Characterization of MGMT and EGFR protein expression in glioblastoma and association with survival.

PURPOSE: Understanding the molecular landscape of glioblastoma (GBM) is increasingly important in the age of targeted therapy. O-6-Methylguanine-DNA methyltransferase (MGMT) promoter methylation and EGFR amplification are markers that may play a role in prognostication, treatment, and/or clinical trial eligibility. Quantification of MGMT and EGFR protein expression may offer an alternative strategy towards understanding GBM. Here, we quantify baseline expression of MGMT and EGFR protein in newly diagnosed GBM samples using mass spectrometry. We correlate findings with MGMT methylation and EGFR amplification statuses and survival. METHODS: We retrospectively identified adult patients with newly diagnosed resected GBM. MGMT and EGFR protein expression were quantified using a selected reaction monitoring mass spectrometry assay. Protein levels were correlated with MGMT methylation and EGFR amplification and survival data. RESULTS: We found a statistically significant association between MGMT protein expression and promoter methylation status (p = 0.02) as well as between EGFR protein expression and EGFR amplification (p < 0.0001). EGFR protein expression and amplification were more tightly associated than MGMT protein expression and methylation. Only MGMT promoter methylation was statistically significantly associated with progression-free and overall survival. CONCLUSIONS: Unlike EGFR protein expression and EGFR amplification which are strongly associated, only a weak association was seen between MGMT protein expression and promoter methylation. Quantification of MGMT protein expression was inferior to MGMT methylation for prognostication in GBM. Discordance was observed between EGFR amplification and EGFR protein expression; additional study is warranted to determine whether EGFR protein expression is a better biomarker than EGFR amplification for clinical decisions and trial enrollment.

Bovine lactoferricin-induced anti-inflammation is, in part, via up-regulation of interleukin-11 by secondary activation of STAT3 in human articular cartilage.

Bovine lactoferricin (LfcinB), a multifunctional peptide, was recently demonstrated to be anti-catabolic and anti-inflammatory in human articular cartilage. LfcinB blocks IL-1-mediated proteoglycan depletion, matrix-degrading enzyme expression, and pro-inflammatory mediator induction. LfcinB selectively activates ERK1/2, p38 (but not JNK), and Akt signaling. However, the relationship between these pathways and LfcinB target genes has never been explored. In this study, we uncovered the remarkable ability of LfcinB in the induction of an anti-inflammatory cytokine, IL-11. LfcinB binds to cell surface heparan sulfate to initiate ERK1/2 signaling and activate AP-1 complexes composed of c-Fos and JunD, which transactivate the IL-11 gene. The induced IL-11 functions as an anti-inflammatory and chondroprotective cytokine in articular chondrocytes. Our data show that IL-11 directly attenuates IL-1-mediated catabolic and inflammatory processes ex vivo and in vitro. Moreover, IL-11 activates STAT3 signaling pathway to critically up-regulate TIMP-1 expression, as a consecutive secondary cellular response after IL-11 induction by LfcinB-ERK-AP-1 axis in human adult articular chondrocytes. The pathological relevance of IL-11 signaling to osteoarthritis is evidenced by significant down-regulation of its cognate receptor expression in osteoarthritic chondrocytes. Together, our results suggest a two-step mechanism, whereby LfcinB induces TIMP-1 through an IL-11-dependent pathway involving transcription factor AP-1 and STAT3.

Use of Deep Learning to Evaluate Tumor Microenvironmental Features for Prediction of Colon Cancer Recurrence.

