNγ and may predict anti-PD1/L1 efficacy in LC.

Glioblastoma: Relationship between Metabolism and Immunosuppressive Microenvironment.

Glioblastoma (GBM) is the most aggressive brain tumor in adults and is characterized by an immunosuppressive microenvironment. Different factors shaping this tumor microenvironment (TME) regulate tumor initiation, progression, and treatment response. Genetic alterations and metabolism pathways are two main elements that influence tumor immune cells and TME. In this manuscript, we review how both factors can contribute to an immunosuppressive state and overview the strategies being tested.

Correlation between PD-L1 expression and MET gene amplification in patients with advanced non-small cell lung cancer and no other actionable oncogenic driver.

Non-small cell lung cancers (NSCLC) are the most common type of lung cancer and can be classified according to the presence of mutually exclusive oncogenic drivers. The majority of NSCLC patients present a non-actionable oncogenic driver, and treatment resistance through the amplification of the MET proto-oncogene (MET) or the expression of programmed cell death protein 1 ligand (PD-L1) is common. Herein, we investigated the relation between MET gene amplification and PD-L1 expression in patients with advanced NSCLC and no other actionable oncogenic driver (i.e., EGFR, ALK, ROS1). Our retrospective observational study analyzed data from 48 patients (78% men, median age 66 years) admitted to the Germans Trias i Pujol Hospital, Spain, between July 2015 and February 2019. Patients presenting MET amplification showed a higher proportion of PD-L1 expression (93% vs. 39%; p < 0.001) and overexpression (64% vs. 27%; p = 0.020) than those with non-amplified MET. PD-L1 expression was not significantly different when analyzed by sex (p = 0.624), smoking history (p = 0.429), and Eastern Cooperative Oncology Group Performance Status (p = 0.597) Overall survival rates were not significantly affected by MET amplification (high and intermediate amplification vs low amplification and non-amplificated) (p = 0.252) nor PD-L1 expression (> vs =< 50%) (p = 0.893). In conclusion, a positive correlation was found between MET gene amplification and PD-L1 expression and highly expressed (above 50%) in patients with NSCLC and no other actionable oncogenic driver. It could be translated as new guided-treatment oportunities for these patients.

Glioblastoma TCGA Mesenchymal and IGS 23 Tumors are Identifiable by IHC and have an Immune-phenotype Indicating a Potential Benefit from Immunotherapy.

PURPOSE: Molecular subtype classifications in glioblastoma may detect therapy sensitivities. IHC would potentially allow the identification of molecular subtypes in routine clinical practice. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded tumor samples of 124 uniformly treated, newly diagnosed patients with glioblastoma were submitted to RNA sequencing, IHC, and immune-phenotyping to identify differences in molecular subtypes associated with treatment sensitivities. RESULTS: We detected high molecular and IHC overlapping of the The Cancer Genome Atlas (TCGA) mesenchymal subtype with instrinsic glioma subtypes (IGS) cluster 23 and of the TCGA classical subtype with IGS cluster 18. IHC patterns, gene fusion profiles, and immune-phenotypes varied across subtypes. IHC revealed that the TCGA classical subtype was identified by high expression of EGFR and low expression of PTEN, while the mesenchymal subtype was identified by low expression of SOX2 and high expression of two antibodies, SHC1 and TCIRG1, selected on the basis of RNA differential transcriptomic expression. The proneural subtype was identified by frequent positive IDH1 expression and high Olig2 and Ki67 expression. Immune-phenotyping showed that mesenchymal and IGS 23 tumors exhibited a higher positive effector cell score, a higher negative suppressor cell score, and lower levels of immune checkpoint molecules. The cell-type deconvolution analysis revealed that these tumors are highly enriched in M2 macrophages, resting memory CD4+ T cells, and activated dendritic cells, indicating that they may be ideal candidates for immunotherapy, especially with anti-M2 and/or dendritic cell vaccination. CONCLUSIONS: There is a subset of tumors, frequently classified as mesenchymal or IGS cluster 23, that may be identified with IHC and could well be optimal candidates for immunotherapy.

ROS1 copy number alterations are frequent in non-small cell lung cancer.

