Injectable platelet-rich fibrin promotes proliferation and trichogenic inductivity of dermal papilla cells through activating TGF-ß/Smad signaling pathway.

Dermal papilla cell (DPC) belongs to a specialized mesenchymal stem cell for hair follicle regeneration. Maintaining the ability of DPCs to stimulate hair in vitro culture is important for hair follicle morphogenesis and regeneration. As the third generation of platelet concentrate, injectable platelet-rich fibrin (i-PRF) is a novel biomaterial containing many growth factors and showing promising effects on tissue reconstruction. We aimed to explore the influences of i-PRF on the proliferative, migratory, as well as trichogenic ability of DPCs and compared the effects of i-PRF and platelet-rich plasma (PRP), the first generation of platelet concentrate. Both PRP and i-PRF facilitated DPCs proliferation, and migration, along with trichogenic inductivity as well as stimulated the TGF-ß/Smad pathway, while the impacts of i-PRF were more significant than PRP. A small molecule inhibitor of TGF-beta receptor I, Galunisertib, was also applied to treat DPCs, and it rescued the impacts of i-PRF on the proliferative, migratory, trichogenic inductivity, and proteins-associated with TGF-ß/Smad pathway in DPCs. These findings revealed that i-PRF had better effects than PRP in enhancing the proliferative, migratory, and hair-inducing abilities of DPCs by the TGF-ß/Smad pathway, which indicated the beneficial role of i-PRF in hair follicle regeneration.

Suppressing MTERF3 inhibits proliferation of human hepatocellular carcinoma via ROS-mediated p38 MAPK activation.

Mitochondrial transcription termination factor 3 (MTERF3) negatively regulates mitochondrial DNA transcription. However, its role in hepatocellular carcinoma (HCC) progression remains elusive. Here, we investigate the expression and function of MTERF3 in HCC. MTERF3 is overexpressed in HCC tumor tissues and higher expression of MTERF3 positively correlates with poor overall survival of HCC patients. Knockdown of MTERF3 induces mitochondrial dysfunction, S-G2/M cell cycle arrest and apoptosis, resulting in cell proliferation inhibition. In contrast, overexpression of MTERF3 promotes cell cycle progression and cell proliferation. Mechanistically, mitochondrial dysfunction induced by MTERF3 knockdown promotes ROS accumulation, activating p38 MAPK signaling pathway to suppress HCC cell proliferation. In conclusion, ROS accumulation induced by MTERF3 knockdown inhibits HCC cell proliferation via p38 MAPK signaling pathway suggesting a promising target in HCC patients.

RRP15 deficiency induces ribosome stress to inhibit colorectal cancer proliferation and metastasis via LZTS2-mediated ß-catenin suppression.

Ribosome biogenesis (RiBi) plays a pivotal role in carcinogenesis by regulating protein translation and stress response. Here, we find that RRP15, a nucleolar protein critical for RiBi and checkpoint control, is frequently upregulated in primary CRCs and higher RRP15 expression positively correlated with TNM stage (P < 0.0001) and poor survival of CRC patients (P = 0.0011). Functionally, silencing RRP15 induces ribosome stress, cell cycle arrest, and apoptosis, resulting in suppression of cell proliferation and metastasis. Overexpression of RRP15 promotes cell proliferation and metastasis. Mechanistically, ribosome stress induced by RRP15 deficiency facilitates translation of TOP mRNA LZTS2 (Leucine zipper tumor suppressor 2), leading to the nuclear export and degradation of ß-catenin to suppress Wnt/ß-catenin signaling in CRC. In conclusion, ribosome stress induced by RRP15 deficiency inhibits CRC cell proliferation and metastasis via suppressing the Wnt/ß-catenin pathway, suggesting a potential new target in high-RiBi CRC patients.

Development and validation of postoperative and preoperative platelets ratio (PPR) to predict the prognosis of patients undergoing surgery for colorectal cancer: A dual-center retrospective cohort study.

