TOX as a new diagnostic marker for T cell large granular lymphocytic leukaemia.

AIMS: T cell large granular lymphocytic leukaemia (T-LGLL) is a rare disorder that may underlie otherwise unexplained cytopenias. The identification of T-LGLL cells in bone marrow biopsies can be a challenge, because a robust immunohistochemistry marker is lacking. The markers currently in use (granzyme B, TIA-1 and CD8) are difficult to interpret or lack specificity. Therefore, we investigated whether immunohistochemistry for thymocyte selection-associated high-mobility group box (TOX), a transcription factor that associates with chronic T cell stimulation, could be a reliable tool for the identification of T-LGLL cells. METHODS AND RESULTS: In this retrospective study, expression of TOX in CD8+ cells in bone marrow biopsies of T-LGLL patients (n = 38) was investigated and compared to bone marrow of controls with reactive T cell lymphocytosis (n = 10). All biopsies were evaluated for TOX staining within the CD8-positive T cell population. The controls were essentially negative for TOX, whereas all T-LGLL cases were positive (median = 80%, range = 10-100%), even when bone marrow involvement was subtle. CONCLUSION: TOX is a highly sensitive marker for the neoplastic cells of T-LGLL and we recommend its use, especially in the diagnostic work-up of patients with unexplained cytopenias.

Toxic Determination of Cry11 Mutated Proteins Obtained Using Rational Design and Its Computational Analysis.

Cry11 proteins are toxic to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. Cry11Aa and Cry11Bb are protoxins, which when activated present their active-toxin form in two fragments between 30 and 35 kDa respectively. Previous studies conducted with Cry11Aa and Cry11Bb genes using DNA shuffling generated variant 8, which presented a deletion in the first 73 amino acids and one at position 572 and 9 substitutions including L553F and L556W. In this study, variant 8 mutants were constructed using site-directed mutagenesis, resulting in conversion of phenylalanine (F) and tryptophan (W) to leucine (L) at positions 553 and 556, respectively, producing the mutants 8F553L, 8W556L, and 8F553L/8W556L. Additionally, two mutants, A92D and C157R, derived from Cry11Bb were also generated. The proteins were expressed in the non-crystal strain BMB171 of Bacillus thuringiensis and subjected to median-lethal concentration (LC50) tests on first-instar larvae of A. aegypti. LC50 analysis showed that the 8F553L, 8W556L, 8F553L/8W556L, and C157R variants lost their toxic activity (>500 ng·mL-1), whereas the A92D protein presented a loss of toxicity of 11.4 times that of Cry11Bb. Cytotoxicity assays performed using variant 8, 8W556L and the controls Cry11Aa, Cry11Bb, and Cry-negative BMB171 on the colorectal cancer cell line SW480 reported 30-50% of cellular viability except for BMB171. Molecular dynamic simulations performed to identify whether the mutations at positions 553 and 556 were related to the stability and rigidity of the functional tertiary structure (domain III) of the Cry11Aa protein and variant 8 showed the importance of these mutations in specific regions for the toxic activity of Cry11 against A. aegypti. This generates pertinent knowledge for the design of Cry11 proteins and their biotechnological applications in vector-borne disease control and cancer cell lines.

Navitoclax Most Promising BH3 Mimetic for Combination Therapy in Hodgkin Lymphoma.

The treatment of young patients with Hodgkin lymphoma (HL) is often successful but a significant proportion of patients suffers from late toxicity. In the current era there are new opportunities for less toxic and more targeted treatment options. In this respect, the anti-apoptotic pathway is an attractive target since Hodgkin tumor cells abundantly express components of this pathway. We measured the effect of BH3 mimetics that interfere with anti-apoptotic proteins in cell lines, also in combination with the standard of care chemotherapeutic doxorubicin and the recently discovered preclinically active tamoxifen. Several anti-apoptotic BCL-2 family proteins were expressed in each case (n = 84) and in HL cell lines (n = 5). Cell lines were checked for sensitivity to BH3 mimetics by BH3 profiling and metabolic assays and monotherapy was only partially successful. Doxorubicin was synergistic with a BCL-XL inhibitor and BCL2/XL/W inhibitor navitoclax. Tamoxifen that targets the estrogen receptor ß present in the mitochondria of the cell lines, could induce cell death, and was synergistic with several BH3 mimetics including/as well as navitoclax. In conclusion, targeting the anti-apoptotic pathway by the triple inhibitor navitoclax in combination with doxorubicin or tamoxifen is a promising treatment strategy in HL.

