HER3 overexpression: a predictive marker for poor prognosis in advanced ALK-positive non-small cell lung cancer treated with ALK inhibitors.

Background: Anaplastic lymphoma kinase (ALK)-targeted tyrosine kinase inhibitors (TKIs) improve patient survival; however, some patients develop ALK-TKI resistance with unidentified mechanisms. We investigated ErbB family and c-MET expression in patients with ALK-positive non-small cell lung cancer (NSCLC) to understand their roles in the ALK-TKI response. Methods: We studied 72 patients with advanced ALK-positive NSCLC with EML4-ALK fusion variant subtyping and immunostaining for c-MET, EGFR, HER2, and HER3 on tissue specimens both pre- (primary) and post-treatment (secondary) with ALK-TKI. We investigated the association of their expression with survival outcomes and assessed the effectiveness of combining ALK and EGFR inhibitors in ALK-positive NSCLC cell lines stimulated with the HER3-specific ligand HRG1. Results: High expression of c-MET, EGFR, HER2, and HER3 was observed in 4.9%, 18.0%, 1.6%, and 25.8% of primary tumors, respectively, and 18.5%, 37.0%, 10.7%, and 35.7% of secondary tumors, respectively. HER3 overexpression in primary tumors showed inferior survival (P=0.132). In the subgroup with EML4-ALK variant 1/2 (V1/V2), HER3 overexpression was significantly associated with inferior survival in both primary and secondary tumors (P=0.022 and P=0.004, respectively). Combination treatment with lorlatinib and erlotinib significantly reduced HRG1-induced activation of RTK signaling in ALK-positive NSCLC cells. Conclusions: HER3 overexpression has potential as a prognostic marker in ALK-positive NSCLCs, including ALK-TKI naïve and treated cases, especially those with EML4-ALK V1/V2. Assessing HER3 expression may be crucial for treatment planning and outcome prediction in these patients.

Overexpression of the NEK9-EG5 axis is a novel metastatic marker in pathologic stage T3 colon cancer.

NEK9 is a key player in the NEK9-EG5 axis for microtubule polymerization, chromosome alignment, and mitosis. In present study, we investigated the altered expression of the NEK9, EG5 and acetyl-α-tubulin as well as common epithelial-mesenchymal transition (EMT) markers (E-cadherin, vimentin, claudin-1, and ß-catenin) through the immunohistochemistry analysis of 138 patients with pathologic T3 (pT3) stage colon cancers, and evaluated their metastatic potential. NEK9 expression showed an association with distant metastasis (P = 0.032) and was an independent predictive factor for distant metastasis (HR = 3.365, P < 0.001) by multivariate analysis, which was more significant than either the regional nodal metastasis (HR = 2.496, P = 0.007) or lymphovascular invasion (HR = 2.090, P = 0.153). Positive correlations were observed between NEK9 and EG5 or acetyl-α-tubulin (r = 0.236 and P = 0.007; r = 0.181 and P = 0.038, respectively) and concordant overexpression of the NEK9-EG5 axis was further confirmed in colon cancer cell lines. These findings collectively suggest that the overexpression of the NEK9-EG5 axis is present and associated with distant metastasis in colon cancer. These biomarkers might be useful for predicting metastatic potential among the patients with pT3 colon cancers.

EML4-ALK Variant 3 Promotes Mitotic Errors and Spindle Assembly Checkpoint Deficiency Leading to Increased Microtubule Poison Sensitivity.

EML4-ALK is an oncogenic fusion protein present in approximately 5% of non-small cell lung cancers (NSCLC). Alternative breakpoints in the gene encoding EML4 result in distinct variants that are linked to markedly different patient outcomes. Patients with EML4-ALK variant 3 (V3) respond poorly to ALK inhibitors and have lower survival rates compared with patients with other common variants, such as V1. Here, we use isogenic Beas-2B bronchial epithelial cell lines expressing EML4-ALK V1 or V3, as well as ALK-positive NSCLC patient cells that express V1 (H3122 cells) or V3 (H2228 cells), to show that EML4-ALK V3 but not V1 leads to hyperstabilized K-fibers in mitosis, as well as errors in chromosome congression and segregation. This is consistent with our observation that EML4-ALK V3 but not V1 localizes to spindle microtubules and that wild-type EML4 is a microtubule stabilizing protein. In addition, cells expressing EML4-ALK V3 exhibit loss of spindle assembly checkpoint control that is at least in part dependent on ALK catalytic activity. Finally, we demonstrate that cells expressing EML4-ALK V3 have increased sensitivity to microtubule poisons that interfere with mitotic spindle assembly, whereas combination treatment with paclitaxel and clinically approved ALK inhibitors leads to a synergistic response in terms of reduced survival of H2228 cells. IMPLICATIONS: This study suggests that combining the microtubule poison, paclitaxel, with targeted ALK inhibitors may provide an effective new treatment option for patients with NSCLC with tumors that express the EML4-ALK V3 oncogenic fusion.

