Clinical practice guidelines for molecular tumor marker, 2nd edition review part 2.

In recent years, rapid advancement in gene/protein analysis technology has resulted in target molecule identification that may be useful in cancer treatment. Therefore, "Clinical Practice Guidelines for Molecular Tumor Marker, Second Edition" was published in Japan in September 2021. These guidelines were established to align the clinical usefulness of external diagnostic products with the evaluation criteria of the Pharmaceuticals and Medical Devices Agency. The guidelines were scoped for each tumor, and a clinical questionnaire was developed based on a serious clinical problem. This guideline was based on a careful review of the evidence obtained through a literature search, and recommendations were identified following the recommended grades of the Medical Information Network Distribution Services (Minds). Therefore, this guideline can be a tool for cancer treatment in clinical practice. We have already reported the review portion of "Clinical Practice Guidelines for Molecular Tumor Marker, Second Edition" as Part 1. Here, we present the English version of each part of the Clinical Practice Guidelines for Molecular Tumor Marker, Second Edition.

A pilot study of on-site evaluation as external quality assessment for ISO 15189 accreditation of laboratories performing next-generation sequencing oncology tests.

BACKGROUND: As next-generation sequencing (NGS) oncology tests vary by platform, application, and target of genes, specific methods for external quality assessment (EQA) have not been universally applied. Hence, we have attempted to implement on-site evaluation as EQA in the accreditation program under ISO 15189 for laboratories that perform NGS oncology tests. METHODS: A total of 10 laboratories that performed NGS oncology tests were enrolled. Two types of EQA samples were prepared (Acrometrix Oncology Hotspot Control DNA and OncoSpan gDNA DNA samples), and the variant allele frequency of targeted genes was assigned. The samples were subjected to NGS oncology tests in participant laboratories according to their routine protocols. Based on the result reports, auditors visited the participant laboratories to perform on-site evaluations and provided feedback regarding possible laboratory process improvement. RESULTS: The participant laboratories identified the targeted variants in the Acrometrix Oncology Hotspot Control DNA and OncoSpan gDNA samples with a success rate of 31-100% and 9.5-100%, respectively, compared with reference information, depending on their sequencing systems, and reported a few lower-variant allele frequencies. Six of the eight evaluated laboratories failed to report at least three pathogenic variants due to errors in wet-lab and/or dry-lab processes. Based on the feedback reports and self-assessment, auditors and laboratory staff discussed potential improvements to processes during on-site evaluations for laboratory accreditations. CONCLUSIONS: On-site evaluation as EQA for NGS oncology tests in the laboratory accreditation program under ISO 15189 was successfully implemented and proved applicable to a broad spectrum of NGS tests.

Virucidal effect of monogalactosyl diacylglyceride from a green microalga, Coccomyxa sp. KJ, against clinical isolates of SARS-CoV-2 as assessed by a plaque assay.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and is capable of human-to-human transmission and rapid global spread. Thus, the establishment of high-quality viral detection and quantification methods, and the development of anti-SARS-CoV-2 agents are critical. METHODS: Here, we present the rapid detection of infectious SARS-CoV-2 particles using a plaque assay with 0.5% agarose-ME (Medium Electroosmosis) as an overlay medium. RESULTS: The plaques were capable of detecting the virus within 36-40 h post-infection. In addition, we showed that a monogalactosyl diacylglyceride isolated from a microalga (Coccomyxa sp. KJ) could inactivate the clinical isolates of SARS-CoV-2 in a time- and concentration-dependent manner. CONCLUSIONS: These results would allow rapid quantification of the infectious virus titers and help develop more potent virucidal agents against SARS-CoV-2.

Attempts to optimize postinduction treatment in childhood acute myeloid leukemia without core-binding factors: A report from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG).

We previously reported that risk-stratified therapy and intensive postremission chemotherapy (PRC) contributed to the improved survival of childhood acute myeloid leukemia (AML) in the AML99 study, which led us to consider a reduction in the number of PRC courses with more restrictive indications for stem cell transplantation (SCT) in the successor AML-05 study. We here report the outcome of AML patients without core-binding factor mutation (non-CBF AML) in the AML-05 study. Two-hundred eighty-nine children (age < 18 years old) with non-CBF AML were eligible. Patients with unfavorable cytogenetics and/or poor bone marrow response to the first induction course were candidates for SCT in the AML-05 study. After two courses of induction, a further three courses of PRC were given in AML-05, while four courses were given in the AML99 study. The 3-year event-free survival (EFS) rate in the AML-05 study (46.7%, 95% CI: 40.6-52.6%) was comparable to that of non-CBF AML in the AML99 study (51.5%, 95% CI: 42.7-59.6%) (P = .16). However, the 3-year overall survival (OS) rate in the AML-05 study (62.9%, 95% CI: 56.3-68.8%) was slightly lower than that in the AML99 study (71.6%, 95% CI: 63.2-78.5%) (P = .060), mainly due to decreased remission induction rate and increased nonrelapsed mortality. In conclusion, reductions in the number of PRC courses from four to three, together with repetitive cycles of high-dose cytarabine, were acceptable for non-CBF childhood AML.