OBJECTIVES: We aimed to determine the prevalence and partners of ROS1 rearrangements, to explore the correlation between FISH and IHC assays, and to investigate clinical implications of ROS1 copy number alterations (CNAs). METHODS: A total of 314 NSCLC patients were screened using ROS1 FISH break-apart probes. Of these, 47 surgical tumors were included in TMAs to analyze ROS1 heterogeneity assessed either by FISH and IHC, and chromosome 6 aneusomy. To characterize ROS1 partners, probes for CD74, EZR, SLC34A2 and SDC3 genes were developed. ROS1 positive FISH cases were screened also by IHC. RESULTS: Five patients were ROS1 positive (1.8%). We identified two known fusion partners in three patients: CD74 and SLC34A2. Four out of five ROS1 rearranged patients were female, never smokers and with adenocarcinoma histology. Rearranged cases were also positive by IHC as well. According to ROS1 CNAs, we found a prevalence of 37.8% gains/amplifications and 25.1% deletions. CONCLUSIONS: This study point out the high prevalence of ROS1 CNAs in a large series of NSCLC. ROS1 gains, amplifications and deletions, most of them due to chromosome 6 polysomy or monosomy, were heterogeneous within a tumor and had no impact on overall survival.

Cystatin C is differentially involved in multiple system atrophy phenotypes.

AIMS: As cystatin C (CysC) is involved in some forms of neurodegeneration, we investigated the possible relationship between CysC and multiple system atrophy (MSA), including its parkinsonian (MSAp) and cerebellar (MSAc) phenotypes. METHODS: Cystatin C gene (CST3) haplotypes were determined by PCR followed by KspI digestion in 50 MSA patients and 108 controls. CST3 and cathepsins B, D and L1 mRNA levels were studied in frozen post-mortem caudate nucleus and cerebellar samples of eight MSAp, four MSAc and 18 control brains and analysed by the ΔΔCt method. CysC immunohistochemistry was performed on three MSAp, three MSAc and three control cerebella. Additionally, determination of CST3 and cathepsins B, D and L1 mRNA levels and immunohistochemistry for CysC were carried out in cerebella from three patients with paraneoplastic cerebellar degeneration, three with spinocerebellar ataxia (type 3, SCA3) and three with cerebellar ischaemia (CI). RESULTS: In the set of blood samples, the CST3 B-haplotype was associated with MSAp (OR 4.86, confidence interval 1.84-13.3). High CST3 mRNA levels were found in MSAp caudate nuclei [expression change: 3.08 (2.98-3.18)] and MSAc cerebella [expression change: 2.44 (2.14-2.88)]. In the latter there was CysC over-expression in Purkinje cells, Bergmann glia and dentate nucleus neurones. No cathepsin increase was detected in MSA cerebella. High mRNA levels of CST3 and cathepsins B and L1 were observed in SCA3 and CI brains. CONCLUSIONS: CysC changes are differentially present in the parkinsonian and cerebellar forms of MSA and may play an important role in the pathogenesis of this neurodegenerative condition.

MYC status determination in aggressive B-cell lymphoma: the impact of FISH probe selection.

AIMS: To assess how hybridization probe design may affect MYC status determination in Burkitt lymphoma and diffuse large B-cell lymphoma. METHODS AND RESULTS: We compared the results obtained with one dual-fusion and two break-apart commercial probes in a retrospective series of 91 aggressive B-cell lymphomas. All three probes were able to detect the IGH-MYC translocation in every case bearing it (13/13). However, seven of 13 (54%) non-IGH-MYC (light-chain immunoglobulin or non-immunoglobulin-MYC) rearrangements were unambiguously detected by just one of the probes tested. On the other hand, when the IGH-MYC dual-fusion probe was used, nine of 15 (60%) cases with a hybridization pattern suggestive of a non-IGH-MYC translocation were attributable to MYC copy gain rather than MYC rearrangement, as demonstrated by both break-apart probes. CONCLUSIONS: Taking into account the prognostic and therapeutic implications of the MYC translocation, probe design and limitations should be particularly kept in mind when MYC hybridization patterns are interpreted. In our experience, detection of 8q24 abnormalities could be optimized by a two-probe approach involving the application of both IGH-MYC dual-fusion and MYC break-apart selected kits.

Fetal-maternal interface: a chronicle of allogeneic coexistence.