BACKGROUND: Platelets occupy a prominent place in tumor proliferation and metastasis, and platelet count is relevant to the prognosis of tumor patients. But preoperative platelet counts cannot be standardized and individualized due to the variability among individuals, instruments, and regions, and the connection between postoperative platelet count and prognosis remains unknown. A standardized indicator of platelet count was designed to forecast the prognosis of colorectal cancer (CRC). METHODS: Five hundred and eighty six patients who suffered radical resection of CRC between 2013 and 2019 were collected. A development-validation cohort of standardized and individualized platelet counts for prognostic assessment of CRC was designed. We first determined the ability of PPR and other peripheral blood count-related indicators to predict the mortality of patients with CRC and validated them in a separate cohort. Kaplan-Meier analysis was executed to evaluate the survival and univariate and multivariate analyses were executed to explore the relevance. Time-dependent ROC was measured to estimate the predictive usefulness. Decision curve analysis was used to verify the clinical net benefit. RESULTS: Important baseline variables showed a similar distribution in two independent queues. In the development cohort, postoperative platelet count and postoperative/preoperative platelets ratio (PPR) were independent predictors of prognosis in CRC patients. PPR showed the largest area under the curve (AUC) in evaluating 1-year and 5-year OS (AUC: 0.702 and 0.620) compared to others. In the validation cohort, platelet/lymphocyte ratio and PPR were validated to be independently concerned about OS of CRC patients and PPR showed the largest AUC in evaluating 1-year and 3-year OS (AUC: 0.663 and 0.673). PPR and joint index of platelet count and PPR showed better predictive value and clinical net benefit. CONCLUSIONS: PPR has been identified and validated to be independently concerned about OS of patients with CRC and was a reliable and economic indicator to evaluate the prognosis.

HOXA13 serves as a biomarker to predict neoadjuvant therapy efficacy in advanced colorectal cancer patients.

Neoadjuvant therapy (NAT) for advanced colorectal cancer (ACRC) is a kind of well-evidenced therapy, yet a portion of ACRC patients have poor therapeutic response. To date, no suitable biomarker used for assessing NAT efficacy has been reported. Here, we collect 72 colonoscopy biopsy tissue specimens from ACRC patients before undergoing NAT and investigate the relationship between HOXA13 expression and NAT efficacy. The results show that HOXA13 expression in pretreated tumor specimens is negatively associated with tumor regression ( P<0.001) and progression-free survival ( P<0.05) in ACRC patients who underwent NAT. Silencing of HOXA13 or its regulator HOTTIP significantly enhances the chemosensitivity of colorectal cancer (CRC) cells, leading to an increase in cell apoptosis and the DNA damage response (DDR) to chemotherapeutic drug treatment. In contrast, HOXA13 overexpression causes a significant increase in chemoresistance in CRC cells. In summary, we find that the HOTTIP/HOXA13 axis is involved in regulating chemotherapeutic sensitivity in CRC cells by modulating the DDR and that HOXA13 serves as a promising marker for NAT efficacy prediction in ACRC patients.

Detection of DNA copy number alterations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of single nucleotide polymorphisms.

OBJECTIVES: Copy number alterations (CNAs) are frequently found in malignant tissues. Different approaches have been used for CNA detection. However, it is not easy to detect a large panel of CNA targets in heterogenous tumors. METHODS: We have developed a CNAs detection approach through quantitatively analyzed allelic imbalance by allelotyping single nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, the copy number changes were quantified by real-competitive PCR (rcPCR) to distinguish loss of heterozygosity (LOH) and genomic amplification. The approach was used to validate the CNA regions detected by next generation sequencing (NGS) in early-stage lung carcinoma. RESULTS: CNAs were detected in heterogeneous DNA samples where tumor DNA is present at only 10% through the SNP based allelotyping. In addition, two different types of CNAs (loss of heterozygosity and chromosome amplification) were able to be distinguished quantitatively by rcPCR. Validation on a total of 41 SNPs from the selected CNA regions showed that copy number changes did occur, and the tissues from early-stage lung carcinoma were distinguished from normal. CONCLUSIONS: CNA detection by MALDI-TOF MS can be used for validating potentially interesting genomic regions identified from next generation sequencing, and for detecting CNAs in tumor tissues consisting of a mixture of neoplastic and normal cells.