Long-term Remission of Acute Myeloid Leukemia Developed From Systemic Mastocytosis by Conventional Chemotherapy.

Systemic mastocytosis (SM) is a disorder characterized by abnormal proliferation of mast cells with KIT mutations, especially in codon 816. The prognosis of patients developing acute myeloid leukemia (AML) from SM is extremely poor, and hematopoietic cell transplantation is recommended. Herein, we describe a case of an 8-year-old female diagnosed with SM developing AML. A KIT M541L variant in SM was identified in leukemic cells, normal hematopoietic cells, and buccal mucosal cells, suggesting a germline polymorphism. The patient has remained in complete remission for 39 months after completion of chemotherapy. SM developing AML without a KIT D816 mutation may be not necessarily associated with a poor prognosis.

[Acute myeloid leukemia evolving from KIT D816-mutated systemic mastocytosis relapsing two months after completion of chemotherapy].

Here, we report the case of a 9-year-old girl with acute myeloid leukemia (AML) developed from systemic mastocytosis (SM). She experienced bladder and rectal disturbance due to an extramedullary nodule in the paraspinal region of the sacrum. Cytogenetic and genetic analyses of leukemic cells revealed the KIT D816Y mutation besides t (8;21) (q22:q22) /RUNX1-RUNX1T1. Despite receiving proton beam therapy after conventional chemotherapy, the patient relapsed after 2 months. As SM-AML with the KIT D816 mutation in adults exhibits a poor prognosis, hematopoietic stem cell transplantation is recommended. Owing to a few reports of SM-AML in children, the standard therapy for pediatric cases has not been established to date. Based on our experience and the related literature, the prognosis of childhood SM-AML could be as poor as in adults. Hence, further investigation, including mutational analyses of the KIT gene, is warranted to establish a risk-oriented strategy for managing childhood SM-AML.

Evaluation of Risk Factors for Clostridium difficile Infection Based on Immunochromatography Testing and Toxigenic Culture Assay.

In recent years, the diagnostic method of choice for Clostridium difficile infection (CDI) is a rapid enzyme immunoassay in which glutamate dehydrogenase (GDH) antigen and C. difficile toxin can be detected (C. diff Quik Chek Complete; Alere Inc.) (Quik Chek). However, the clinical significance remains unclear in cases that demonstrate a positive result for GDH antigen and are negative for toxin. In this study, we used the Quik Chek test kit on fecal samples, with an additional toxin detection step using a toxigenic culture assay for the aforementioned cases. CDI risk factors were assessed among the 3 groups divided by the Quik Chek test results. The study involved 1,565 fecal samples from patients suspected to have CDI who were hospitalized during the period of April 2012 to March 2014. The 3 groups were defined as follows: both GDH antigen positive and toxin positive (by Quik Chek test) (toxin-positive [TP] group, n = 109), both GDH antigen and toxin negative (toxin-negative [TN] group, n = 111), and positive only for GDH antigen but toxin positive with subsequent toxigenic culture (toxigenic culture [TC] group, n = 72). The gender, age, number of hospitalization days, white blood cell (WBC) counts, serum albumin levels, body mass index (BMI), fecal consistency, and use of antibacterials and proton pump inhibiters (PPIs) were analyzed. The positive rate for the fecal direct Quik Chek test was 7.0% (109/1,565 cases). However, toxigenic culture assays using the Quik Chek test for only the GDH-antigen-positive/toxin-negative samples were 35.3% positive (72/204 cases). As a result, the true positive rate for C. difficile toxin detection was estimated to be 11.6% (181/1,565 cases). Moreover, significant differences (P < 0.05) in the number of hospitalization days (>50 days), WBC counts (>10,000 WBCs/µl), and use of PPIs comparing the TN, TP, and TC groups, were observed. The odds ratios (ORs) for the development of CDI were 1.61 (95% confidence interval [CI], 0.94 to 2.74) and 2.98 (95% CI, 1.59 to 5.58) for numbers of hospitalization days, 2.16 (95% CI, 1.24 to 3.75) and 2.24 (95% CI, 1.21 to 4.14) for WBC counts, and 9.03 (95% CI, 4.9 to 16.6) and 9.15 (95% CI, 4.59 to 18.2) for use of PPIs in the TP and TC groups, respectively. These findings demonstrated that the use of PPIs was a significant risk factor for CDI development. Moreover, antibacterials such as carbapenems, cephalosporins, and fluoroquinolones were demonstrated to be risk factors. In conclusion, identification of the TC group of patients is thought to be important, as this study demonstrates that this group bears the same high risk of developing CDI as the TP group.

Evaluation of high-dose cytarabine in induction therapy for children with de novo acute myeloid leukemia: a study protocol of the Japan Children's Cancer Group Multi-Center Seamless Phase II-III Randomized Trial (JPLSG AML-12).