The existence of allogeneic cells within an individual has been demonstrated in multiple fields such as hematopoietic stem cell or solid organ transplantation, non-depleted blood transfusions and the most common form which is bidirectional maternal-fetal cell trafficking, whereby cells from the fetus pass through the placental barrier. In order to graphically illustrate this early natural phenomenon that initiates the journey of a child's cells within the mother's blood and other tissues, we used a new procedure in microscopy imaging generating Large Scale Panoramic Pictures (LSPP). This technique can also be extended to explore a broad diversity of experimental models.

Improved clonality detection in Hodgkin lymphoma using the BIOMED-2-based heavy and kappa chain assay: a paraffin-embedded tissue study.

AIMS: Although BIOMED-2 polymerase chain reaction (PCR) standardization protocols allow clonality detection in nearly 100% of non-Hodgkin B cell lymphomas, they have not been widely validated for Hodgkin lymphoma (HL). Our aim was to assess BIOMED-2 protocol sensitivity when using non-microdissected, formalin-fixed, paraffin-embedded (FFPE) tissue from HL cases. METHODS AND RESULTS: We studied 69 consecutive HL cases, of which 61 corresponded to classic HL (cHL) and eight to nodular lymphocyte-predominant HL (NLPHL). CD30-positive cell numbers (<10, 10-25 or >25 per ×200 field), background CD20-positive cell density (low or high) and tumour cell immunophenotype were evaluated. IGH and IGK clonality was assessed on FFPE tissue following BIOMED-2 protocols. Of the 58 assessable cHL cases, 15 (25.9%) exhibited IGH and/or IGK clonality; IGH clonality was shown by nine (15.5%) and IGK clonality by 12 (20.7%). Clonality detection rates in cHL improved as CD30-positive Reed-Sternberg (RS) cell density increased and CD20-positive B cell density decreased, although these correlations did not reach statistical significance. Of the eight NLPHL cases studied, none showed clonal rearrangement. CONCLUSIONS: Combined study of IGH and IGK rearrangement according to BIOMED-2 protocols improves clonality detection rate (up to 25% of cases) in HL, even when working on non-microdissected FFPE tissue.

New brain-specific beta-synuclein isoforms show expression ratio changes in Lewy body diseases.

Lewy body diseases (LBDs) include dementia with Lewy bodies (DLB) and Parkinson disease (PD). Alpha-synuclein (AS) aggregation is a key event in the pathogenesis of LBDs and beta-synuclein (BS) inhibits AS aggregation in vitro and in vivo. Recently, BS has been shown to interact directly with AS regulating its functionality and preventing its oligomerization, and a molecular subgroup of pure DLB lacks BS in cortical regions. In this study, we characterized four new BS transcript variants and analyzed their expression in neuronal and non-neuronal tissue, and their differential expression in frozen samples of three areas from brains of patients with pure Lewy body pathology (LBP), common LBP, Alzheimer pathology, and of controls. Relative mRNA expression was determined by real-time PCR with neuron-specific enolase 2 and synaptophysin as housekeeping genes, and expression changes were evaluated by the ΔΔCt method. Two main findings are in concordance with earlier studies. First, all BS isoforms are drastically diminished in the cortex of patients with pure LBP that had presented clinically as DLB but not PD with dementia. Second, an important shift of the isoform expression ratio was observed in the temporal cortex of all LBD cases, and the minor isoforms, normally absent in the midbrain, were detected in the caudate nucleus of all DLB samples. Our results provide further evidence for the role of minor transcript variants in the development of complex diseases and provide new insights into the pathogenesis of LBDs that may be important for the understanding of molecular mechanisms involved in these complex diseases.

Immunohistochemical detection of MYC protein correlates with MYC gene status in aggressive B cell lymphomas.

AIMS: MYC gene translocation entails a bad prognosis and a poor response to rituximab-cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) in diffuse large B cell lymphomas (DLBCL), and more intensive chemotherapy regimens could be more effective in those cases. Its evaluation requires cytogenetic or fluorescence in-situ hybridization (FISH) studies, which are expensive and not widely available. The aim of this work was to find an immunohistochemical marker able to be used as a screening tool to identify MYC translocations. METHODS AND RESULTS: Aggressive B cell lymphomas in which MYC status was assessed during their diagnostic work-up between 2007 and 2010 were collected, their immunophenotype was re-evaluated, and were stratified according to the Hans algorithm. Two tissue microarrays were built in order to evaluate MYC protein expression with a commercially available antibody. The study was performed on 56 specimens: nine Burkitt lymphomas (eight translocated), 45 DLBCLs (nine translocated) and two lymphomas with intermediate features (both translocated). Only MYC protein expression detected by immunohistochemistry correlated with MYC translocation. No relationship was seen between MYC gene copies and protein expression. CONCLUSIONS: MYC protein expression detected by immunohistochemistry using a commercially available antibody correlates with MYC gene translocation, and could be used as a screening tool to select those cases in which confirmatory genetic testing is mandatory.