Direct adenylation from 5'-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase.

5'-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5'-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.

Kif4A mediates resistance to neoadjuvant chemoradiotherapy in patients with advanced colorectal cancer via regulating DNA damage response.

More and more patients with advanced colorectal cancer (CRC) have benefited from surgical resection or ablation following neoadjuvant chemoradiotherapy (nCRT), but nCRT may be ineffective and have potential risks to some patients. Therefore, it is necessary to discover effective biomarkers for predicting the nCRT efficacy in CRC patients. Chromokinesin Kif4A plays a critical role in mitosis, DNA damage repair and tumorigenesis, but its relationship with nCRT efficacy in advanced CRC remains unclear. Here, we find that Kif4A expression in pretreated tumor tissue is positively correlated with poorer tumor regression after receiving nCRT ( P=0.005). Knockdown of endogenous Kif4A causes an increased sensitivity of CRC cells to chemotherapeutic drugs 5-fluorouracil (5-FU) and Cisplatin (DDP), while overexpression of Kif4A enhances resistance of CRC cells to the chemotherapeutic drugs. Furthermore, depending on its motor domain and tail domain, Kif4A regulates DNA damage response (DDR) induced by 5-FU or DDP treatment in CRC cells. In conclusion, we demonstrate that Kif4A may be a potential independent biomarker for predicting the nCRT efficacy in advanced CRC patients, and Kif4A regulates chemosensitivity of CRC cells through controlling DDR.

Dynamic changes of platelets before and after surgery predict the prognosis of patients with operable non-small cell lung cancer.

Purpose: To determine the prognostic significance of postoperative platelet/preoperative platelet ratio (PPR) in patients with operable non-small cell lung cancer (NSCLC), and assess its prognostic benefit compared to models relying solely preoperative platelet counts (PLT). Materials and Methods: A retrospective analysis of 403 patients who underwent radical resection of NSCLC in our institution from 2013 to 2018 was conducted to assess the prognostic significance of PLT and PPR. Progression-free survival (PFS) and overall survival (OS) were performed by the Kaplan-Meier method. Single-factor and multi-factor COX regression models were used to determine factors that affect long-term outcomes. Time-dependent ROC was used to evaluate the value of PPR in predicting the prognosis. Results: A significant association between high PLT and PPR and poor long-term patient survival outcomes was observed. The median PFS and OS of NSCLC patients with high PLT were 25 months and 29 months, which was significantly shorter than that of patients with low PLT (30 months and 33 months) (both P = 0.002). In addition, the median PFS and OS of NSCLC patients with high PPR were 18 months and 26.5 months, which was significantly shorter than that of patients with low PPR (33 months and 35 months) (both P<0.001). Univariate and Multivariate analysis using Cox regression model showed that PLT and PPR were independent factors affecting PFS and OS. Time-dependent ROC showed that the predictive capability of PLT and PPR preserved well when they were compared over time following surgery. The AUCs of PLT and PPR to predict 1-year PFS and OS, 3-year PFS and OS, 5-year PFS and OS stabilized between 0.528-0.607. PPR showed significantly higher accuracy than PLT in the prediction of 1-year and 3-year PFS and OS. Conclusions: Elevated PPR is significantly related to the adverse outcomes of patients with NSCLC. PPR can stably predict the long-term prognosis of patients, and can be used as a reliable indicator for evaluating the prognosis of patients with operable NSCLC.

Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy.

Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery.

Serum lipocalin-2 is a potential biomarker for the clinical diagnosis of nonalcoholic steatohepatitis.