Site-Directed Mutants of Parasporin PS2Aa1 with Enhanced Cytotoxic Activity in Colorectal Cancer Cell Lines.

Parasporin 2 has cytotoxic effects against numerous colon cancer cell lines, making it a viable alternative to traditional treatments. However, its mechanism of action and receptors remain unknown. In this study, site-directed mutagenesis was used to obtain PS2Aa1 mutants with variation in domain I at positions 256 and 257. Variants 015, 002, 3-3, 3-35, and 3-45 presented G256A, G256E, G257A, G257V, and G257E substitutions, respectively. Cytotoxicity tests were performed for the cell viability of cell lines SW480, SW620, and CaCo-2. Mutants 3-3, 3-35, and 3-45 efficiently killed the cell lines. It was found that the activated forms of caspase-3 and PARP were in higher abundance as well as increased production of γH2AX when 3-35 was used to treat CaCo-2 and SW480. To assess possible membrane-binding receptors involved in the interaction, an APN receptor blocking assay showed reduced activity of some parasporins. Hence, we performed molecular docking and molecular dynamics simulations to analyze the stability of possible interactions and identify the residues that could be involved in the protein-protein interaction of PS2Aa1 and APN. We found that residues 256 and 257 facilitate the interaction. Parasporin 3-35 is promising because it has higher cytotoxicity than PS2Aa1.

Prognostic value of TARC and quantitative PET parameters in relapsed or refractory Hodgkin lymphoma patients treated with brentuximab vedotin and DHAP.

Risk-stratified treatment strategies have the potential to increase survival and lower toxicity in relapsed/refractory classical Hodgkin lymphoma (R/R cHL) patients. This study investigated the prognostic value of serum (s)TARC, vitamin D and lactate dehydrogenase (LDH), TARC immunohistochemistry and quantitative PET parameters in 65 R/R cHL patients who were treated with brentuximab vedotin (BV) and DHAP followed by autologous stem-cell transplantation (ASCT) within the Transplant BRaVE study (NCT02280993). At a median follow-up of 40 months, the 3-year progression free survival (PFS) was 77% (95% CI: 67-88%) and the overall survival was 95% (90-100%). Significant adverse prognostic markers for progression were weak/negative TARC staining of Hodgkin Reed-Sternberg cells in the baseline biopsy, and a high standard uptake value (SUV)mean or SUVpeak on the baseline PET scan. After one cycle of BV-DHAP, sTARC levels were strongly associated with the risk of progression using a cutoff of 500 pg/ml. On the pre-ASCT PET scan, SUVpeak was highly prognostic for progression post-ASCT. Vitamin D, LDH and metabolic tumor volume had low prognostic value. In conclusion, we established the prognostic impact of sTARC, TARC staining, and quantitative PET parameters for R/R cHL, allowing the use of these parameters in prospective risk-stratified clinical trials. Trial registration: NCT02280993.

Cytochrome P450 27C1 Level Dictates Lung Cancer Tumorigenicity and Sensitivity towards Multiple Anticancer Agents and Its Potential Interplay with the IGF-1R/Akt/p53 Signaling Pathway.

Cytochrome P450 enzymes (CYP450s) exert mighty catalytic actions in cellular metabolism and detoxication, which play pivotal roles in cell fate determination. Preliminary data shows differential expression levels of CYP27C1, one of the "orphan P450s" in human lung cancer cell lines. Here, we study the functions of CYP27C1 in lung cancer progression and drug endurance, and explore its potential to be a diagnostic and therapeutic target for lung cancer management. Quantitative real-time PCR and immunoblot assays were conducted to estimate the transcription and protein expression level of CYP27C1 in human lung cancer cell lines, which was relatively higher in A549 and H1975 cells, but was lower in H460 cells. Stable CYP27C1-knockdown A549 and H1975 cell lines were established, in which these cells showed enhancement in cell proliferation, colony formation, and migration. In addition, aberrant IGF-1R/Akt/p53 signal transduction was also detected in stable CYP27C1-knockdown human lung cancer cells, which exhibited greater tolerance towards the treatments of anticancer agents (including vinorelbine, picropodophyllin, pacritinib, and SKLB610). This work, for the first time, reveals that CYP27C1 impacts lung cancer cell development by participating in the regulation of the IGF-1R/Akt/p53 signaling pathway, and the level of CYP27C1 plays indispensable roles in dictating the cellular sensitivity towards multiple anticancer agents.

