Usefulness of blood biomarkers for predicting venous thromboembolism in Japanese patients with cancer.

Prospective studies on risk factors for the occurrence of venous thromboembolism (VTE) in Asian patients with cancer are limited. Therefore, the present study assessed risk factors for VTE, including multiple blood biomarkers and risk scores consisting of several risk factors, in Japanese patients receiving anticancer drug therapy. In this single-center, prospective, observational study, 200 patients with six types of cancer were enrolled and followed for 1 year to observe the occurrence of symptomatic or asymptomatic VTE. The present study evaluated risk factors, Khorana and Vienna cancer and thrombosis study (CATS) scores at enrollment, and longitudinal data on various blood biomarkers. A Vienna CATS score of ≥3 was significantly associated with VTE occurrence (HR, 2.8; 95% CI, 0.9-8.7; P=0.045). In multivariable analysis, there was a significant association between VTE and the presence of pancreatic cancer (HR, 3.2; 95% CI, 1.1-8.8; P=0.028) and high soluble fibrin (HR, 3.7; 95% CI, 1.1-7.8; P=0.036). Covariate analysis using the propensity score also showed a significant association with hemoglobin dichotomized at <100 g/l (HR, 3.9; 95% CI, 1.1-14.0; P=0.034). Longitudinal data indicated that VTE was associated with soluble fibrin baseline values and an increase in D-dimer levels over time. The present results suggested that blood biomarkers are beneficial for predicting the risk of VTE in Japanese patients with cancer. The present study also provided novel evidence for the importance of measuring soluble fibrin in patients with cancer.

Anti-cancer effect of afatinib, dual inhibitor of HER2 and EGFR, on novel mutation HER2 E401G in models of patient-derived cancer.

BACKGROUND: Precision medicine with gene panel testing based on next-generation sequencing for patients with cancer is being used increasingly in clinical practice. HER2, which encodes the human epidermal growth factor receptor 2 (HER2), is a potentially important driver gene. However, therapeutic strategies aimed at mutations in the HER2 extracellular domain have not been clarified. We therefore investigated the effect of EGFR co-targeted therapy with HER2 on patient-derived cancer models with the HER2 extracellular domain mutation E401G, based on our previous findings that this mutation has an epidermal growth factor receptor (EGFR)-mediated activation mechanism. METHODS: We generated a xenograft (PDX) and a cancer tissue-originated spheroid (CTOS) from a patient's cancer containing an amplified HER2 E401G mutation. With these platforms, we compared the efficacy of afatinib, a tyrosine kinase inhibitor having anti-HER2 and anti-EGFR activity, with two other therapeutic options: lapatinib, which has similar properties but weaker EGFR inhibition, and trastuzumab plus pertuzumab, for which evidence exists of treatment efficacy against cancers with wild-type HER2 amplification. Similar experiments were also performed with H2170, a cell line with wild-type HER2 amplification, to contrast the characteristics of these drug's efficacies against HER2 E401G. RESULTS: We confirmed that PDX and CTOS retained morphological and immunohistochemical characteristics and HER2 gene profiles of the original tumor. In both PDX and CTOS, afatinib reduced tumor size more than lapatinib or trastuzumab plus pertuzumab. In addition, afatinib treatment resulted in a statistically significant reduction in HER2 copy number at the end of treatment. On the other hand, in H2170 xenografts with wild-type HER2 amplification, trastuzumab plus pertuzumab was most effective. CONCLUSIONS: Afatinib, a dual inhibitor of HER2 and EGFR, showed a promising effect on cancers with amplified HER2 E401G, which have an EGFR-mediated activation mechanism. Analysis of the activation mechanisms of mutations and development of therapeutic strategies based on those mechanisms are critical in precision medicine for cancer patients.

Absence of copy number gain of EGFR: A possible predictive marker of long-term response to afatinib.