A Polytherapy Strategy Using Vincristine and ALK Inhibitors to Sensitise EML4-ALK-Positive NSCLC.

The oncogenic fusion of EML4-ALK is present in about 4-6% of non-small cell lung cancer (NSCLC). A targeted approach with ALK tyrosine kinase inhibitors (TKIs) has been proven highly effective in ALK-positive NSCLC patients. However, despite the initial responses, the outcome of the treatment is variable. Previous studies have shown that the differential response depends in part on the type of EML4-ALK variant. Here, we examined the combination of ALK inhibitors and microtubule poison, vincristine, in cells expressing EML4-ALK V1 and V3, the two most common variants in NSCLC. We showed that combination therapy of ALK-TKIs with vincristine had anti-proliferative effects and blocked RAS/MAPK, PI3K/AKT and JAK/STAT3 signalling pathways in EML4-ALK V1 but not V3 cells. Our results demonstrate that high levels of tubulin acetylation are associated with poor response to vincristine in EML4-ALK V3 cells. Additionally, we demonstrated differences in microtubule stability between the two EML4-ALK fusions. EML4-ALK V3 cells exhibited dynamic microtubules that confer poor response to vincristine compared to V1 cells. Hence, we suggested that the portion of EML4 in the fusion has an important role for the outcome of the combination treatment.

Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation.

Variants of the oncogenic EML4-ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4-ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4-ALK V3 but not V1 protein re-localises to microtubules, an effect recapitulated in a catalytically inactive EML4-ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild-type and catalytically inactive EML4-ALK V3 proteins, but not a Lys-Glu salt bridge mutant. We propose that EML4-ALK foci formation occurs as a result of transient association of stable EML4-ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4-ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors.

Distinct mutational profile and immune microenvironment in microsatellite-unstable and POLE-mutated tumors.

INTRODUCTION: Mismatch repair (MMR)-deficient and DNA polymerase epsilon (POLE)-mutated tumors exhibit a high tumor mutation burden (TMB) and have been proven to be associated with good responses to immune checkpoint inhibitor treatments. However, the relationship between mutational characteristics of MMR-deficient and POLE-mutated tumors and the spatial architecture of tumor-infiltrating lymphocytes (TILs) has not been fully evaluated. METHODS: We retrieved microsatellite instability-high (MSI-high, N=20) and POLE-mutated (N=47) cases from the clinical next-generation sequencing cohort at Asan Medical Center. Whole-slide immunostaining for CD3, CD4, CD8, FoxP3 and PD-1 were performed with tissue samples of colorectal and gastric cancer (N=24) and the tumor-positive TIL cell densities were correlated with the tumor's mutational features. The findings were compared with the results of similar analyses in The Cancer Genome Atlas-Colorectal Adenocarcinoma (TCGA-COADREAD) cohort (N=592). RESULTS: The MSI-high group showed significantly higher overall TMBs with a number of insertion/deletion (indel) mutations relative to the POLE-mutated group (median TMB; 83.6 vs 12.5/Mb). Oncogenic/likely-oncogenic POLE mutations were identified with ultrahypermutations (≥100 mutations/Mb) (2/47, 4.3%). Concurrent POLE mutations of unknown significance and MSI-high cases were identified in eight cases (8/67, 11%), and two of these colorectal cancers had multiple POLE mutations, showing an ultramutated phenotype (378.1 and 484.4/Mb) and low indel mutation burdens with complete loss of MSH-6 or PMS-2, which was similar to the mutational profile of the POLE-inactivated tumors. Intratumoral CD3-positive, CD4-positive, CD8-positive, FoxP3-positive and PD-1-positive TIL cell densities were more strongly correlated with the indel mutation burden than with the total TMB (correlation coefficient, 0.61-0.73 vs 0.23-0.38). In addition, PI3K/AKT/mTOR pathway mutations were commonly found in MSI-high tumors (75%) but not in POLE-mutated tumors. CONCLUSIONS: Indel mutation burden rather than total TMB could serve as a predictor of high TILs in both MSI-high and POLE-mutated tumors. Multiple uncharacterized/non-pathogenic POLE mutations occurring via MMR deficiency within MSI-high tumors may have combined pathogenic roles. A mutated PI3K/AKT/mTOR pathway may be a biomarker that can be used to stratify patients with advanced MSI-high tumors for immune therapy.