Validation of Novel Molecular Imaging Targets Identified by Functional Genomic mRNA Profiling to Detect Dysplasia in Barrett's Esophagus.

Barrett's esophagus (BE) is the precursor of esophageal adenocarcinoma (EAC). Dysplastic BE (DBE) has a higher progression risk to EAC compared to non-dysplastic BE (NDBE). However, the miss rates for the endoscopic detection of DBE remain high. Fluorescence molecular endoscopy (FME) can detect DBE and mucosal EAC by highlighting the tumor-specific expression of proteins. This study aimed to identify target proteins suitable for FME. Publicly available RNA expression profiles of EAC and NDBE were corrected by functional genomic mRNA (FGmRNA) profiling. Following a class comparison between FGmRNA profiles of EAC and NDBE, predicted, significantly upregulated genes in EAC were prioritized by a literature search. Protein expression of prioritized genes was validated by immunohistochemistry (IHC) on DBE and NDBE tissues. Near-infrared fluorescent tracers targeting the proteins were developed and evaluated ex vivo on fresh human specimens. In total, 1976 overexpressed genes were identified in EAC (n = 64) compared to NDBE (n = 66) at RNA level. Prioritization and IHC validation revealed SPARC, SULF1, PKCι, and DDR1 (all p < 0.0001) as the most attractive imaging protein targets for DBE detection. Newly developed tracers SULF1-800CW and SPARC-800CW both showed higher fluorescence intensity in DBE tissue compared to paired non-dysplastic tissue. This study identified SPARC, SULF1, PKCι, and DDR1 as promising targets for FME to differentiate DBE from NDBE tissue, for which SULF1-800CW and SPARC-800CW were successfully ex vivo evaluated. Clinical studies should further validate these findings.

Anti-CD37 radioimmunotherapy with 177Lu-NNV003 synergizes with the PARP inhibitor olaparib in treatment of non-Hodgkin's lymphoma in vitro.

BACKGROUND AND PURPOSE: PARP inhibitors have been shown to increase the efficacy of radiotherapy in preclinical models. Radioimmunotherapy results in selective radiation cytotoxicity of targeted tumour cells. Here we investigate the combined effect of anti-CD37 ß-emitting 177Lu-NNV003 radioimmunotherapy and the PARP inhibitor olaparib, and gene expression profiles in CD37 positive non-Hodgkin's lymphoma cell lines. MATERIALS AND METHODS: The combined effect of 177Lu-NNV003 and olaparib was studied in seven cell lines using a fixed-ratio ray design, and combination index was calculated for each combination concentration. mRNA was extracted before and after treatment with the drug combination. After RNA-sequencing, hierarchical clustering was performed on basal gene expression profiles and on differentially expressed genes after combination treatment from baseline. Functional gene annotation analysis of significant differentially expressed genes after combination treatment was performed to identify enriched biological processes. RESULTS: The combination of olaparib and 177Lu-NNV003 was synergistic in four of seven cell lines, antagonistic in one and both synergistic and antagonistic (conditionally synergistic) in two, depending on the concentration ratio between olaparib and 177Lu-NNV003. Cells treated with the combination significantly overexpressed genes in the TP53 signalling pathway. However, cluster analysis did not identify gene clusters that correlate with the sensitivity of cells to single agent or combination treatment. CONCLUSION: The cytotoxic effect of the combination of the PARP inhibitor olaparib and the ß-emitting radioimmunoconjugate 177Lu-NNV003 was synergistic in the majority of tested lymphoma cell lines.

Identification of the estrogen receptor beta as a possible new tamoxifen-sensitive target in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype. Despite the proven efficacy of combined immunochemotherapy (R-CHOP) in the majority of patients, ~40% of DLBCL patients do not respond or will relapse and consequently have a very poor prognosis. The development of targeted therapies has not improved patient survival, underscoring the need for new treatment approaches. Using an unbiased genome-wide CD20 guilt-by-association approach in more than 1800 DLBCL patients, we previously identified the estrogen receptor beta (ERß) as a new target in DLBCL. Here, we demonstrate that ERß is expressed at significantly higher levels in DLBCL compared to normal B cells, and ERß plays a role in the protection against apoptosis in DLBCL. Targeting of the ERß with the selective estrogen receptor modulator tamoxifen reduces cell viability in all tested DLBCL cell lines. Tamoxifen-induced cell death was significantly decreased in an ERß knock-out cell line. The activity of tamoxifen was confirmed in a xenograft human lymphoma model, as tumor growth decreased, and survival significantly improved. Finally, tamoxifen-treated breast cancer (BC) patients showed a significantly reduced risk of 38% for DLBCL compared to BC patients who did not receive tamoxifen. Our findings provide a rationale to investigate tamoxifen, a hormonal drug with a good safety profile, in DLBCL patients.

