Chiho Nakashima

SPARC is a possible predictive marker for albumin-bound paclitaxel in non-small-cell lung cancer

Objectives Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) produced good tumor response in cases with lung squamous cell carcinoma, one of the most difficult cancers to treat. Secreted protein acidic and rich in cysteine (SPARC) binds to albumin, suggesting that SPARC plays an important role in tumor uptake of nab-paclitaxel. There is as yet no predictive marker for cytotoxic agents against non-small-cell lung cancer (NSCLC), and hence we believed that SPARC expression might be associated with tumor response to nab-paclitaxel. Patients and methods We studied stromal SPARC reactivity and its association with clinicopathological characteristics in 200 cases of NSCLC using a custom tissue microarray fabricated in our laboratory by immunohistochemical staining. We also investigated the relationship between stromal SPARC reactivity and tumor response to nab-paclitaxel using biopsy or surgical specimens obtained from advanced or recurrent lung cancer patients. Results High SPARC stromal reactivity (>50% of optical fields examined) was detected in 16.5% of cases and intermediate SPARC reactivity (10%–50%) in 56% of cases. High expression in cancer cells was rare (five cases). Stromal SPARC level was correlated with smoking index, squamous cell carcinoma, and vessel invasion. Furthermore, patients with high stromal SPARC reactivity in biopsy specimens such as transbronchial lung biopsy or surgical specimens tended to respond better to nab-paclitaxel. Conclusion Stromal SPARC was detected by immunohistochemical staining in ∼70% of NSCLC cases, and good tumor response to nab-paclitaxel was correlated with high stromal SPARC reactivity. SPARC may be a useful predictive marker for selecting patients likely to respond favorably to nab-paclitaxel treatment.

Functional analysis of Discoidin domain receptor 2 mutation and expression in squamous cell lung cancer

OBJECTIVES Discoidin domain receptor (DDR) 2 mutations have recently been reported to be candidate targets of molecular therapy in lung squamous cell carcinoma (SQCC). However, the status of DDR2 expression and mutations, as well as their precise roles in lung SQCC, have not been clarified. We here report DDR2 mutation and expression status in clinical samples and its role of lung SQCC. MATERIALS AND METHODS We investigated DDR2 expression and mutation status in 44 human clinical samples and 7 cell lines. Biological functions of DDR2 were assessed by in vitro cell invasion assay and animal model experiments. RESULTS Endogenous DDR2 protein expression levels were high in one cell line, PC-1, and immunohistochemistry of lung cancer tissue array showed high levels of DDR2 protein in 29% of lung SQCC patients. A mutation (T681I) identified in lung SQCC and the cell line EBC-1 was detected among 44 primary lung SQCC samples and 7 lung SQCC cell lines. Although Forced expression of DDR2 and its mutant (T681I) led to induce SQCC cell invasion in vitro, only wild type DDR2 enhanced lung metastasis in an animal model. We also found that ectopic expression of DDR2 induced MMP-1 mRNA expression accompanied by phosphorylation of c-Jun after treatment with its ligand, collagen type I, but DDR2 with the T681I mutation did not, suggesting that T681I mutation is an inactivating mutation. CONCLUSION Overexpression of DDR2 might contribute to tumor progression in lung SQCC. The overexpression of DDR2 could be potential molecular target of lung SQCC.

Automated DNA extraction using cellulose magnetic beads can improve EGFR point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments

The clinical utility of plasma DNA for detecting cancer-specific mutations has rapidly achieved recognition, but reliability has not been established because of relatively low mutation-detection rates compared with those from tissue re-biopsy. To address this shortcoming we examined efficiency, in terms of mutation detection, of an automated DNA extraction system that uses cellulose magnetic beads. A fully automated, highly sensitive point-mutation-detection method, mutation-biased PCR and quenching probe (MBP-QP) system, was used for this study. Plasma DNA was extracted from 61 plasma samples collected from patients with advanced non-small cell lung cancer. Extraction was performed manually with 200 μl plasma (200-M) by using a silica membrane spin column system or an automated system using 200 μl (200-A) or 1000 μl (1000-A) plasma. Median DNA yield quantified by real-time PCR was 4.4, 4.5, and 17.3 ng with the three methods, respectively. Sensitivity for detecting epidermal growth factor receptor (EGFR) L858R point mutation was 36.6%, 58.5%, and 77.5%, and specificity was 93.3%, 100%, and 96.7%, respectively. Concordance rates were 60.6%, 76.1%, and 85.7%. The size distribution of plasma DNA with automated extraction was bimodal with modes at about 170 bp and 5 Kb, and plasma DNA of both sizes included tumor-derived DNA. In this report, we demonstrate that automated DNA extraction using cellulose magnetic beads can improve mutation-detection rates with plasma DNA in association with two overall sizes of DNA fragments recovered by this DNA isolation system. Examining the biological characteristics of these fragments will be the subject of further investigation.

Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

Liquid biopsy with circulating free DNA (cfDNA) is a recommended alternative method of re-biopsy. Quality control with cfDNA is indispensable for precise examinations, and it is desirable to achieve high-quality cfDNA separation. We investigated two issues: the influence of pre-analytical procedures on cfDNA analysis performed as a routine procedure in a standard clinical laboratory, and the extent of deterioration of cfDNA quality due to long-term storage. Comparisons among blood collection tube types, storage temperatures, and periods of blood separation were performed in terms of cfDNA quantification, cfDNA size distribution, and detection of EGFR mutations. Quality of cfDNA was better with collection tubes containing 3.2% sodium citrate than with those containing EDTA 2K, and was maintained with storage at 4° C for up to 72 h after blood collection, equivalent to results with cell-stabilizing blood collection tubes. Analysis of cfDNA stored for 7 years showed that samples with low allele frequency (AF) deteriorated more readily than samples with high AF. Despite the same storage period and extraction method, AF of plasma stored for 7 years was remarkably lower than that of cfDNA. However, deterioration due to long-term plasma storage was overcome by changing the DNA extraction method from a silica membrane spin column to a cellulose magnetic beads system. These results can guide the establishment of standardized pre-analytical procedures for liquid biopsy with cfDNA.