Deep learning may detect biologically important signals embedded in tumor morphologic features that confer distinct prognoses. Tumor morphologic features were quantified to enhance patient risk stratification within DNA mismatch repair (MMR) groups using deep learning. Using a quantitative segmentation algorithm (QuantCRC) that identifies 15 distinct morphologic features, we analyzed 402 resected stage III colon carcinomas [191 deficient (d)-MMR; 189 proficient (p)-MMR] from participants in a phase III trial of FOLFOX-based adjuvant chemotherapy. Results were validated in an independent cohort (176 d-MMR; 1,094 p-MMR). Association of morphologic features with clinicopathologic variables, MMR, KRAS, BRAFV600E, and time-to-recurrence (TTR) was determined. Multivariable Cox proportional hazards models were developed to predict TTR. Tumor morphologic features differed significantly by MMR status. Cancers with p-MMR had more immature desmoplastic stroma. Tumors with d-MMR had increased inflammatory stroma, epithelial tumor-infiltrating lymphocytes (TIL), high-grade histology, mucin, and signet ring cells. Stromal subtype did not differ by BRAFV600E or KRAS status. In p-MMR tumors, multivariable analysis identified tumor-stroma ratio (TSR) as the strongest feature associated with TTR [HRadj 2.02; 95% confidence interval (CI), 1.14-3.57; P = 0.018; 3-year recurrence: 40.2% vs. 20.4%; Q1 vs. Q2-4]. Among d-MMR tumors, extent of inflammatory stroma (continuous HRadj 0.98; 95% CI, 0.96-0.99; P = 0.028; 3-year recurrence: 13.3% vs. 33.4%, Q4 vs. Q1) and N stage were the most robust prognostically. Association of TSR with TTR was independently validated. In conclusion, QuantCRC can quantify morphologic differences within MMR groups in routine tumor sections to determine their relative contributions to patient prognosis, and may elucidate relevant pathophysiologic mechanisms driving prognosis. SIGNIFICANCE: A deep learning algorithm can quantify tumor morphologic features that may reflect underlying mechanisms driving prognosis within MMR groups. TSR was the most robust morphologic feature associated with TTR in p-MMR colon cancers. Extent of inflammatory stroma and N stage were the strongest prognostic features in d-MMR tumors. TIL density was not independently prognostic in either MMR group.

Bovine lactoferricin is anti-inflammatory and anti-catabolic in human articular cartilage and synovium.

Bovine lactoferricin (LfcinB) is a multi-functional peptide derived from proteolytic cleavage of bovine lactoferrin. LfcinB was found to antagonize the biological effects mediated by angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2) in endothelial cells. However, the effect of LfcinB on human articular cartilage remained unknown. Here, our findings demonstrate that LfcinB restored the proteoglycan loss promoted by catabolic factors (interleukin-1ß) IL-1ß and FGF-2 in vitro and ex vivo. Mechanistically, LfcinB attenuated the effects of IL-1ß and FGF-2 on the expression of cartilage-degrading enzymes (MMP-1, MMP-3, and MMP-13), destructive cytokines (IL-1ß and IL-6), and inflammatory mediators (iNOS and TLR2). LfcinB induced protective cytokine expression (IL-4 and IL-10), and downregulated aggrecanase basal expression. LfcinB specifically activated ERK MAPK and Akt signaling pathways, which may account for its anti-inflammatory activity. We also revealed that LfcinB exerted similar protective effects on human synovial fibroblasts challenged by IL-1ß, with minimal cytotoxicity. Collectively, our results suggest that LfcinB possesses potent anti-catabolic and anti-inflammatory bioactivities in human articular tissues, and may be utilized for the prevention and/or treatment of OA in the future.

Lactoferricin mediates anti-inflammatory and anti-catabolic effects via inhibition of IL-1 and LPS activity in the intervertebral disc.

The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production, and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future.

The pathophysiologic role of the protein kinase Cδ pathway in the intervertebral discs of rabbits and mice: in vitro, ex vivo, and in vivo studies.

OBJECTIVE: Protein kinase Cδ (PKCδ) activation has been shown to be a principal rate-limiting step in matrix-degrading enzyme production in human articular chondrocytes. The aim of this study was to assess the role of the PKC pathways, specifically PKCδ, in intervertebral disc tissue homeostasis. METHODS: Using in vitro, ex vivo, and in vivo techniques, we evaluated the pathophysiologic role of the PKCδ pathway by examining 1) proteoglycan deposition, 2) matrix-degrading enzyme production and activity, 3) downstream signaling pathways regulated by PKCδ, and 4) the effect on in vivo models of disc degeneration in genetically engineered PKCδ-knockout mice. RESULTS: Studies of pathway-specific inhibitors revealed a vital role of the PKCδ/MAPK (ERK, p38, JNK) axis and NF-κB in disc homeostasis. Accordingly, in an in vivo model of disc injury, PKCδ-knockout mice were markedly resistant to disc degeneration. CONCLUSION: Suppression of the PKCδ pathway may be beneficial in the prevention and/or treatment of disc degeneration. The results of this study provide evidence for a potential therapeutic role of pathway-specific inhibitors of the PKCδ cascade in the future.