CD4+ T cells in classical Hodgkin lymphoma express exhaustion associated transcription factors TOX and TOX2: Characterizing CD4+ T cells in Hodgkin lymphoma.

In classical Hodgkin lymphoma (cHL), the highly abundant CD4+ T cells in the vicinity of tumor cells are considered essential for tumor cell survival, but are ill-defined. Although they are activated, they consistently lack expression of activation marker CD26. In this study, we compared sorted CD4+CD26- and CD4+CD26+ T cells from cHL lymph node cell suspensions by RNA sequencing and T cell receptor variable gene segment usage analysis. This revealed that although CD4+CD26- T cells are antigen experienced, they have not clonally expanded. This may well be explained by the expression of exhaustion associated transcription factors TOX and TOX2, immune checkpoints PDCD1 and CD200, and chemokine CXCL13, which were amongst the 100 significantly enriched genes in comparison with the CD4+CD26+ T cells. Findings were validated in single-cell RNA sequencing data from an independent cohort. Interestingly, immunohistochemistry revealed predominant and high frequency of staining for TOX and TOX2 in the T cells attached to the tumor cells. In conclusion, the dominant CD4+CD26- T cell population in cHL is antigen experienced, polyclonal, and exhausted. This population is likely a main contributor to the very high response rates to immune checkpoint inhibitors in cHL.

A gene expression-based model predicts outcome in children with intermediate-risk classical Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) is a common malignancy in children and adolescents. Although cHL is highly curable, treatment with chemotherapy and radiation often come at the cost of long-term toxicity and morbidity. Effective risk-stratification tools are needed to tailor therapy. Here, we used gene expression profiling (GEP) to investigate tumor microenvironment (TME) biology, to determine molecular correlates of treatment failure, and to develop an outcome model prognostic for pediatric cHL. A total of 246 formalin-fixed, paraffin-embedded tissue biopsies from patients enrolled in the Children's Oncology Group trial AHOD0031 were used for GEP and compared with adult cHL data. Eosinophil, B-cell, and mast cell signatures were enriched in children, whereas macrophage and stromal signatures were more prominent in adults. Concordantly, a previously published model for overall survival prediction in adult cHL did not validate in pediatric cHL. Therefore, we developed a 9-cellular component model reflecting TME composition to predict event-free survival (EFS). In an independent validation cohort, we observed a significant difference in weighted 5-year EFS between high-risk and low-risk groups (75.2% vs 90.3%; log-rank P = .0138) independent of interim response, stage, fever, and albumin. We demonstrate unique disease biology in children and adolescents that can be harnessed for risk-stratification at diagnosis. This trial was registered at www.clinicaltrials.gov as #NCT00025259.

Genetic Modification Approaches for Parasporins Bacillus thuringiensis Proteins with Anticancer Activity.

Bacillus thuringiensis (Bt) is a bacterium capable of producing Cry toxins, which are recognized for their bio-controlling actions against insects. However, a few Bt strains encode proteins lacking insecticidal activity but showing cytotoxic activity against different cancer cell lines and low or no cytotoxicity toward normal human cells. A subset of Cry anticancer proteins, termed parasporins (PSs), has recently arisen as a potential alternative for cancer treatment. However, the molecular receptors that allow the binding of PSs to cells and their cytotoxic mechanisms of action have not been well established. Nonetheless, their selective cytotoxic activity against different types of cancer cell lines places PSs as a promising alternative treatment modality. In this review, we provide an overview of the classification, structures, mechanisms of action, and insights obtained from genetic modification approaches for PS proteins.

Computational study, synthesis and evaluation of active peptides derived from Parasporin-2 and spike protein from Alphacoronavirus against colorectal cancer cells.

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and ß-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.

Soluble PD-L1 is a promising disease biomarker but does not reflect tissue expression in classic Hodgkin lymphoma.