Current Status and Problems of T790M Detection, a Molecular Biomarker of Acquired Resistance to EGFR Tyrosine Kinase Inhibitors, with Liquid Biopsy and Re-biopsy

Background/Aim: The purpose of this study was to consider appropriate application of liquid and re-biopsy through analysis of current status in practice. Patients and Methods: We performed a retrospective analysis of 22 patients with epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer who exhibited 1st/2nd generation EGFR-tyrosine kinase inhibitors resistance. The cobas® method was used to detect T790M with re-biopsy and the mutation-biased PCR and quenched probe method was used with liquid biopsy. Results: T790M detection rate was 52% with re-biopsy and 58% with liquid biopsy. The concordance between tissue and plasma was 58%. One patient who was T790M-positive with liquid biopsy showed heterogeneity among metastatic lesions in terms of osimertinib efficacy, as revealed by T790M detection with re-biopsy. Conclusion: Liquid biopsy reflects the whole body, whereas re-biopsy is useful for spatial diagnosis. Considering these characteristics, a combination of liquid and re-biopsy contribute to enhanced treatment.

Chemotherapy for primary mediastinal yolk sac tumor in a patient undergoing chronic hemodialysis: a case report

BackgroundThe safety and efficacy of chemotherapy for patients undergoing concomitant hemodialysis have not been fully established and optimal doses of anti-cancer drugs and best timing of hemodialysis remains unclear. Although chemosensitive cancers, such as germ cell tumors, treated with chemotherapy should have sufficient dose intensity maintained to achieve the desired effect, many patients with cancer undergoing hemodialysis might be under-treated because the pharmacokinetics of anti-cancer drugs in such patients remains unknown.Case presentationWe describe a 31-year-old Japanese man with a mediastinal yolk sac tumor treated with surgery followed by five cycles of chemotherapy containing cisplatin and etoposide while concomitantly undergoing hemodialysis. The doses of these agents used in the first cycle were 50% of the standard dose of cisplatin (10 mg/m2) and 60% of the standard dose of etoposide (60 mg/m2) on days 1 through to 5; the doses were subsequently escalated to 75% with both agents. Hemodialysis was started 1 hour after infusions of these agents. Severe hematological toxicities were observed despite successful treatment. During treatment with concurrent hemodialysis, pharmacokinetic analysis of cisplatin was performed and its relationship with adverse effects was assessed. Compared with patients with normal renal function, the maximum drug concentration was higher, and concentration increased in the interval between hemodialysis and the subsequent cisplatin infusion, resulting in a higher area under the curve despite a reduction in the dose to 75% of the standard regimen.ConclusionsBecause of the altered pharmacokinetics pharmacodynamics status of patients with renal dysfunction undergoing hemodialysis, pharmacokinetics pharmacodynamics analysis is deemed to be helpful for effective and safe management of chemotherapy in patients undergoing hemodialysis.

Abstract 2751: Automatic DNA extraction system can improve the EGFR point mutation detection rate of liquid biopsy

The usefulness of liquid biopsy to detect mutations from cancer patients has been well recognized today. However, because the mutation detection rates from plasma DNA were relatively lower than those of tissue re-biopsy, its clinical utility has not been confirmed yet. As previously we reported, we have developed fully automatic high-sensitive point mutation detecting system named mutation-biased PCR and quenched probe (MBP-QP) system for liquid biopsy. Recently, the importance of pre-analytical procedures for plasma DNA anazysis has been highlighted. In this study, we examined whether the automatic DNA extraction system can improve the mutation detection rate in our MBP-QP system. Sixty-one plasma samples were obtained from advanced non-small cell lung cancer patients, and plasma DNA extraction was performed from 200μl plasma by manually (200-M), and 200μl (200-A), 1000μl (1000-A) plasma by automatically. We used silica membrane spin column system for manual DNA extraction, and magnet beads system for automatic DNA extraction procedure. The median DNA concentrations quantified by quantitative real-time PCR of 200-M, 200-A, 1000-A were 4.92, 6.00, 20.1 ng/mL plasma, respectively. In terms of the epidermal growth factor receptor (EGFR) L858R point mutation detection, the sensitivity of 200-M, 200-A, 1000-A were 36.6%, 58.5%, 77.5%, that of the specificity were 93.3%, 100%, 96.7%, and the concordance rates were 60.6%, 76.1%, 85.7%, respectively. The size distribution of automatically extracted plasma DNA represented two peaks characteristics at 170 bp and 5 kb. In this study, we indicate the automatic DNA extraction can improve mutation detection rates in plasma DNA. Citation Format: Chiho Nakashima, Akemi Sato, Tomonori Abe, Tomomi Nakamura, Kazutoshi Komiya, Eisaburo Sueoka, Shinya Kimura, Naoko Sueoka-Aragane, Junichi Kato, Mitsuharu Hirai. Automatic DNA extraction system can improve the EGFR point mutation detection rate of liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2751. doi:10.1158/1538-7445.AM2017-2751