Treatment efficacy of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is diverse even in non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. Extraordinary long-term responses sustained over 3 years among NSCLC patients treated with afatinib, an EGFR-TKI, have been reported, but how to predict such long survivors has not been clarified. A multi-institutional prospective observational study, based on comprehensive genomic examination performed with next-generation sequencing of circulating tumor DNA (ctDNA), was conducted to identify potential predictive markers of long-term response to afatinib. Twenty-nine patients with advanced stage NSCLC and EGFR driver mutations detected by standard techniques were enrolled in the study. ctDNA from plasma collected before afatinib treatment was analyzed by Guardant360. ctDNA was detected in 25 of the 29 samples. Median progression-free survival was shorter in patients whose tumors had EGFR copy number gain (7.0 vs 23.0 months, p = 0.022). The impact of EGFR copy number on cell proliferation and the antitumor effect of afatinib were evaluated using genome-editing lung cancer cell lines. HCC827 with EGFR amplification was relatively resistant to afatinib at concentrations below 0.5 nM, but genome-edited derivatives of HCC827 with decreased EGFR copy number demonstrated growth inhibition with 0.1 nM afatinib. The absence of EGFR copy number gain detected in ctDNA may be a predictive marker of long-term response to afatinib. Comprehensive genomic analysis could lead to a more accurate prediction of EGFR-TKI efficacy.

Abnormal White Matter Microstructure in the Limbic System Is Associated With Tuberous Sclerosis Complex-Associated Neuropsychiatric Disorders.

Objective: Tuberous sclerosis complex (TSC) is a genetic disease that arises from TSC1 or TSC2 abnormalities and induces the overactivation of the mammalian/mechanistic target of rapamycin pathways. The neurological symptoms of TSC include epilepsy and tuberous sclerosis complex-associated neuropsychiatric disorders (TAND). Although TAND affects TSC patients' quality of life, the specific region in the brain associated with TAND remains unknown. We examined the association between white matter microstructural abnormalities and TAND, using diffusion tensor imaging (DTI). Methods: A total of 19 subjects with TSC and 24 age-matched control subjects were enrolled. Tract-based spatial statistics (TBSS) were performed to assess group differences in fractional anisotropy (FA) between the TSC and control groups. Atlas-based association analysis was performed to reveal TAND-related white matter in subjects with TSC. Multiple linear regression was performed to evaluate the association between TAND and the DTI parameters; FA and mean diffusivity in seven target regions and projection fibers. Results: The TBSS showed significantly reduced FA in the right hemisphere and particularly in the inferior frontal occipital fasciculus (IFOF), inferior longitudinal fasciculus (ILF), superior longitudinal fasciculus (SLF), uncinate fasciculus (UF), and genu of corpus callosum (CC) in the TSC group relative to the control group. In the association analysis, intellectual disability was widely associated with all target regions. In contrast, behavioral problems and autistic features were associated with the limbic system white matter and anterior limb of the internal capsule (ALIC) and CC. Conclusion: The disruption of white matter integrity may induce underconnectivity between cortical and subcortical regions. These findings suggest that TANDs are not the result of an abnormality in a specific brain region, but rather caused by connectivity dysfunction as a network disorder. This study indicates that abnormal white matter connectivity including the limbic system is relevant to TAND. The analysis of brain and behavior relationship is a feasible approach to reveal TAND related white matter and neural networks. TAND should be carefully assessed and treated at an early stage.

Clinical utility of comprehensive genomic profiling in Japan: Result of PROFILE-F study.

INTRODUCTION: Clinical sequencing has provided molecular and therapeutic insights into the field of clinical oncology. However, despite its significance, its clinical utility in Japanese patients remains unknown. Here, we examined the clinical utility of tissue-based clinical sequencing with FoundationOne® CDx and FoundationOne® Heme. Between August 2018 and August 2019, 130 Japanese pretreated patients with advanced solid tumors were tested with FoundationOne® CDx or FoundationOne® Heme. RESULTS: The median age of 130 patients was 60.5 years (range: 3 to 84 years), and among them, 64 were males and 66 were females. Major cancer types were gastrointestinal cancer (23 cases) and hepatic, biliary, and pancreatic cancer (21 cases). A molecular tumor board had been completed on all 130 cases by October 31, 2019. The median number of gene alterations detected by Foundation testing, excluding variants of unknown significance (VUS) was 4 (ranged 0 to 21) per case. Of the 130 cases, one or more alterations were found in 123 cases (94.6%), and in 114 cases (87.7%), actionable alterations with candidates for therapeutic agents were found. In 29 (22.3%) of them, treatment corresponding to the gene alteration was performed. Regarding secondary findings, 13 cases (10%) had an alteration suspected of a hereditary tumor. Of the 13 cases, only one case received a definite diagnosis of hereditary tumor. CONCLUSIONS: Our study showed that clinical sequencing might be useful for detecting gene alterations in various cancer types and exploring treatment options. However, many issues still need to be improved.