Individually, tissue and soluble markers involved in the programmed cell death protein 1/programmed death-ligand (PD-1/PD-L) axis have been described as biomarkers with clinical value in classical Hodgkin lymphoma (cHL). In the context of the success of immune checkpoint blockade therapy in cHL, it is interesting to discover whether plasma levels of proteins in the PD-1/PD-L axis are a reflection of expression by the corresponding tissue. Paired tissue and plasma samples of cHL patients were collected and analysed for PD-1, PD-L1 and PD-L2 levels. In addition, vascular endothelial growth factor (VEGF) and CD83, molecules regarded to influence the expression of PD-1, PD-L1 and/or PD-L2, were included. PD-L1 was upregulated in the plasma of cHL patients compared to healthy controls and correlated well with several clinical parameters. Strong PD-L1 expression in the tumour microenvironment contributed to high soluble (s)PD-L1 levels, although there was no direct correlation between plasma PD-L1 levels and total expression of PD-L1 in corresponding cHL tissue. Interestingly, we observed a positive correlation between VEGF and PD-1 levels in both tissue and plasma. In conclusion, although PD-L1 is a promising soluble biomarker in cHL, its levels do not reflect the total tissue expression. Future studies focusing on PD-L1 as a predictor for immune checkpoint treatment response, should include both biopsy and plasma samples.

Interaction between ERAP Alleles and HLA Class I Types Support a Role of Antigen Presentation in Hodgkin Lymphoma Development.

Genetic variants in the HLA region are the strongest risk factors for developing Hodgkin lymphoma (HL), suggesting an important role for antigen presentation. This is supported by another HL-associated genomic region which contains the loci of two enzymes that process endogenous proteins to peptides to be presented by HLA class I, i.e., endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2. We hypothesized that ERAP and HLA class I type interact in HL susceptibility, as shown previously for several autoimmune diseases. We detected ERAP1 and ERAP2 expression in tumor cells and cells in the microenvironment in primary HL tissue samples. Seven ERAP SNPs and ERAP1 haplotypes showed strong associations with RNA and protein levels of ERAP1 and ERAP2 in LCLs and HL cell lines. Analysis of HLA class I types, ERAP SNPs and ERAP haplotypes by direct genotyping or imputation from genome-wide association data in 390 HL patients revealed significant interactions between HLA-A11, rs27038 and the rs27038 associated ERAP haplotype, as well as between HLA-Cw2 and rs26618. In conclusion, our results show that ERAP and HLA class I interact in genetic susceptibility to HL, providing further evidence that antigen presentation is an important process in HL susceptibility and pathogenesis.

Rosetting T cells in Hodgkin lymphoma are activated by immunological synapse components HLA class II and CD58.

A unique feature of Hodgkin lymphoma (HL) is the presence of CD4+ T cells that surround, protect, and promote survival of tumor cells. The adhesion molecules involved in this so-called T-cell rosetting are important components of the immunological synapse (IS). However, it is unknown whether this synapse is fully assembled and leads to T-cell activation by enabling interaction between the T-cell receptor (TCR) and human leukocyte antigen class II (HLA-II). We established a novel rosetting model by coculturing HLA-II-matched peripheral blood mononuclear cells with HL cell lines and showed IS formation with activation of rosetting T cells. HLA-II downregulation by class II transactivator knockout did not affect the extent of rosetting, but almost completely abrogated T-cell activation. Intriguingly, the level of CD58 expression correlated with the extent of rosette formation, and CD58 knockout or CD2 blockade reduced both rosette formation and T-cell activation. The extension of our findings to primary HL tissue by immunohistochemistry and proximity ligation assays showed interaction of CD2 with CD58 and of TCR-associated CD4 with HLA-II. In conclusion, T-cell rosetting in HL is established by formation of the IS, and activation of rosetting T cells critically depends on the interaction of both TCR-HLA-II and CD2-CD58.

Enrichment of the tumour immune microenvironment in patients with desmoplastic colorectal liver metastasis.

BACKGROUND: Patients with resected colorectal liver metastasis (CRLM) who display only the desmoplastic histopathological growth pattern (dHGP) exhibit superior survival compared to patients with any non-desmoplastic growth (non-dHGP). The aim of this study was to compare the tumour microenvironment between dHGP and non-dHGP. METHODS: The tumour microenvironment was investigated in three cohorts of chemo-naive patients surgically treated for CRLM. In cohort A semi-quantitative immunohistochemistry was performed, in cohort B intratumoural and peritumoural T cells were counted using immunohistochemistry and digital image analysis, and in cohort C the relative proportions of individual T cell subsets were determined by flow cytometry. RESULTS: One hundred and seventeen, 34, and 79 patients were included in cohorts A, B, and C, with dHGP being observed in 27%, 29%, and 15% of patients, respectively. Cohorts A and B independently demonstrated peritumoural and intratumoural enrichment of cytotoxic CD8+ T cells in dHGP, as well as a higher CD8+/CD4+ ratio (cohort A). Flow cytometric analysis of fresh tumour tissues in cohort C confirmed these results; dHGP was associated with higher CD8+ and lower CD4+ T cell subsets, resulting in a higher CD8+/CD4+ ratio. CONCLUSION: The tumour microenvironment of patients with dHGP is characterised by an increased and distinctly cytotoxic immune infiltrate, providing a potential explanation for their superior survival.