Immune Marker Spatial Distribution and Clinical Outcome after PD-1 Blockade in Mismatch Repair-deficient, Advanced Colorectal Carcinomas.

PURPOSE: Targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) interaction has led to durable responses in fewer than half of patients with mismatch repair-deficient (MMR-d) advanced colorectal cancers. Immune contexture, including spatial distribution of immune cells in the tumor microenvironment (TME), may predict immunotherapy outcome. EXPERIMENTAL DESIGN: Immune contexture and spatial distribution, including cell-to-cell distance measurements, were analyzed by multiplex immunofluorescence (mIF) in primary colorectal cancers with d-MMR (N = 33) from patients treated with anti-PD-1 antibodies. By digital image analysis, density, ratio, intensity, and spatial distribution of PD-L1, PD-1, CD8, CD3, CD68, LAG3, TGFßR2, MHC-I, CD14, B2M, and pan-cytokeratin were computed. Feature selection was performed by regularized Cox regression with LASSO, and a proportional hazards model was fitted to predict progression-free survival (PFS). RESULTS: For predicting survival among patients with MMR-d advanced colorectal cancer receiving PD-1 blockade, cell-to-cell distance measurements, but not cell densities or ratios, achieved statistical significance univariately. By multivariable feature selection, only mean number of PD-1+ cells within 10 µm of a PD-L1+ cell was significantly predictive of PFS. Dichotomization of this variable revealed that those with high versus low values had significantly prolonged PFS [median not reached (>83 months) vs. 8.5 months (95% confidence interval (95% CI), 4.7-NR)] with a median PFS of 28.4 months for all patients [adjusted HR (HRadj) = 0.14; 95% CI, 0.04-0.56; P = 0.005]. Expression of PD-1 was observed on CD8+ T cells; PD-L1 on CD3+ and CD8+ T lymphocytes, macrophages (CD68+), and tumor cells. CONCLUSIONS: In d-MMR colorectal cancers, PD-1+ to PD-L1+ receptor to ligand proximity is a potential predictive biomarker for the effectiveness of PD-1 blockade.

Associations between AI-Assisted Tumor Amphiregulin and Epiregulin IHC and Outcomes from Anti-EGFR Therapy in the Routine Management of Metastatic Colorectal Cancer.

PURPOSE: High tumor production of the EGFR ligands, amphiregulin (AREG) and epiregulin (EREG), predicted benefit from anti-EGFR therapy for metastatic colorectal cancer (mCRC) in a retrospective analysis of clinical trial data. Here, AREG/EREG IHC was analyzed in a cohort of patients who received anti-EGFR therapy as part of routine care, including key clinical contexts not investigated in the previous analysis. EXPERIMENTAL DESIGN: Patients who received panitumumab or cetuximab ± chemotherapy for treatment of RAS wild-type mCRC at eight UK cancer centers were eligible. Archival formalin-fixed paraffin-embedded tumor tissue was analyzed for AREG and EREG IHC in six regional laboratories using previously developed artificial intelligence technologies. Primary endpoints were progression-free survival (PFS) and overall survival (OS). RESULTS: A total of 494 of 541 patients (91.3%) had adequate tissue for analysis. A total of 45 were excluded after central extended RAS testing, leaving 449 patients in the primary analysis population. After adjustment for additional prognostic factors, high AREG/EREG expression (n = 360; 80.2%) was associated with significantly prolonged PFS [median: 8.5 vs. 4.4 months; HR, 0.73; 95% confidence interval (CI), 0.56-0.95; P = 0.02] and OS [median: 16.4 vs. 8.9 months; HR, 0.66 95% CI, 0.50-0.86; P = 0.002]. The significant OS benefit was maintained among patients with right primary tumor location (PTL), those receiving cetuximab or panitumumab, those with an oxaliplatin- or irinotecan-based chemotherapy backbone, and those with tumor tissue obtained by biopsy or surgical resection. CONCLUSIONS: High tumor AREG/EREG expression was associated with superior survival outcomes from anti-EGFR therapy in mCRC, including in right PTL disease. AREG/EREG IHC assessment could aid therapeutic decisions in routine practice. See related commentary by Randon and Pietrantonio, p. 4021.