BACKGROUND/AIMS: Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease (NAFLD) characterized by hepatic steatosis, inflammation, hepatocellular injury, and fibrosis. We aimed to investigate the usefulness of a key biomarker, lipocalin-2 (LCN2), for the detection of NASH progression. METHODS: A mouse NASH model was established using a high-fat diet and a high-sugar drinking water. Gene expression profile of the NASH model was analyzed using RNA sequencing. Moreover, 360 NAFLD patients (steatosis, 83; NASH, 277), 40 healthy individuals, and 87 patients with alcoholic fatty liver disease were recruited. RESULTS: Inflammatory infiltration, focal necrosis in the leaflets, steatosis, and fibrosis were documented in the mouse liver. In total, 504 genes were differentially expressed in the livers of NASH mice, and showed significant functional enrichment in the inflammation-related category. Upregulated liver LCN2 was found to be significantly interactive with various interleukins and toll-like receptors. Serum LCN2 levels were significantly increased in NAFLD patients. Serum LCN2 levels were correlated with steatosis, intralobular inflammation, semiquantitative fibrosis score, and nonalcoholic fatty liver disease activity score. The area under the curve of serum LCN2 was 0.987 with a specificity of 100% and a sensitivity of 93.5% for NASH diagnosis, and 0.977 with almost the same specificity and sensitivity for steatosis. CONCLUSION: LCN2 might be involved in the transition from NAFL to NASH by mediating inflammation. Serum LCN2 levels might be a novel biomarker for the diagnosis of NASH.

Highly multiplexed quantifications of 299 somatic mutations in colorectal cancer patients by automated MALDI-TOF mass spectrometry.

BACKGROUND: Detection of somatic mutations in tumor tissues helps to understand tumor biology and guide treatment selection. Methods such as quantitative PCR can analyze a few mutations with high efficiency, while next generation sequencing (NGS) based methods can analyze hundreds to thousands of mutations. However, there is a lack of cost-effective method for quantitatively analyzing tens to a few hundred mutations of potential biological and clinical significance. METHODS: Through a comprehensive database and literature review we selected 299 mutations associated with colorectal cancer. We then designed a highly multiplexed assay panel (8-wells covering 299 mutations in 109 genes) based on an automated MADLI-TOF mass spectrometry (MS) platform. The multiplex panel was tested with a total of 319 freshly frozen tissues and 92 FFPE samples from 229 colorectal cancer patients, with 13 samples also analyzed by a targeted NGS method covering 532 genes. RESULTS: Multiplex somatic mutation panel based on MALDI-TOF MS detected and quantified at least one somatic mutation in 142 patients, with KRAS, TP53 and APC being the most frequently mutated genes. Extensive validation by both capillary sequencing and targeted NGS demonstrated high accuracy of the multiplex MS assay. Out of 35 mutations tested with plasmid constructs, sensitivities of 5 and 10% mutant allele frequency were achieved for 19 and 16 mutations, respectively. CONCLUSIONS: Automated MALDI-TOF MS offers an efficient and cost-effective platform for highly multiplexed quantitation of 299 somatic mutations, which may be useful in studying the biological and clinical significance of somatic mutations with large numbers of cancer tissues.

A systematic evaluation of stool DNA preparation protocols for colorectal cancer screening via analysis of DNA methylation biomarkers.