B Cells as Prognostic Biomarker After Surgery for Colorectal Liver Metastases.

Background: The aim of this study was to identify more accurate variables to improve prognostication of individual patients with colorectal liver metastases (CRLM). Clinicopathological characteristics only partly explain the large range in survival rates. Methods: MessengerRNA expression profiles of resected CRLM of two patient groups were analysed by mRNA sequencing: poor survivors (death from recurrent disease <30 months after surgery) and good survivors (no recurrent disease >60 months after surgery). Tumour and adjacent liver parenchyma samples were analysed. Results: MessengerRNA expression profiling of the tumour samples identified 77 genes that were differentially expressed between the two survival groups at a False Discovery Rate (FDR) <0.1. In the adjacent liver parenchyma samples only one gene, MTRNR2L1, showed significantly higher expression in the good survivors. Pathway analysis showed higher expression of immune-related and stroma-related genes in tumour samples from good survivors. Expression data was then validated by immunohistochemistry in two cohorts comprising a total of 125 patients. Immunohistochemical markers that showed to be associated with good survival in the total cohort were: high K/L+ infiltration in tumour stroma [p = 0.029; OR 2.500 (95% CI 1.100-5.682)] and high CD79A+ infiltration in tumour stroma [p = 0.036; OR 2.428 (95%CI 1.062-5.552)]. Conclusions: A high stromal infiltration of CD79A+ B cells and K/L+ plasma cells might be favourable prognostic biomarkers after surgery for CRLM.

Interim thymus and activation regulated chemokine versus interim 18 F-fluorodeoxyglucose positron-emission tomography in classical Hodgkin lymphoma response evaluation.

Serum thymus and activation regulated chemokine (TARC) levels reflect classical Hodgkin lymphoma (cHL) disease activity and correspond with treatment response. We compared mid-treatment interim TARC (iTARC) with interim 18 F-fluorodeoxyglucose positron-emission tomography (iPET) imaging to predict modified progression-free survival (mPFS) in a group of 95 patients with cHL. High iTARC levels were found in nine and positive iPET in 17 patients. The positive predictive value (PPV) of iTARC for a 5-year mPFS event was 88% compared to 47% for iPET. The negative predictive value was comparable at 86% for iTARC and 85% for iPET. Serum iTARC levels more accurately reflect treatment response with a higher PPV compared to iPET.

WEE1 inhibition synergizes with CHOP chemotherapy and radiation therapy through induction of premature mitotic entry and DNA damage in diffuse large B-cell lymphoma.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, characterized by high levels of genomic instability and the activation of DNA damage repair pathways. We previously found high expression of the cell cycle regulator WEE1 in DLBCL cell lines. Here, we investigated the combination of the WEE1 inhibitor, AZD1775, with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) and radiation therapy (RT), with the aim of improving first-line treatment. METHODS: Cell viability experiments were performed to determine synergistic combinations. Levels of DNA damage were established using flow cytometry for γH2AX and protein analysis for DNA damage response proteins CHK1 and CHK2. Flow cytometry analysis for cell cycle and pH3 were performed to determine cell cycle distribution and premature mitotic entry. RESULTS: Treatment with either RT or CHOP led to enhanced sensitivity to AZD1775 in several DLBCL cell lines. Treatment of cells with AZD1775 induced unscheduled mitotic progression, resulting in abnormal cell cycle distribution in combination with RT or CHOP treatment. In addition, a significant increase in DNA damage was observed compared with CHOP or RT alone. Of the single CHOP components, doxorubicin showed the strongest effect together with AZD1775, reducing viability and increasing DNA damage. CONCLUSION: In conclusion, the combination of RT or CHOP with AZD1775 enhances sensitivity to WEE1 inhibition through unscheduled G2/M progression, leading to increased DNA damage. Based on these results, WEE1 inhibition has great potential together with other G2/M arresting or DNA damaging (chemo) therapeutic compounds and should be further explored in clinical trials.