EML4-ALK V3 oncogenic fusion proteins promote microtubule stabilization and accelerated migration through NEK9 and NEK7.

EML4-ALK is an oncogenic fusion present in ∼5% of non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants of EML4-ALK with different patient outcomes. Here, we show that, in cell models, EML4-ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. EML4-ALK V3 also recruits the NEK9 and NEK7 kinases to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but does not require ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4-ALK patients. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through NEK9 and NEK7, leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4-ALK lung cancer.

High Grade Transformation in Mucoepidermoid Carcinoma of the Minor Salivary Gland with Polyploidy of the Rearranged MAML2 Gene.

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor of the salivary gland. However, reports of high grade transformation in MEC are extremely rare, and only two cases have so far been described. Recent development of salivary gland pathology revealed recurrent gene rearrangements in many kinds of tumors, including MAML2 fusion of MEC. To date, the MAML2 status of high grade transformed MEC has not been studied. Here we report the first case of minor salivary gland origin high grade transformation in MEC with a MAML2 break apart FISH study. A 73-year-old woman presented with a 1-month history of left mandibular area swelling, and computed tomography and magnetic resonance imaging revealed a mass in the hard palate with various-sized lymphadenopathy of the neck. The resected tumor histologically consisted of two carcinomatous components. Approximately 30% of the tumor showed a conventional MEC feature, while 70% was comprised of a high grade transformed component. In the conventional MEC component, FISH revealed MAML2 rearrangement. High grade transformed cells showed multiple split signals, and the results were interpreted as rearrangement and polyploidy after comparison with 1p/19q FISH as validation. The patient received adjuvant radiation therapy after wide resection with neck dissection and retropharyngeal dissection. Nevertheless, as the remaining tumor grew up rapidly and metastatic lymph nodes were newly revealed, the patient expired 7 months after the diagnosis. We first report regarding a high grade transformation in MEC with polyploidy of the rearranged MAML2 gene and aggressive biological behavior.

Therapeutic effect of mesenchymal stem cells derived from human umbilical cord in rabbit temporomandibular joint model of osteoarthritis.

Osteoarthritis (OA) is a degenerative condition of the temporomandibular joint (TMJ) characterised by chronic inflammation and damage to joint structures. Because of the complexity of TMJ-OA, only symptomatic treatments are currently available. Recent reports have shown that many of stem cells can exert anti-inflammatory and tissue-regenerating effects. In this study, we investigated the potential cartilage-regenerating and anti-inflammatory effects of human umbilical cord matrix-mesenchymal stem cells (hUCM-MSCs) for the treatment of TMJ-OA. hUCM-MSC lines, isolated from different donors, which showed different activities in vitro. Using a selected cell line, we used different concentrations of hUCM-MSCs to assess therapeutic effects in a rabbit model of monosodium iodoacetate-induced TMJ-OA. Compared with the untreated control group, the potential regenerative result and anti-inflammatory effects of hUCM-MSCs were evident at all the tested concentrations in rabbits with induced TMJ-OA. The median dose of hUCM-MSCs showed the prominent cartilage protective effect and further cartilage regeneration potential. This effect occurred via upregulated expression of growth factors, extracellular matrix markers, and anti-inflammatory cytokines, and reduced expression of pro-inflammatory cytokines. The anti-inflammatory effect of hUCM-MSCs was comparable to that of dexamethasone (DEX). However, only hUCM-MSCs showed potential chondrogenesis effects in this study. In conclusion, our results indicate that hUCM-MSCs may be an effective treatment option for the treatment of TMJ-OA.

Umbilical cord-derived mesenchymal stem cell extracts ameliorate atopic dermatitis in mice by reducing the T cell responses.