Objectives: Colorectal cancer (CRC) screening using stool samples is now in routine use where tumor DNA methylation analysis for leading markers such as NDRG4 and SDC2 is an integral part of the test. However, processing stool samples for reproducible and efficient extraction of human genomic DNA remains a bottleneck for further research into better biomarkers and assays. Methods: We systematically evaluated several factors involved in the processing of stool samples and extraction of DNA. These factors include: stool processing (solid and homogenized samples), preparation of DNA from supernatant and pellets, and DNA extraction with column and magnetic beads-based methods. Furthermore, SDC2 and NDRG4 methylation levels were used to evaluate the clinical performance of the optimal protocol. Results: The yield of total and human genomic DNA (hgDNA) was not reproducible when solid stool scraping is used, possibly due to sampling variations. More reproducible results were obtained from homogenized stool samples. Magnetic beads-based DNA extraction using the supernatant from the homogenized stool was chosen for further analysis due to better reproducibility, higher hgDNA yield, lower non-hgDNA background, and the potential for automation. With this protocol, a combination of SDC2 and NDRG4 methylation signals with a linear regression model achieved a sensitivity and specificity of 81.82 and 93.75%, respectively. Conclusions: Through the systematic evaluation of different stool processing and DNA extraction methods, we established a reproducible protocol for analyzing tumor DNA methylation markers in stool samples for colorectal cancer screening.

Strict Assembly Restriction of Peptides from Rabbit Hemorrhagic Disease Virus Presented by Rabbit Major Histocompatibility Complex Class I Molecule RLA-A1.

Rabbits are pivotal domestic animals for both the economy and as an animal model for human diseases. A large number of rabbits have been infected by rabbit hemorrhagic disease virus (RHDV) in natural and artificial pandemics in the past. Differences in presentation of antigenic peptides by polymorphic major histocompatibility complex (MHC) molecules to T-cell receptors (TCR) on T lymphocytes are associated with viral clearance in mammals. Here, we screened and identified a series of peptides derived from RHDV binding to the rabbit MHC class I molecule, RLA-A1. The small, hydrophobic B and F pockets of RLA-A1 capture a peptide motif analogous to that recognized by human class I molecule HLA-A*0201, with more restricted aliphatic anchors at P2 and PΩ positions. Moreover, the rabbit molecule is characterized by an uncommon residue combination of Gly53, Val55, and Glu56, making the 310 helix and the loop between the 310 and α1 helices closer to the α2 helix. A wider A pocket in RLA-A1 can induce a special conformation of the P1 anchor and may play a pivotal role in peptide assembly and TCR recognition. Our study broadens the knowledge of T-cell immunity in domestic animals and also provides useful insights for vaccine development to prevent infectious diseases in rabbits.IMPORTANCE We screened rabbit MHC class I RLA-A1-restricted peptides from the capsid protein VP60 of rabbit hemorrhagic disease virus (RHDV) and determined the structures of RLA-A1 complexed with three peptides, VP60-1, VP60-2, and VP60-10. From the structures, we found that the peptide binding motifs of RLA-A1 are extremely constraining. Thus, there is a generally restricted peptide selection for RLA-A1 compared to that for human HLA-A*0201. In addition, uncommon residues Gly53, Val55, and Glu56 of RLA-A1 are located between the 310 helix and α1 helix, which makes the steric position of the 310 helix in RLA-A1 much closer to the α2 helix than that found in other mammalian MHC class I molecules. This special conformation between the 310 helix and α1 helix plays a pivotal role in rabbit MHC class I assembly. Our results provide new insights into MHC class I molecule assembly and peptide presentation of domestic mammals. Furthermore, these data also broaden our knowledge on T-cell immunity in rabbits and may also provide useful information for vaccine development to prevent infectious diseases in rabbits.

Dynamics of Plasma EGFR T790M Mutation in Advanced NSCLC: A Multicenter Study.