Integrated approach to functional analysis of an ERBB2 variant of unknown significance detected by a cancer gene panel test.

PURPOSE: Dealing with variants of unknown significance (VUS) is an important issue in the clinical application of NGS-based cancer gene panel tests. We detected a novel ERBB2 extracellular domain VUS, c.1157A > G p.(E401G), in a cancer gene panel test. Since the mechanisms of activation by ERBB2 extracellular domain (ECD) variants are not fully understood, we aimed to clarify those mechanisms and the biological functions of ERBB2 E401G. METHODS: ERBB2 E401G was selected as VUS for analysis because multiple software tools predicted its pathogenicity. We prepared ERBB2 expression vectors with the E401G variant as well as vectors with S310F and E321G, which are known to be activating mutations. On the basis of wild-type ERBB2 or mutant ERBB2 expression in cell lines without ERBB2 amplification or variants, we evaluated the phosphorylation of human epidermal growth factor receptor 2 and related proteins, and investigated with molecular dynamics (MD) simulation the mechanisms conferred by the variants. The biological effects of ERBB2 E401G were also investigated, both in vitro and in vivo. RESULTS: We found that ERBB2 E401G enhances C-terminal phosphorylation in a way similar to S310F. MD simulation analysis revealed that these variants maintain the stability of the EGFR-HER2 heterodimer in a ligand-independent manner. Moreover, ERBB2 E401G-transduced cells showed an increased invasive capacity in vitro and an increased tumor growth capacity in vivo. CONCLUSION: Our results provide important information on the activating mechanisms of ERBB2 extracellular domain (ECD) variants and illustrate a model workflow integrating wet and dry bench processes for the analysis of VUS detected with cancer gene panel tests.

Clinical Characteristics and Predictors Related to the Progression of Multinodular Goiter Causing Tracheal Compression and Deviation: A Report of Two Cases and Review of the Literature.

We describe the clinical course of two patients who developed tracheal compression and deviation by multinodular goiter (MNG). Case 1: A 66-year-old woman presented with thyroid swelling. Five years after the initial admission, she was diagnosed with hyperthyroidism by Graves' disease and increased bilateral thyroid lobes compressing the trachea. Thyroglobulin was elevated from 210 to 472 ng/mL. Case 2: A 52-year-old woman presented with thyroid swelling. Five years after the initial admission, the increased right lobe deviated the trachea and compressed the right recurrent laryngeal nerve. Thyroglobulin was elevated from 122 to 392 ng/mL. Two cases and literature review indicated that MNG with >50 mm, solid components, and extension to the mediastinum or paralarynx were risk factors of tracheal compression and deviation. Monitoring thyroglobulin elevation can help predict the clinical course.

Origin of circulating free DNA in patients with lung cancer.

Liquid biopsy has become widely applied in clinical medicine along with the progress in innovative technologies, such as next generation sequencing, but the origin of circulating tumor DNA (ctDNA) has not yet been precisely established. We reported bimodal peaks of long fragment circulating free DNA (cfDNA) of 5 kb and short fragment cfDNA of 170 bp in patients with advanced lung cancer, and both contained ctDNA. In this paper, we demonstrate that the total amount of cfDNA is higher when patients with lung cancer have extrathoracic metastases, and the amount of long fragment cfDNA is significantly higher in those patients. To investigate the origin of long fragment cfDNA, conditioned media isolated from lung cancer cell lines was fractionated. Long fragment cfDNA was found concomitant with extracellular vesicles (EVs), but short fragment cfDNA was not observed in any fractions. However, in peripheral blood from a metastatic animal model both fragments were detected even with those same lung cancer cell lines. In human plasma samples, long fragment cfDNA was observed in the same fraction as that from conditioned media, and short fragment cfDNA existed in the supernatant after centrifugation at 100,000g. Concentration of ctDNA in the supernatant was two times higher than that in plasma isolated by the conventional procedure. Long fragment cfDNA associated with tumor progression might therefore be released into peripheral blood, and it is possible that the long fragment cfDNA escapes degradation by co-existing with EVs. Examination of the biological characteristics of long fragment cfDNA is a logical subject of further investigation.