Mesenchymal stem cells derived from Wharton's jelly of the umbilical cord (UC-MSCs) have immunomodulatory properties. The aim of this study was to explore whether extracts of MSCs (MSC-Ex) could augment the low therapeutic efficacy of the whole cells in an Aspergillus fumigatus (Af)-induced atopic dermatitis (AD) model. LPS- or TNF-α/IFN-γ-stimulated keratinocytes (HaCaT cells) were treated with MSC-Ex, and the Af-induced AD model was established in BALB/c mice. In HaCaT cells, MSC-Ex treatment significantly reduced the inflammatory cytokine (IL-6, IL-1ß, IL-4, IL-5 and TNF-α), iNOS and NF-κB levels, and upregulated the anti-inflammatory cytokines (IL-10 and TGF-ß1). In the AD mice, the MSC-Ex group showed greatly reduced dermatitis, and lower clinical symptom scores and IgE levels. The histological dermatitis scores were also markedly lower in the MSC-Ex-treated animals compared with the MSC-treated group. Decreased levels of IFN-γ (Th1) and IL-17 (Th17), IL-4 and IL-13 (Th2) were detected in T cells and the skin tissue from the MSC-Ex treated AD mice. The therapeutic capacity of MSC-Ex was preserved after lyophilization and reconstitution. MSC-Ex treatment reproducibly suppresses dermatitis and inhibits the induction of inflammatory cytokines in the skin of AD mice. MSC-Ex is therefore a potential new treatment agent for AD.

Correction to: A novel histone deacetylase inhibitor, CG200745, potentiates anticancer effect of docetaxel in prostate cancer via decreasing Mcl-1 and Bcl-XL.

The blots of control and docetaxel for caspase-9, caspase-3, caspase-8, Bcl-XL, and tubulin in the Figure 4f were reused from Figure 4 of our previous paper published in Journal of Urology in 2010 ( https://doi.org/10.1016/j.juro.2010.07.035 ).

Characterization of Hepatocellular Carcinoma Patients with FGF19 Amplification Assessed by Fluorescence in situ Hybridization: A Large Cohort Study.

BACKGROUND: FGF19 amplification is a relatively novel type of genetic aberration that has been proposed to be a driver of hepatocarcinogenesis. Selective inhibitors of FGFR4, a receptor of FGF19, have been developed as targeted therapies for hepatocellular carcinoma (HCC). Despite the role of FGF19 in mediating HCC progression, the clinicopathological characterization of patients exhibiting FGF19 amplification remains unclear. Immunohistochemical staining is the simplest and most widely used method of identifying aberrations in the FGF19 gene, although its specificity is very low. METHODS: This study investigated the prognostic significance of FGF19 amplification in a large cohort of 989 HCC patients using fluorescence in situ hybridization (FISH), which has a high degree of specificity. In addition, FISH data from formalin-fixed, paraffin-embedded sections were compared with copy number variation (CNV) data obtained from fresh frozen sections to validate the use of FISH as a diagnostic tool. RESULTS: FGF19 amplifications were detected by FISH in 51 (5.15%) of the 989 patients, and were independently associated with poor survival and a higher risk of tumor recurrence, as well as with poor prognostic factors such as a high α-fetoprotein level, hepatitis B or C virus infection, a large tumor size, microvascular invasion, and necrosis. In addition, FGF19 amplification was associated with TP53 mutation, and was mutually exclusive with CTNNB1 mutation. The results of the FISH and CNV analyses exhibited a significant concordance rate of 96% (κ = 0.618, p < 0.001). CONCLUSIONS: These data indicate that FGF19 amplification represents a unique molecular subtype associated with poor prognostic characteristics, which supports the hypothesis that the FGF19-FGFR4 signaling pathway plays an important role in hepatocarcinogenesis. We have also demonstrated that FISH is a viable alternative to CNV analysis, offering a number of advantages in the clinical setting.

Correlation between progression-free survival, tumor burden, and circulating tumor DNA in the initial diagnosis of advanced-stage EGFR-mutated non-small cell lung cancer.