BACKGROUND: Droplet digital polymerase chain reaction (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, a ddPCR assay for the epidermal growth factor receptor (EGFR) p.Thr790Met (T790M) mutation suitable for clinical use remains to be established with analytical and clinical validations. OBJECTIVE: We aimed to develop and validate a new ddPCR assay to quantify the T790M mutation in plasma for monitoring and predicting the progression of advanced non-small-cell lung cancer (NSCLC). METHODS: Specificity of the ddPCR assay was evaluated with genomic DNA samples from healthy individuals. The inter- and intraday variations of the assay were evaluated using mixtures of plasmid DNA containing wild-type EGFR and T790M mutation sequences. We assessed the clinical utility of the T790M assay in a multicenter prospective study in patients with advanced NSCLC receiving tyrosine kinase inhibitor (TKI) treatment by analyzing longitudinal plasma DNA samples. RESULTS: We set the criteria for a positive call when the following conditions were satisfied: (1) T790M mutation frequency > 0.098% (3 standard deviations above the background signal); (2) at least two positive droplets in duplicate ddPCR reactions. Among the 62 patients with advanced NSCLC exhibiting resistance to TKI treatment, 15 had one or more serial plasma samples that tested positive for T790M. T790M mutation was detected in the plasma as early as 205 days (median 95 days) before disease progression, determined by imaging analysis. Plasma T790M concentrations also correlated with intervention after disease progression. CONCLUSIONS: We developed a ddPCR assay to quantify the T790M mutation in plasma. Quantification of longitudinal plasma T790M mutation may allow noninvasive assessment of drug resistance and guide follow-up treatment in TKI-treated patients with NSCLC. TRIAL REGISTRATION: Clinical Trials.gov identifier: NCT02804100.

Divergent Peptide Presentations of HLA-A*30 Alleles Revealed by Structures With Pathogen Peptides.

Human leukocyte antigen (HLA) alleles have a high degree of polymorphism, which determines their peptide-binding motifs and subsequent T-cell receptor recognition. The simplest way to understand the cross-presentation of peptides by different alleles is to classify these alleles into supertypes. A1 and A3 HLA supertypes are widely distributed in humans. However, direct structural and functional evidence for peptide presentation features of key alleles (e.g., HLA-A*30:01 and -A*30:03) are lacking. Herein, the molecular basis of peptide presentation of HLA-A*30:01 and -A*30:03 was demonstrated by crystal structure determination and thermostability measurements of complexes with T-cell epitopes from influenza virus (NP44), human immunodeficiency virus (RT313), and Mycobacterium tuberculosis (MTB). When binding to the HIV peptide, RT313, the PΩ-Lys anchoring modes of HLA-A*30:01, and -A*30:03 were similar to those of HLA-A*11:01 in the A3 supertype. However, HLA-A*30:03, but not -A*30:01, also showed binding with the HLA*01:01-favored peptide, NP44, but with a specific structural conformation. Thus, different from our previous understanding, HLA-A*30:01 and -A*30:03 have specific peptide-binding characteristics that may lead to their distinct supertype-featured binding peptide motifs. Moreover, we also found that residue 77 in the F pocket was one of the key residues for the divergent peptide presentation characteristics of HLA-A*30:01 and -A*30:03. Interchanging residue 77 between HLA-A*30:01 and HLA-A*30:03 switched their presented peptide profiles. Our results provide important recommendations for screening virus and tumor-specific peptides among the population with prevalent HLA supertypes for vaccine development and immune interventions.

Rapid symptomatic relief of HER2-positive gastric cancer leptomeningeal carcinomatosis with lapatinib, trastuzumab and capecitabine: a case report.

BACKGROUND: Gastric cancer patients with widespread metastasis, especially meningeal metastases, have an extremely prognosis and limited therapeutic choices. CASE PRESENTATION: We reported the case of a 39-year-old male patient with HER2-positive gastric cancer with bone and meningeal metastases. He presented with multiple bone metastases and received 3 cycles of docetaxel plus S1. However, he complained with headache and imaging examinations revealed leptomeningeal carcinomatosis. FISH revealed that tumor cells in the cerebrospinal fluid were HER-positive. Herceptin was added to the regimen, but the symptoms were not relieved, the patient suffered from dizziness and nausea. The chemotherapy regimen was switched d to lapatinib (orally at 1250 mg/day, every day), capecitabine (orally at 1000 mg/m2, bid for 2 weeks, followed by a 1-week rest interval, as 1 cycle) and Herceptin (390 mg/3 weeks). After 3 weeks of the new treatment, all the symptoms relieved. The clinical complete response was maintained for 3 months. CONCLUSIONS: Lapatinib/Capecitabine combination therapy is an alternative treatment strategy for leptomeningeal carcinomatosis of HER2-positive gastric cancer in which trastuzumab and/or chemotherapy essentially has no effect.