Lactoferricin mediates anabolic and anti-catabolic effects in the intervertebral disc.

Lactoferricin (LfcinB) antagonizes biological effects mediated by angiogenic and catabolic growth factors, in addition to pro-inflammatory cytokines and chemokines in human endothelial cells and tumor cells. However, the effect of LfcinB on intervertebral disc (IVD) cell metabolism has not yet been investigated. Using bovine nucleus pulposus (NP) cells, we analyzed the effect of LfcinB on proteoglycan (PG) accumulation, PG synthesis, and anabolic gene expression. We assessed expression of genes for matrix-degrading enzymes such as matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family), as well as their endogenous inhibitors, tissue inhibitor of metalloproteases (TIMPs). In order to understand the specific molecular mechanisms by which LfcinB exerts its biological effects, we investigated intracellular signaling pathways in NP cells. LfcinB increased PG accumulation mainly via PG synthesis in a dose-dependent manner. Simultaneously, LfcinB dose-dependently downregulated catabolic enzymes. LfcinB's anti-catabolic effects were further demonstrated by a dose-dependent increase in multiple TIMP family members. Our results demonstrate that ERK and/or p38 mitogen-activated protein kinase pathways are the key signaling cascades that exert the biological effects of LfcinB in NP cells, regulating transcription of aggrecan, SOX-9, TIMP-1, TIMP-2, TIMP-3, and iNOS. Our results suggest that LfcinB has anabolic and potent anti-catabolic biological effects on bovine IVD cells that may have considerable promise in the treatment of disc degeneration in the future.

Biological effects of the plant-derived polyphenol resveratrol in human articular cartilage and chondrosarcoma cells.

The natural phytoestrogen resveratrol (RSV) may have therapeutic potential for arthritic conditions. RSV is chondroprotective for articular cartilage in rabbit models for arthritis, but its biological effects on human articular cartilage and chondrosarcoma cells are unknown. Effects of RSV on human articular cartilage homeostasis were studied by assessing production of matrix-degrading enzymes (MMP-13, ADAMTS4, and ADAMTS5), as well as proteoglycan production and synthesis. The counteractions of RSV against catabolic factors (e.g., FGF-2 or IL-1ß) were examined by in vitro and ex vivo using monolayer, three-dimensional alginate beads and cartilage explants cultures, respectively. RSV improves cell viability of articular chondrocytes and effectively antagonizes cartilage-degrading protease production that was initiated by catabolic and/or anti-anabolic cytokines in human articular chondrocytes. RSV significantly also enhances BMP7-promoted proteoglycan synthesis as assessed by (35) S-sulfate incorporation. Protein-DNA interaction arrays suggest that RSV inhibits the activation of transcription factors involved in inflammation and cartilage catabolic signaling pathways, including direct downstream regulators of MAPK (e.g., AP-1, PEA3) and NFκB. RSV selectively compromises survival of human chondrosarcoma cells, but not primary articular chondrocytes, revealing cell-specific activity of RSV on non-tumorigenic versus tumor-derived cells. We propose that RSV exerts its chondroprotective functions, in part, by deactivating p53-induced apoptosis in human primary chondrocytes, but not human chondrosarcoma. Our findings suggest that RSV has potential as a unique biologic treatment for both prevention and treatment of cartilage degenerative diseases.