BACKGROUND: This study was conducted to identify whether the presence of circulating tumor DNA (ctDNA) in plasma before treatment with EGFR-tyrosine kinase inhibitors (TKIs) is associated with clinical outcomes. METHODS: Fifty-seven pairs of tissues and plasma samples were obtained from patients with NSCLC adenocarcinoma harboring activating EGFR mutations before the administration of EGFR-TKI treatment. ctDNA mutation was identified using the PANAMutyper EGFR mutation kit. Both qualitative and quantitative analyzes of the data were performed. RESULTS: Concordance rates with tissue biopsy were 40.4% and 59.6% for the qualitative and quantitative methods, respectively. Bone metastasis showed a statistically significant correlation with ctDNA detection (odds ratio 3.985, 95% confidence interval [CI] 1.027-15.457; P = 0.046). Progression-free survival (PFS) was significantly shorter in the group detected with ctDNA than in the undetected ctDNA group (median PFS 9.8 vs. 20.7 months; hazard ratio [HR] 2.30, 95% CI 1.202-4.385; P = 0.012). Detection of ctDNA before treatment with EGFR-TKIs (HR 2.388, 95% CI 1.138-5.014; P = 0.021) and extra-thoracic lymph node metastasis (HR 13.533, 95% CI 2.474-68.747; P = 0.002) were independently associated with PFS. Six of 11 patients (45.5%) monitored by serial sampling showed a dynamic change in ctDNA prior to disease progression. CONCLUSION: Quantitative testing can increase the sensitivity of the ctDNA detection test. Patients with detectable ctDNA had significantly shorter PFS after receiving EGFR-TKIs than those with undetectable ctDNA. Tumor burden may be associated with plasma ctDNA detection. A shorter PFS was associated with detection of ctDNA and extra-thoracic lymph node metastasis. Dynamic changes in the ctDNA level may help predict clinical outcomes.

A Review and Update on Papillary Immature Metaplasia of the Uterine Cervix: A Distinct Subset of Low-Grade Squamous Intraepithelial Lesion, Proposing a Possible Cell of Origin.

CONTEXT: - Papillary immature metaplasia (PIM) is a known papillary cervical lesion associated with low-risk human papillomavirus (LR-HPV). OBJECTIVE: - To evaluate additional clinicopathologic features and the HPV genotypes of PIM and discuss the presumptive cell of origin. DESIGN: - A total of 26 PIM cases were evaluated by p16INK4a, cytokeratin (CK) 7, and CK17 immunohistochemical stainings. Human papillomavirus genotyping was performed, by using HPV DNA Chip, HPV polymerase chain reaction (PCR), and real-time PCR. RESULTS: - Histologically, PIM forms either a papillary mass (n = 21 of 26, 81%) or a slightly elevated/flat plaque (n = 5, 19%). All cases contain variable amounts of mucinous epithelia within the lesions. Koilocytosis was identified in 15 of the 26 cases (58%). Sixteen cases (61%) were associated with LR-HPV (types 6, 11, or 42), but 3 cases (12%) with high-risk (HR) HPV (16, 16/18, and 33), 2 cases (8%) with mixed LR- and HR-HPV (6/16 and 11/58), while 2 cases (8%) were negative, but p16INK4a immunostaining showed nonblock positivity in all cases. Eight (31%) had high-grade squamous intraepithelial lesion (HSIL) in the adjacent mucosa, 4 (50%) of which showed direct continuity. Identical HPV subtypes were confirmed in separately microdissected cases from PIM and adjacent HSIL. Most lesions (n = 24, 92%) expressed CK17 (reserve cell marker) in a bottom-heavy pattern and CK7 (squamocolumnar junction [SCJ] marker) in a top-heavy pattern, while most cases of low-grade squamous intraepithelial lesion (LSIL) were negative for both markers. CONCLUSIONS: - Our results suggest that PIM is a distinct subset of LSIL showing a productive HPV infection, but PIM involves the transformation zone and is proximal to SCJ, while LSIL is mostly from ectocervix or distal to the SCJ.

Comparison of Immunohistochemistry and Direct Sanger Sequencing for Detection of the BRAF(V600E) Mutation in Thyroid Neoplasm.