Targeting aberrant DNA hypermethylation as a driver of ATL leukemogenesis by using the new oral demethylating agent OR-2100.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+ T cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Most HTLV-1-infected individuals are asymptomatic, and only 3% to 5% of carriers develop ATL. Here, we describe the contribution of aberrant DNA methylation to ATL leukemogenesis. HTLV-1-infected T-cells and their uninfected counterparts were separately isolated based on CADM1 and CD7 expression status, and differentially methylated positions (DMPs) specific to HTLV-infected T cells were identified through genome-wide DNA methylation profiling. Accumulation of DNA methylation at hypermethylated DMPs correlated strongly with ATL development and progression. In addition, we identified 22 genes downregulated because of promoter hypermethylation in HTLV-1-infected T cells, including THEMIS, LAIR1, and RNF130, which negatively regulate T-cell receptor (TCR) signaling. Phosphorylation of ZAP-70, a transducer of TCR signaling, was dysregulated in HTLV-1-infected cell lines but was normalized by reexpression of THEMIS. Therefore, we hypothesized that DNA hypermethylation contributes to growth advantages in HTLV-1-infected cells during ATL leukemogenesis. To test this idea, we investigated the anti-ATL activities of OR-1200 and OR-2100 (OR21), novel decitabine (DAC) prodrugs with enhanced oral bioavailability. Both DAC and OR21 inhibited cell growth, accompanied by global DNA hypomethylation, in xenograft tumors established by implantation of HTLV-1-infected cells. OR21 was less hematotoxic than DAC, whereas tumor growth inhibition was almost identical between the 2 compounds, making it suitable for long-term treatment of ATL patient-derived xenograft mice. Our results demonstrate that regional DNA hypermethylation is functionally important for ATL leukemogenesis and an effective therapeutic target.

Phase II trial of gefitinib plus pemetrexed after relapse using first-line gefitinib in patients with non-small cell lung cancer harboring EGFR gene mutations.

OBJECTIVES: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (i.e., EGFR-TKIs) improve the survival of lung cancer patients harboring EGFR mutations. Despite the initial efficacy of EGFR-TKIs, the disease progression caused by acquired resistance to these inhibitors is inevitable. T790M mutations represent a major resistance mechanism to EGFR-TKIs but can be overcome using osimertinib. The IMPRESS trial revealed that the continuation of EGFR-TKI beyond progressive disease (PD) concurrent with platinum-doublet chemotherapy was not beneficial. However, various clinical trials have suggested that EGFR-TKI beyond PD plus single-agent chemotherapy may be a possible treatment strategy. MATERIALS AND METHODS: This study was a single-arm phase II trial. Patients with EGFR-activating mutations (del19 and L858R) that progressed using first-line gefitinib treatment were enrolled and treated with gefitinib beyond PD plus pemetrexed 500 mg/m2 q3w. The primary endpoint was progression-free survival (PFS). Mutation-biased polymerase chain reaction quenching probe, which is the original method for detecting T790M mutations in cell-free plasma DNA, was used prior to treatment. RESULTS: Thirty-six patients were enrolled between May 1, 2013, and March 31, 2016. One patient was excluded before starting the treatment. Among the 35 patients, 15 patients had del19 mutations, and 20 patients had L858R mutations; 33 patients were evaluable for response by using radiographic findings. The median PFS was 6.7 months (95% confidence interval: 4.4-7.7 months). Nineteen patients were T790M positive. No significant difference in PFS was found in a subgroup analysis of EGFR mutation status and T790M positivity. All toxicities were tolerable. CONCLUSION: Gefitinib plus pemetrexed treatment following relapse using gefitinib in patients with Non-small cell lung cancer harboring EGFR mutations demonstrated preferable PFS with mild toxicity. This combination therapy may be considered for platinum-unfit patients without T790M with disease progression using first-line gefitinib. (This clinical trial was registered in UMIN-CTGR as UMIN000010709).