Fibroblast growth factor-2 promotes catabolism via FGFR1-Ras-Raf-MEK1/2-ERK1/2 axis that coordinates with the PKCδ pathway in human articular chondrocytes.

Fibroblast growth factor 2 (FGF-2) has been found to play an anti-anabolic and/or a catabolic role in adult human articular cartilage via regulation of multiple signaling pathways. Upon FGF-2 stimulation, a molecular crosstalk between the mitogen activated protein kinase (MAPK) and protein kinase C δ (PKCδ) pathways are initiated, where PKCδ positively regulates downstream MAPK signaling. In this study, we explored the relationship between fibroblast growth factor receptor 1 (FGFR1), Ras, and PKCδ in FGF-2 signaling in human articular chondrocytes. Pathway-specific inhibition using both chemical inhibitors and siRNA targeting FGFR1 demonstrated that, upon FGF-2 stimulation, FGFR1 controlled both Ras and PKCδ activation, which converged on the Raf-MEK1/2-ERK1/2 axis. No crosstalk was observed between Ras and PKCδ. Quantitative PCR analyses revealed that both Ras and PKCδ contributed to FGF-2-mediated upregulation of MMP-13, ADAMTS5, and repression of aggrecan gene. Correspondingly, FGF-2-mediated proteoglycan loss was effectively reversed by individual pathway-specific inhibitor of Ras, PKCδ, and ERK1/2 in both 3-dimensional alginate bead culture and cartilage organ culture systems. Our findings suggest that FGFR1 interacts with FGF-2 and then activates Ras and PKCδ, which concertedly drive MAPK signaling to mediate biological effects of FGF-2. Such an integration of dual inputs constitutes a novel mechanism of FGF-2 signaling cascade in human articular chondrocytes.

Species-specific biological effects of FGF-2 in articular cartilage: implication for distinct roles within the FGF receptor family.

Existing literature demonstrates that fibroblast growth factor-2 (FGF-2) exerts opposing, contradictory biological effects on cartilage homeostasis in different species. In human articular cartilage, FGF-2 plays a catabolic and anti-anabolic role in cartilage homeostasis, driving homeostasis toward degeneration and osteoarthritis (OA). In murine joints, however, FGF-2 has been identified as an anabolic mediator as ablation of the FGF-2 gene demonstrated increased susceptibility to OA. There have been no previous studies specifically addressing species-specific differences in FGF-2-mediated biological effects. In this study, we provide a mechanistic understanding by which FGF-2 exerts contradictory biological effects in human versus murine tissues. Using human articular cartilage (ex vivo) and a medial meniscal destabilization (DMM) animal model (in vivo), species-specific expression patterns of FGFR receptors (FGFRs) are elucidated between human and murine articular cartilage. In the murine OA model followed by intra-articular injection of FGF-2, we further correlate FGFR profiles to changes in behavioral pain perception, proteoglycan content in articular cartilage, and production of inflammatory (CD11b) and angiogenic (VEGF) mediators in synovium lining cells. Our results suggest that the fundamental differences in cellular responses between human and murine tissues may be secondary to distinctive expression patterns of FGFRs that eventually determine biological outcomes in the presence of FGF-2. The complex interplay of FGFRs and the downstream signaling cascades induced by FGF-2 in human cartilage should add caution to the use of this particular growth factor for biological therapy in the future.

Comprehensive Analysis and Summary of the Value of Immunophenotypes of Mature NK Cell Tumors for Differential Diagnosis, Treatment, and Prognosis.