BACKGROUND: The BRAF(V600E) mutation is the most common genetic alteration identified in papillary thyroid carcinoma (PTC). Because of its costs effectiveness and sensitivity, direct Sanger sequencing has several limitations. The aim of this study was to evaluate the efficiency of immunohistochemistry (IHC) as an alternative method to detect the BRAF(V600E) mutation in preoperative and postoperative tissue samples. METHODS: We evaluated 71 patients who underwent thyroid surgery with the result of direct sequencing of the BRAF(V600E) mutation. IHC staining of the BRAF(V600E) mutation was performed in 49 preoperative and 23 postoperative thyroid specimens. RESULTS: Sixty-two patients (87.3%) had PTC, and of these, BRAF(V600E) was confirmed by direct sequencing in 57 patients (91.9%). In 23 postoperative tissue samples, the BRAF(V600E) mutation was detected in 16 samples (70%) by direct sequencing and 18 samples (78%) by IHC. In 24 fine needle aspiration (FNA) samples, BRAF(V600E) was detected in 18 samples (75%) by direct sequencing and 16 samples (67%) by IHC. In 25 core needle biopsy (CNB) samples, the BRAF(V600E) mutation was detected in 15 samples (60%) by direct sequencing and 16 samples (64%) by IHC. The sensitivity and specificity of IHC for detecting the BRAF(V600E) mutation were 77.8% and 66.7% in FNA samples and 99.3% and 80.0% in CNB samples. CONCLUSION: IHC could be an alternative method to direct Sanger sequencing for BRAF(V600E) mutation detection both in postoperative and preoperative samples. However, application of IHC to detect the BRAF(V600E) mutation in FNA samples is of limited value compared with direct sequencing.

Protein Phosphatase Magnesium-Dependent 1δ (PPM1D) Expression as a Prognostic Marker in Adult Supratentorial Diffuse Astrocytic and Oligodendroglial Tumors.

BACKGROUND: Protein phosphatase magnesium-dependent 1δ (PPM1D) is a p53-induced serine/threonine phosphatase, which is overexpressed in various human cancers. A recent study reported that a mutation in the PPM1D gene is associated with poor prognosis in brainstem gliomas. In this study, we evaluated the utility of PPM1D as a prognostic biomarker of adult supratentorial diffuse astrocytic and oligodendroglial tumors. METHODS: To investigate PPM1D protein expression, mRNA expression, and copy number changes, immunohistochemistry, RNAscope in situ hybridization, and fluorescence in situ hybridization were performed in 84 adult supratentorial diffuse gliomas. We further analyzed clinical characteristics and overall survival (OS) according to PPM1D protein expression, and examined its correlation with other glioma biomarkers such as isocitrate dehydrogenase (IDH) mutation, and p53 expression. RESULTS: Forty-six cases (54.8%) were PPM1D-positive. PPM1D expression levels were significantly correlated with PPM1D transcript levels (p= .035), but marginally with PPM1D gene amplification (p=.079). Patients with high-grade gliomas showed a higher frequency of PPM1D expression than those with low-grade gliomas (p <.001). Multivariate analysis demonstrated that PPM1D expression (hazard ratio [HR], 2.58; p=.032), age over 60 years (HR, 2.55; p=.018), and IDH1 mutation (HR, 0.18; p=.002) were significantly independent prognostic factors; p53 expression had no prognostic significance (p=.986). The patients with tumor expressing PPM1D showed a shorter OS (p=.003). Moreover, patients with tumor harboring wild-type IDH1 and PPM1D expression had the worst OS (p<.001). CONCLUSIONS: Our data suggest that a subset of gliomas express PPM1D; PPM1D expression is a significant marker of poor prognosis in adult supratentorial diffuse astrocytic and oligodendroglial tumors.

Umbilical cord-derived mesenchymal stem cell extracts reduce colitis in mice by re-polarizing intestinal macrophages.

Human umbilical cord mesenchymal stem cells (hUC-MSCs), originating in Wharton's jelly, are multipotent stem cells that home to damaged tissues and can modulate the immune system. We examined whether administering extracts of MSCs (MSC-Ex) instead of MSCs could augment the beneficial effects of MSC therapy by overcoming the low homing efficiency of MSCs systemically administered in inflammatory bowel diseases (IBD). Dextran sodium sulfate-induced colitis model was established in C57BL/6 mice, and MSC-Ex was administered intraperitoneally. MSC-Ex reduced colitis, disease activity index (DAI), and histological colitis scores, and increased the body weight. Treatment with MSC-Ex completely blocked the induction of inflammatory cytokines, which were strongly detected in mice with colitis. MSC-Ex shifted the macrophage functional phenotype from M1 to M2 by decreasing the levels of MCP1, CXCL9, and iNOS, but increasing the levels of IL-10, LIGHT, CCL1, and Arg-1. MSC-Ex recovered the destruction of the epithelial barrier in the differentiated Caco-2 cells in vitro. Treatment with MSC-Ex was more potent than that with MSC in reducing DAI, the histological score, and nitrite levels. These data strongly support that MSC-Ex treatment can be a potent approach to overcome severe refractory IBD.