Multisite and bidirectional exonic splicing enhancer in CD44 alternative exon v3.

The human CD44 gene encodes multiple isoforms of a transmembrane protein that differ in their extracellular domains as a result of alternative splicing of its variable exons. Expression of CD44 is tightly regulated according to the type and physiological status of a cell, with expression of high molecular weight isoforms by inclusion of variable exons and low molecular weight isoforms containing few or no variable exons. Human CD44 variable exon 3 (v3) can follow a specific alternative splicing route different from that affecting other variable exons. Here we map and functionally describe the splicing enhancer element within CD44 exon v3 which regulates its inclusion in the final mRNA. The v3 splicing enhancer is a multisite bipartite element consisting of a tandem nonamer, the XX motif, and an heptamer, the Y motif, located centrally in the exon. Each of the three sites of this multisite enhancer partially retains its splicing enhancing capacity independently from each other in CD44 and shows full enhancing function in gene contexts different from CD44. We further demonstrate that these motifs act cooperatively as at least two motifs are needed to maintain exon inclusion. Their action is differential with respect to the splice-site target abutting v3. The first X motif acts on the 3' splice site, the second X motif acts on both splice sites (as a bidirectional exonic splicing enhancer), and the Y motif acts on the 5' splice site. We also show that the multisite v3 splicing enhancer is functional irrespective of flanking intron length and spatial organization within v3.

Exonization of Alu-generated splice variants in the survivin gene of human and non-human primates.

Survivin is a member of the inhibitor apoptosis family that is overexpressed in many malignancies. It has five known alternative splice forms, some of which differ in their antiapoptotic properties and expression levels in human cancers. Here we describe a novel donor splice site (DSS), 2B+32 DSS, which is used in conjunction with survivin alternative exon 2B, resulting in the inclusion of 32 additional nucleotides from intron 2 at the 3' end of this exon. Sequence analysis showed that both the classical exon 2B DSS and 2B+32 are provided by an Alu sequence, which is inserted in intron 2 downstream of a functional acceptor splice site, leading to the exonization of part of the repetitive element. Minor transcripts including the 2B+32 alternative exon, or retaining the whole intronic region comprised between exons 2B and 3, were detected in several human cell lines and in some human tissues. Survivin 2B+32 containing variants acquire a premature stop codon (PTC) and may therefore be degraded by the nonsense mediated decay pathway. The implication of these novel isoforms, as well as other PTC+ survivin variants, in the overall regulation of survivin expression is discussed. Sequence analysis of intron 2 which contains the Alu Y element was performed on different primate species in order to trace its insertion and exonization during primate evolution.

JC virus early protein detection by immunohistochemistry in progressive multifocal leukoencephalopathy: a comparative study with in situ hybridization and polymerase chain reaction.

In situ hybridization (ISH) for JC virus (JCV) is generally applied for the diagnosis of progressive multifocal leukoencephalopathy (PML). To explore the usefulness of immunohistochemistry (IHC) for JCV early proteins, 14 paraffin-embedded postmortem brain specimens with histologic features compatible with PML were tested for the presence of JCV by means of DNA-DNA ISH with a biotinylated probe corresponding to the entire JCV genome, for JCV early proteins IHC with both PAb 2003 and anti-SV40 large T antigen monoclonal antibodies, and polymerase chain reaction (PCR) amplification of JCV virion protein 3 (VP3) and transcriptional control region (TCR) sequences. ISH was positive in 13 cases and IHC in all 14 cases, the number of IHC-positive cells generally being far in excess of ISH-positive cells. Of the 2 monoclonal antibodies used, PAb 2003 proved to be more sensitive than anti-SV40 large T antigen. Occasional neuronal nuclei were positive for JCV early proteins in 5 cases. As for PCR, VP3 was amplified in all 14 cases and TCR in 9 cases. Consequently, PAb 2003 IHC for JCV early proteins seems to be a powerful tool for viral demonstration in PML and may well become the diagnostic recourse of choice in this setting.