Background: Few studies have been performed to comprehensively analyze and summarize the immunophenotype and differential diagnosis of mature NK cell tumors, and there is often overlap between tumorigenic and reactive NK cell phenotypes. Furthermore, the impact of different phenotypes on patient prognosis has rarely been reported. Methods: The degree of expression of extracellular and intracellular markers of NK cells in each group was compared by FCM, and the differences in expression of various markers among different disease groups and their impact on prognosis have been analyzed and summarized. Results: Compared with normal NK cells, tumor cells of ANKL and ENKTL had characteristics of being more activated and progressive with larger FSC, in contrast to NK-CLPD and RNKL. Differential diagnoses with RNKL, ANKL, and ENKTL have broader FCM clues. In contrast, the phenotypes of NK-CLPD and RNKL are not significantly different, and consistent phenotypic abnormalities require ongoing monitoring to confirm malignant clones. The sensitivity of differentiating malignant NK cells from reactive NK cells by KIRs alone was poor. The clustering results showed that CD5, CD16, CD56, CD57, CD94, CD45RA, CD45RO, HLA-DR, KIRs, Granzyme B, Perforin and Ki-67 were differentially distributed in the expression of three NK cell tumors and reactive NK cell hyperplasia, so a comprehensive judgment using a wide range of antibody combinations is required in disease staging diagnosis. The tumor cell loads in BM and PB were also compared, and there was a clear correlation between the two. Moreover, the sensitivity of PB for monitoring tumor cells was up to 87.10%, suggesting that PB could be used as an alternative to BM for the diagnosis and screening of NK cell tumors. Analysis of the phenotypic impact of ENKTL patients on prognosis showed that those with CD7 and CD45RO expression had a poor prognosis, while those with positive KIRs had a better prognosis. Conclusion: This study systematically characterized the FCM of mature NK cell tumors, emphasizing the importance and clinical value of accurate immunophenotyping in diagnosing, classifying, determining prognosis, and guiding treatment of the disease.

Differential diagnosis and identification of prognostic markers for peripheral T-cell lymphoma subtypes based on flow cytometry immunophenotype profiles.

We compared the differential expression of 15 markers in PTCL (Peripheral T-cell lymphoma) subtypes and T-CUS (T-cell clones of uncertain significance), and summarized the specific immunophenotype profiles of each subtype and its impact on prognosis. PD-1 and CD10 are diagnostic markers for AITL (angioimmunoblastic T-cell lymphoma). To avoid confusion with T-CUS of benign clones, it is recommended to define AITL as bounded by PD-1+%>38.01 and/or CD10+%>7.46. T cell-derived ENKTL-N (extranodal NKT cell lymphoma) specifically expresses CD56. ALCL (anaplastic large cell lymphoma) characteristically expresses CD30 and HLA-DR. PTCL-NOS (peripheral T-cell lymphoma unspecified) still lacks a relatively specific phenotype and is prone to loss of basic lineage markers CD3, CD5, and CD7. The determination of T-CUS can be verified by the overall assessment of the bone marrow and a certain period of follow-up. The clustering results showed that the expression of 8 specific markers was significantly different among the 5 groups, suggesting that a combination of related markers can be analyzed in the identification of PTCLs subtypes. The study explores the advantages of TRBC1 combined with CD45RA/CD45RO in detecting T cell clonality, which can efficiently and sensitively analyze multiple target T cell populations at the same time. The sensitivity of PB to replace BM to monitor the tumor burden or MRD (minimal residual disease) of PTCLs is as high as 85.71%, which can relieve the huge pressure of clinical sampling and improve patient compliance. CD7, CD38, and Ki-67 are prognostic indicators for AITL. CD3 and CD8 on PTCL-NOS, and CD56 and HLA-DR on ENKTL-N have prognostic role. This study supports and validates the current classification of PTCL subtypes and establishes an immunophenotypic profile that can be used for precise diagnosis. The important clinical value of PTCLs immunophenotype in routine classification diagnosis, clonality confirmation, prognosis prediction, and treatment target selection was emphasized.

Immune microenvironment in patients with mismatch-repair-proficient oligometastatic colorectal cancer exposed to chemotherapy: the randomized MIROX GERCOR cohort study.