Competitive evolution of NSCLC tumor clones and the drug resistance mechanism of first-generation EGFR-TKIs in Chinese NSCLC patients.

PURPOSE: Although many studies have reported on the resistance mechanism of first-generation EGFR TKIs (1st EGFR TKIs) treatment, large-scale dynamic ctDNA mutation analysis based on liquid biopsy for non-small cell lung cancer (NSCLC) in the Chinese population is rare. Using in-depth integration and analysis of ctDNA genomic mutation data and clinical data at multiple time points during the treatment of 53 NSCLC patients, we described the resistance mechanisms of 1st EGFR TKIs treatment more comprehensively and dynamically. The resulting profile of the polyclonal competitive evolution of the tumor provides some new insights into the precise treatment of NSCLC. EXPERIMENTAL DESIGN: A prospective study was conducted in patients with advanced NSCLC with acquired resistance to erlotinib, gefitinib or icotinib. By liquid biopsy, we detected mutations in 124 tumor-associated genes in the context of drug resistance. These 124 genes covered all tumor therapeutic targets and related biological pathways. During the entire course of treatment, the interval between two liquid biopsies was two months. RESULTS: Unlike the common mutations tested in tissue samples, our data showed a higher coverage of tumor heterogeneity (32.65%), more complex patterns of resistance and some new resistance mutation sites, such as EGFR p.V769M and KRAS p.A11V. The major resistance-associated mutations detected were still EGFR p.T790M (45.28%), other point mutations in EGFR (33.9%), and KRAS and NRAS mutations (15.09%). These mutation ratios might be considered as a preliminary summary of the characteristics of Chinese patients. In addition, starting from the two baseline mutations of the EGFR gene (19del vs. L858R), we first described the detailed mutation profile of the EGFR gene. Although there was no significant difference in the number of patients with EGFR p.19del and EGFR p.L858R baseline mutations (24% vs. 16%, P = 0.15), patients from the EGFR p.19del baseline group were much more likely to develop EGFR p.T790M resistance mutations (62.1% vs. 19.3%, P = 0.007). Through careful integration of gene mutation information and clinical phenotype information, an interesting phenomenon was found. Although the variant allele fraction (VAF) of the EGFR p.T790M mutation was significantly linearly correlated with that of the EGFR drug-sensitive mutation (r = 0.68, P = 0.00025), neither VAF was associated with the tumor volume at the advanced stage. It was shown that other tumor clones might contribute more to the resistance to 1st EGFR TKIs treatment than tumor clones carrying the EGFR p.T790M mutation when resistance developed. By further analysis, we found that, in some patients, when the primary tumor clones detected were those carrying EGFR-/- mutations (both types the EGFR p.19del/p.L858R and EGFR p.T790M mutation types were missing), most of them showed a poor prognosis and ineffective late treatment, indicating that EGFR-/- played a more important role than EGFR p.T790M in the process of NSCLC drug resistance in these patients. From the perspective of the clonal evolution of NSCLC tumor cells, these phenomena could be explained by the competitive evolution between different tumor clones. In addition, two new mutations, KRAS p.A11V and EGFR p.V769M, emerged significantly during drug resistance in NSCLC patients and had shown obvious competitive clonal evolution characteristics. Combined with clear clinical drug resistance phenotypic information, we believed that these two new mutations might be related to new drug resistance mechanisms and deserve further study. We have also seen an interesting phenomenon. In some patients undergoing 1st EGFR TKIs treatment, the EGFR p.T790M mutation appeared, disappeared, and reappeared, and this spatial and temporal diversity of the EGFR p.T790M mutation was regulated by targeted drug and chemotherapy and was correlated with the individual tumor mutation profile. CONCLUSIONS: The constitution and competitive evolution of the tumor clones have a decisive influence on treatment and can be regulated by targeted drugs and chemotherapy. Additionally, EGFR p.T790M spatial and temporal diversity during treatment warrants more attention, and this spatial and temporal diversity may be useful for the choice of treatment strategies for certain NSCLC patients. Through longitudinal cfDNA sample analysis, the resistance mechanism and dynamic clinical features of Chinese NSCLC patients are systematically established as reliable and meaningful to understand acquired resistance and make further personalized treatment decisions dynamically. Two new potential drug resistance-associated mutations in EGFR and KRAS have been found and are worthy of further study. Finally, our research shows that the evolutionary process of tumor cloning can be artificially regulated and intervened, possibly providing a new way to treat tumors.