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.

Identification of the BRAF V600E mutation in Japanese patients with hairy cell leukemia and related diseases using a quenching probe method.

Hairy cell leukemia (HCL) is a rare B-cell lymphoid malignancy that is difficult to distinguish from other morphological variants. The frequency of HCL has not been determined accurately in Japan. Recent studies revealed that the BRAF V600E mutation is the causal genetic event in HCL. We assessed the BRAF mutation in Japanese patients with HCL and related diseases using the quenching probe (QP) method, a single-nucleotide polymorphism detection system, and evaluated the incidence rate of HCL among Japanese patients with chronic lymphocytic leukemia, and related diseases. We identified 18 cases (33.3%) harboring the BRAF mutation among 54 patients diagnosed with, or suspected of having HCL. Of BRAF V600E-positive patients, 7 were only detected using the QP method, not by direct sequencing, whereas 11 were positive using both tests. In a larger cohort of Japanese patients diagnosed with chronic lymphoid leukemia or related diseases, the frequency of HCL was 4%. Patients with the BRAF V600E mutation had a significantly higher frequency of neutropenia, thrombocytopenia, and elevated soluble interleukin-2 receptor and common B-cell surface markers than patients without the mutation. Our results confirm that BRAF V600E-positive HCL is a relatively rare disorder in the Japanese leukemia patient population.

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.

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.

Plasma T790M and HGF as potential predictive markers for EGFR-TKI re-challenge.

Re-challenge with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) has been suggested to potentially improve survival in certain populations of patients with advanced lung cancer, but predictive markers for the success of EGFR-TKI re-challenge have not been identified. The present study analyzed 16 re-challenges with EGFR-TKI undertaken in 12 patients with lung adenocarcinoma by investigating T790M and hepatocyte growth factor (HGF) in plasma coupled with clinical characteristics. EGFR mutations in plasma DNA were detected using the wild inhibiting PCR and quenched probe system for exon 19 deletions, and T790M and L858R were detected using the mutation-biased PCR and quenched probe system. HGF levels in the plasma were measured by enzyme-linked immunosorbent assay, and the ratio of HGF levels prior to re-challenge to those prior to the previous EGFR-TKI treatment was calculated. Two re-challenges demonstrated partial response, six remained as stable disease and eight had progressive disease (PD). A total of 4 of the 5 patients with a history of T790M positivity based on plasma DNA levels had PD. A total of 7 of the 8 patients who had ≥1.5-fold elevation of HGF prior to re-challenge with EGFR-TKI suffered PD. Elevation of the HGF ratio to ≥1.5 was significantly associated with poor response to EGFR-TKI re-challenge. Having no history of T790M and an HGF ratio <1.5 was significantly associated with a positive response to EGFR-TKI re-challenge. A combination of T790M detection and HGF quantification using plasma is a potentially useful assay system for predicting the effect of EGFR-TKI re-challenge. Future prospective studies are required to confirm the predictive validity of these markers.

Detection of KRAS Mutations in Plasma DNA Using a fully Automated Rapid Detection System in Colorectal Cancer Patients.

KRAS mutations have been recognized as predictive markers of primary resistance to anti-EGFR-antibodies in colorectal cancer patients. In addition, newly detected KRAS mutations have been reported to be related with acquired resistance to chemotherapy containing anti-EGFR antibody. Considering this evidence, monitoring of KRAS mutations is indispensable for making treatment decisions, and the method should be non-invasive allowing repeated examinations. Recently, we established a novel automated sensitive detection system for KRAS mutations, named mutation-biased PCR quenching probe system (MBP-QP). The goal of our study was to investigate the potential for monitoring KRAS mutations during treatment with anti-EGFR antibodies. The detection limit of MBP-QP using a control plasmid containing KRAS mutations was 1-9 copies, and 0.05-0.3% mutant plasmid was detectable in a mixture of wild type and mutants. One-hundred twenty colorectal cancer patients were genotyped for KRAS mutations with MBP-QP as well as polymerase chain reaction reverse sequence-specific oligonucleotide (PCR-rSSO), which has already been applied to cancer tissue samples in the clinical setting. Concordance rates between plasma DNA and cancer tissues were 68% with MBP-QP and 66% with PCR-rSSO, indicating that these systems are equivalent in terms of detecting KRAS mutations with plasma DNA. KRAS mutations in plasma DNA were frequently observed in systemic metastatic cancer patients, and in three patients KRAS mutations appeared after chemotherapy containing anti-EGFR antibody. A prospective study is needed for clarifying whether KRAS mutations detected in plasma DNA are predictive markers of treatment efficacy with anti-EGFR antibody.