In the era of immune checkpoint inhibitors, understanding the metastatic microenvironment of proficient mismatch repair/microsatellite stable (pMMR/MSS) colorectal cancer (CRC) is of paramount importance to both prognostication and the development of more effective novel therapies. In this study, primary and paired metastasis tissue samples were collected from patients with resectable metastatic CRC treated with adjuvant FOLFOX or peri-operative chemotherapy in the MIROX phase III prospective study. In total, 74 cancer tissues were stained for CD3, CD8, Forkhead box protein 3 (FOXP3), programmed cell death protein-1 (PD-1, invasive front, stromal, intra-epithelial compartments), and programmed death-ligand 1 (PD-L1, tumor, immune cells). The immune profiling of primary CRC had a limited value to predict the immune context of paired metastases for all markers but CD3+. The expression of CD8 and PD-L1 was higher in metastases after neoadjuvant FOLFOX. In metastases, both CD3 T cells at the invasive front and PD-L1 expressions on immune cells were predictive of better disease-free survival. These results show that the effect of FOLFOX on modifying the immune microenvironment in resected CRC metastases and measurement of PD-L1 expression and tumor-infiltrating CD8 T cells in pMMR/MSS metastatic tissue samples could improve treatment strategies of metastatic CRC patients.

Alteration of sensory neurons and spinal response to an experimental osteoarthritis pain model.

OBJECTIVE: To verify the biologic links between progressive cellular and structural alterations within knee joint components and development of symptomatic chronic pain that are characteristic of osteoarthritis (OA), and to investigate the molecular basis of alterations in nociceptive pathways caused by OA-induced pain. METHODS: An animal model of knee joint OA pain was generated by intraarticular injection of mono-iodoacetate (MIA) in Sprague-Dawley rats, and symptomatic pain behavior tests were performed. Relationships between development of OA with accompanying pain responses and gradual alterations in cellular and structural knee joint components (i.e., cartilage, synovium, meniscus, subchondral bone) were examined by histologic and immunohistologic analysis, microscopic examination, and microfocal computed tomography. Progressive changes in the dynamic interrelationships between peripheral knee joint tissue and central components of nociceptive pathways caused by OA-induced pain were examined by investigating cytokine production and expression in sensory neurons of the dorsal root ganglion and spinal cord. RESULTS: We observed that structural changes in components of the peripheral knee joint correlate with alterations in the central compartments (dorsal root ganglia and the spinal cord) and symptomatic pain assessed by behavioral hyperalgesia. Our comparative gene expression studies revealed that the pain pathways in MIA-induced knee OA may overlap, at least in part, with neuropathic pain mechanisms. Similar results were also observed upon destabilization of the knee joint in the anterior cruciate ligament transection and destabilization of the medial meniscus models of OA. CONCLUSION: Our results indicate that MIA-induced joint degeneration in rats generates an animal model that is suitable for mechanistic and pharmacologic studies on nociceptive pain pathways caused by OA, and provide key in vivo evidence that OA pain is caused by central sensitization through communication between peripheral OA nociceptors and the central sensory system. Furthermore, our data suggest a mechanistic overlap between OA-induced pain and neuropathic pain.

Assessment of MYC/PTEN Status by Gene-Protein Assay in Grade Group 2 Prostate Biopsies.

This study leveraged a gene-protein assay to assess MYC and PTEN status at prostate cancer biopsy and examined the association with adverse outcomes after surgery. MYC gain and PTEN loss were simultaneously assessed by chromogenic in situ hybridization and immunohistochemistry, respectively, using 277 Grade Group 2 needle biopsies that were followed by prostatectomy. The maximal size of cribriform Gleason pattern 4 carcinoma (CRIB), the presence of intraductal carcinoma (IDC), and percentage of Gleason pattern 4 carcinoma at biopsy were also annotated. MYC gain or PTEN loss was present in 19% and 18% of biopsies, respectively, whereas both alterations were present in 9% of biopsies. Tumors with one or both alterations were significantly more likely to have non-organ-confined disease (NOCD) at radical prostatectomy. In logistic regression models, including clinical stage, tumor volume on biopsy, and presence of CRIB/IDC, cases with MYC gain and PTEN loss remained at higher risk for NOCD (odds ratio, 6.23; 95% CI, 1.74-24.55; P = 0.005). The area under the curve for a baseline model using CAPRA variables (age, prostate-specific antigen, percentage of core involvement, clinical stage) was increased from 0.68 to 0.69 with inclusion of CRIB/IDC status and to 0.75 with MYC/PTEN status. Dual MYC/PTEN status can be assessed in a single slide and is independently associated with increased risk of NOCD for Grade Group 2 biopsies.