Induction of immunoglobulin transcription factor 2 and resistance to MEK inhibitor in melanoma cells.

Primary or acquired resistance to MEK inhibitors has been a barrier to successful treatment with MEK inhibitors in many tumors. In this study, we analyzed genome-wide gene expression profiling data from 6 sensitive and 6 resistant cell lines to identify candidate genes whose expression changes are associated with responses to a MEK inhibitor, selumetinib (AZD6244). Of 62 identified differentially expressed genes, we selected Immunoglobulin Transcription Factor 2, also known as transcription factor 4 as a potential drug resistance marker for further analysis. This was because the ITF-2 expression increase in resistant cell lines was relatively high and a previous study has suggested that ITF-2 functions as an oncogene in human colon cancers. We also established an AZD6244 resistant cell line (M14/AZD-3) from an AZD6244 sensitive M14 cell line. The expression of the ITF-2 was elevated both in primary AZD6244 resistant cell line, LOX-IMVI and acquired resistant cell line, M14/AZD-3. Targeted silencing of ITF-2 by siRNA significantly enhanced susceptibility to AZD6244 in resistant cells. Wnt/ß-catenin pathway was activated through direct interaction of p-ERK and GSK3ß. Our results suggest that up-regulation of the ITF-2 gene expression is associated with cellular resistance to MEK inhibitors, and activation of Wnt signaling pathway through interaction of p-ERK and GSK3ß seems to be a mechanism for increase of ITF-2.

SHP2 is induced by the HBx-NF-κB pathway and contributes to fibrosis during human early hepatocellular carcinoma development.

The non-receptor tyrosine phosphatase SHP2 has scaffolding functions in signal transduction cascades downstream of growth receptors. A recent study suggested that SHP2 acts as a tumor suppressor during hepatocellular carcinoma (HCC) development. Herein we examined whether SHP2 links the HBx-NF-κB pathway to EGFR signaling during HCC development. The overexpression of HBx or NF-κB led to increased SHP2 expression via NF-κB binding to the Shp2 promoter. EGF treatment induced ERK activation as well as the rapid assembly of SHP2, EGFR, and Gab1. Upon LPS stimulation, NF-κB-SHP2-ERK activation and phosphorylated STAT3 levels exhibited a negative correlation in vitro. By contrast, in patients with HBV-associated HCC, NF-κB-SHP2-ERK and IL-6-JAK-STAT3 pathway activity levels were concomitantly higher in adjacent non-neoplastic tissues than in HCC tissues. The immunohistochemical analysis of 162 tissues of patients with HCC revealed that SHP2 levels were significantly higher in non-neoplastic background tissues than in corresponding HCC tissues and considerably increased in background liver tissues with advanced fibrosis (P < 0.001). SHP2 expression increased gradually from normal liver to chronic hepatitis, cirrhosis, and background liver with a dysplastic nodule, but was decreased or lost in dysplastic nodules and HCC. This is the first report to describe the existence of the HBx-NF-κB-SHP2 pathway, linking HBV infection to the EGFR-RAS-RAF-MAPK pathway in the liver. SHP2 depletion from the negative crosstalk between NF-κB and STAT3 accelerates HCC development.

Good Laboratory Standards for Clinical Next-Generation Sequencing Cancer Panel Tests.

Next-generation sequencing (NGS) has recently emerged as an essential component of personalized cancer medicine due to its high throughput and low per-base cost. However, no sufficient guidelines for implementing NGS as a clinical molecular pathology test are established in Korea. To ensure clinical grade quality without inhibiting adoption of NGS, a taskforce team assembled by the Korean Society of Pathologists developed laboratory guidelines for NGS cancer panel testing procedures and requirements for clinical implementation of NGS. This consensus standard proposal consists of two parts: laboratory guidelines and requirements for clinical NGS laboratories. The laboratory guidelines part addressed several important issues across multistep NGS cancer panel tests including choice of gene panel and platform, sample handling, nucleic acid management, sample identity tracking, library preparation, sequencing, analysis and reporting. Requirements for clinical NGS tests were summarized in terms of documentation, validation, quality management, and other required written policies. Together with appropriate pathologist training and international laboratory standards, these laboratory standards would help molecular pathology laboratories to successfully implement NGS cancer panel tests in clinic. In this way, the oncology community would be able to help patients to benefit more from personalized cancer medicine.