Mutation Profile of B-Raf Gene Analyzed by fully Automated System and Clinical Features in Japanese Melanoma Patients.

BRAF gene mutations have been observed in 30-50 % of malignant melanoma patients. Recent development of therapeutic intervention using BRAF inhibitors requires an accurate and rapid detection system for BRAF mutations. In addition, the clinical characteristics of the melanoma associated with BRAF mutations in Japanese patients have not been investigated on a large scale evaluation. We recently established quenching probe system (QP) for detection of an activating BRAF mutation, V600E and evaluated 113 melanoma samples diagnosed in Saga University Hospital from 1982 to 2011. The QP system includes fully automated genotyping, based on analysis of the probe DNA melting curve, which binds the target mutated site using a fluorescent guanine quenched probe. BRAF mutations were detected in 54 of 115 (47 %) including 51 of V600E and 3 of V600 K in Japanese melanoma cases. Among clinical subtypes of melanoma, nodular melanoma showed high frequency (12 of 15; 80 %) of mutation followed by superficial spreading melanoma (13 of 26; 50 %). The QP system is a simple and sensitive method to determine BRAF V600E mutation, and will be useful tool for patient-oriented therapy with BRAF inhibitors.

A case of a warfarinized renal cancer patient monitored for prothrombin time-international normalized ratio during methadone introduction.

BACKGROUND: Warfarin, a widely used anticoagulant, interacts with various agents used in palliative care, such as oxycodone, morphine, acetaminophen, and non-steroidal anti-inflammatory drugs (NSAIDs); however, there are no reports of its interaction with methadone. We report a case of a patient receiving warfarin when methadone was introduced for pain control with monitoring of the prothrombin time-international normalized ratio (PT-INR) and deduced the pharmacological background. CASE PRESENTATION: A 60-year-old male was emergently admitted to our university hospital for the sudden onset of severe back pain. Abdominal CT imaging revealed that the vertebral body of the ninth thoracic vertebra was occupied by bone metastasis and crushed, which caused his back pain. He received warfarin 3.5 mg/day for atrial fibrillation and tapentadol 100 mg p.o. daily for pain relief. The prothrombin time-international normalized ratio (PT-INR) was maintained at >2.2. The patient's history included diabetes mellitus and hypertension, but his laboratory test was unremarkable with the exception that his eGFR was 34 ml/min.Initially, a fentanyl dermal patch was used instead of tapentadol to avoid interactions with warfarin. We started concomitant administration of oxycodone and 2.4 g/day of acetaminophen while monitoring the PT-INR because acetaminophen increased the PT-INR to 2.93. A continuous intravenous infusion of oxycodone was introduced, in increments of the dose, resulting in an increase of the PT-INR to 3.41, which is required to reduce the dose of warfarin to 1.5 mg. Because of the lack of effective pain relief, methadone was introduced and the dose was gradually increased. The PT-INR was not changed and the dose of warfarin was not changed. An infusion of oxycodone and oral methadone was used to allow the patient to walk in his room, and he was later transferred to the palliative hospital. CONCLUSIONS: In an oral warfarinized patient, methadone seemed to undergo different metabolism than oxycodone. When warfarin and methadone are used together, we have to consider their interaction by comparing the competitive inhibition of CYP2C9 to the induction of CYP3A4 by methadone, because CYP3A4 metabolize various drugs including oxycodone.

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.