Artificial Intelligence-Assisted Amphiregulin and Epiregulin IHC Predicts Panitumumab Benefit in RAS Wild-Type Metastatic Colorectal Cancer.

PURPOSE: High tumor mRNA levels of the EGFR ligands amphiregulin (AREG) and epiregulin (EREG) are associated with anti-EGFR agent response in metastatic colorectal cancer (mCRC). However, ligand RNA assays have not been adopted into routine practice due to issues with analytic precision and practicality. We investigated whether AREG/EREG IHC could predict benefit from the anti-EGFR agent panitumumab. EXPERIMENTAL DESIGN: Artificial intelligence algorithms were developed to assess AREG/EREG IHC in 274 patients from the PICCOLO trial of irinotecan with or without panitumumab (Ir vs. IrPan) in RAS wild-type mCRC. The primary endpoint was progression-free survival (PFS). Secondary endpoints were RECIST response rate (RR) and overall survival (OS). Models were repeated adjusting separately for BRAF mutation status and primary tumor location (PTL). RESULTS: High ligand expression was associated with significant PFS benefit from IrPan compared with Ir [8.0 vs. 3.2 months; HR, 0.54; 95% confidence interval (CI), 0.37-0.79; P = 0.001]; whereas low ligand expression was not (3.4 vs. 4.4 months; HR, 1.05; 95% CI, 0.74-1.49; P = 0.78). The ligand-treatment interaction was significant (P interaction = 0.02) and remained significant after adjustment for BRAF-mutation status and PTL. Likewise, RECIST RR was significantly improved in patients with high ligand expression (IrPan vs. Ir: 48% vs. 6%; P < 0.0001) but not those with low ligand expression (25% vs. 14%; P = 0.10; P interaction = 0.01). The effect on OS was similar but not statistically significant. CONCLUSIONS: AREG/EREG IHC identified patients who benefitted from the addition of panitumumab to irinotecan chemotherapy. IHC is a practicable assay that may be of use in routine practice.

STAT3 and STAT6 Signaling Pathways Synergize to Promote Cathepsin Secretion from Macrophages via IRE1α Activation.

Tumor-associated macrophages play critical roles during tumor progression by promoting angiogenesis, cancer cell proliferation, invasion, and metastasis. Cysteine cathepsin proteases, produced by macrophages and cancer cells, modulate these processes, but it remains unclear how these typically lysosomal enzymes are regulated and secreted within the tumor microenvironment. Here, we identify a STAT3 and STAT6 synergy that potently upregulates cathepsin secretion by macrophages via engagement of an unfolded protein response (UPR) pathway. Whole-genome expression analyses revealed that the TH2 cytokine interleukin (IL)-4 synergizes with IL-6 or IL-10 to activate UPR via STAT6 and STAT3. Pharmacological inhibition of the UPR sensor IRE1α blocks cathepsin secretion and blunts macrophage-mediated cancer cell invasion. Similarly, genetic deletion of STAT3 and STAT6 signaling components impairs tumor development and invasion in vivo. Together, these findings demonstrate that cytokine-activated STAT3 and STAT6 cooperate in macrophages to promote a secretory phenotype that enhances tumor progression in a cathepsin-dependent manner.

The anti-catabolic role of bovine lactoferricin in cartilage.

Bovine lactoferricin (LfcinB) is a multifunctional peptide derived from bovine lactoferrin that demonstrates antibacterial, antifungal, antiviral, antitumor, and immunomodulatory activities. Recently, studies have focused on the anti-catabolic and anti-inflammatory potential of LfcinB. LfcinB is able to modulate the effects cytokines such as IL-1 and fibroblast growth factor 2 as well as promote specific cartilage anabolic factors. These properties are particularly important in maintaining cartilage homeostasis and preventing a catabolic state, which leads to clinical pathology. This review focuses on the recent literature elucidating the role of LfcinB in preventing cartilage degradation.