Diagnostic accuracy of CD44V6 for osteosarcoma: a meta-analysis.

BACKGROUND: Recently, more and more evidences have revealed the association between CD44V6 and osteosarcoma (OS), but whether it can be used as a clinical biomarker is still unknown. The purpose of this study is to assess the diagnostic value of CD44V6 in OS by conducting a meta-analysis. METHODS: All relevant electronic literatures were collected from seven international databases together with three Chinese databases up to April 23, 2016. Eligible studies were selected through multiple search strategies and the quality was assessed by QUADAS. Data was extracted from studies according to the key statistics index. All analyses were performed using STATA 12 and Meta-DiSc 1.4 statistical software. RESULTS: According to the exclusion and inclusion criteria, 8 literatures were retrieved, accounting for 463 cases and 188 controls. For discriminating OS from benign bone tumor or healthy controls, the area under the receiver operating characteristic curve (AUC) was 0.91 (95 % CI 0.88-0.93). Overall, the results showed pooled sensitivity of 0.743 (95 % CI 0.606-0.844) and specificity of 0.897 (95 % CI 0.818-0.945), respectively. Substantial heterogeneity was detected in this study (I 2 = 90 %). The publication bias was assessed by using Deeks' asymmetry test (p = 0.795). No evidence of heterogeneity from threshold effects was detected by the Spearman correlation coefficient (-0.506, p = 0.201). Meta-regression was performed to mining the source of heterogeneity, and subgroup analysis showed that neither the cut-off values nor the control groups were the source of heterogeneity. CONCLUSIONS: The present results suggest that promoted CD44V6 expression levels are associated with OS and CD44V6 may be used as a diagnostic marker for OS.

Prognostic significance of CD44V6 expression in osteosarcoma: a meta-analysis.

Numerous individual studies evaluating the relationship between CD44V6 over-expression and prognostic impact in patients with osteosarcoma (OS) have yielded in conclusive results. This meta-analysis aimed to determine the value of cell adhesion molecule CD44V6 in prognosis of OS by conducting a systematic review and meta-analysis. A comprehensive search was conducted using PubMed (medline), Embase, ISI Web of Knowledge, Springer, the Cochrane Library, Scopus, BioMed Central, ScienceDirect, Wanfang, Weipu, and China National Knowledge Internet (CNKI) databases from inception through May 26, 2015. All available articles written in English or Chinese that investigated the expression of CD44V6 and the prognosis of OS were included. The quantity of the studies was evaluated according to the critical review checklist of the Dutch Cochrane Centre proposed by MOOSE. Finally, a total of eight studies with 486 OS patients were involved and the results indicated that the positive expression of CD44V6 predicts neoplasm metastasis (RR = 1.76, 95 % CI 1.38-2.25, p < 0.00001), and poor survival in OS with the pooled HR of 1.53 (95 % CI 1.25-1.88, p < 0.0001). No significant heterogeneity was observed among all studies. In conclusion, the present meta-analysis and systematic review strongly suggest that CD44V6 over-expression is associated with overall survival rate and metastasis in OS, and may be used as a prognostic biomarker to guide the clinical therapy for OS.