The purpose of this study is to compare the efficacy and safety of combination therapy (HD-ECM) including high-dose cytarabine in initial induction therapy with that of combination therapy (ECM) involving the continuous administration of cytarabine for previously untreated, newly diagnosed patients with AML at <18 years of age. This is a seamless Phase II-III clinical trial, consisting of Phase II and III parts. In the Phase II part, the safety of the experimental treatment (HD-ECM) will be examined. Subsequently, the Phase III study will compare the efficacy and safety of HD-ECM with that of standard ECM. The primary endpoint of the Phase II study is the early mortality rate. The primary endpoints of the Phase III study are the 3-year event-free survival rate and the positive minimal residual disease rate by flow cytometry after initial induction therapy. This trial has been registered at the UMIN Clinical Trials Registry (UMIN000013288).

Hypodysfibrinogenemia with a Heterozygous Mutation of γCys326Ser by the Novel Transversion of TGT to TCT in a Patient with Pulmonary Thromboembolism and Right Ventricular Thrombus.

We encountered a 45-year-old Japanese man who suffered from pulmonary thromboembolism and huge right ventricular thrombus after inferior vena cava (IVC) filter implantation without apparent thrombus in either the deep veins or inside the IVC filter. The biochemical data showed a discrepancy in the level of fibrinogen between the immunological and thrombin time methods, suggesting hypodysfibrinogenemia. The sequencing of the fibrinogen γ-chain gene (FGG) revealed a novel heterozygous missense mutation in exon 8 - a TGT to TCT transversion in codon 326 - resulting in an amino acid substitution of serine for cysteine (γCys326Ser). The characterization of the protein did not show known mechanisms for thrombosis in dysfibrinogenemia, such as dimer or albumin-binding complex formation. In summary, the current case with a life-threatening thrombotic event was found to have a novel heterozygous missense mutation resulting in γCys326Ser, which was suggested as a predisposing factor of the thrombosis. Known mechanisms responsible for thrombosis in the current case were not demonstrated, suggesting other mechanisms including superimposing inherited and/or acquired risk factors. When a patient presents with unusual thrombosis such as breakthrough pulmonary embolism and huge thrombus in the right ventricle, as in the current case, the laboratory process for heritable thrombophilia should be considered.

[Evolution of BCR-ABL1 Testing for Chronic Myeloid Leukemia Under Tyrosine Kinase Inhibitor Treatment].

The advent and long-term use of tyrosine kinase inhibitors in molecular target therapy for chronic myeloid leukemia have resulted in a marked improvement of treatment outcomes. This has changed'the algorithm of the laboratory process for the diagnosis and therapeutic monitoring of chronic myeloid leukemia. It includes defining the molecular typing of BCR-ABL1 to establish the diagnosis, and a quantitative and/or high- sensitivity assay for minimal residual disease to evaluate the treatment response. Along with improved long-term survival outcomes, new issues have arisen regarding the best index to use for the improved clinical outcomes, such as treatment-free remission. To this end, clinical and laboratory monitoring of CML patients has been investigated. Novel methodologies and technologies have been applied to improve decision-making on patient care, tailoring the treatment to the individual characteristics of each patient, including an early index for the treatment response and deeper molecular response to realize treatment-free survival, and BCR-ABLI kinase domain mutation screening for refractory disease. In response to the advancement and applications of these emerging technologies, proper laboratory practice with the quality assurance of testing is expected. [Review].

High event-free survival rate with minimum-dose-anthracycline treatment in childhood acute promyelocytic leukaemia: a nationwide prospective study by the Japanese Paediatric Leukaemia/Lymphoma Study Group.

We evaluated the efficacy of treatment using reduced cumulative doses of anthracyclines in children with acute promyelocytic leukaemia (APL) in the Japanese Paediatric Leukaemia/Lymphoma Study Group AML-P05 study. All patients received two and three subsequent courses of induction and consolidation chemotherapy respectively, consisting of all-trans retinoic acid (ATRA), cytarabine and anthracyclines, followed by maintenance therapy with ATRA. Notably, a single administration of anthracyclines was introduced in the second induction and all consolidation therapies to minimize total doses of anthracycline. The 3-year event-free (EFS) and overall survival rates for 43 eligible children were 83·6% [95% confidence interval (CI): 68·6-91·8%] and 90·7% (95% CI: 77·1-96·4%), respectively. Although two patients died of intracranial haemorrhage or infection during induction phases, no cardiac adverse events or treatment-related deaths were observed during subsequent phases. Patients not displaying M1 marrow after the first induction therapy, or those under 5 years of age at diagnosis, showed inferior outcomes (3-year EFS rate; 33·3% (95% CI: 19·3-67·6%) and 54·6% (95% CI: 22·9-78·0%), respectively). In conclusion, a single administration of anthracycline during each consolidation phase was sufficient for treating childhood APL. In younger children, however, conventional ATRA and chemotherapy may be insufficient so that alternative therapies should be considered.

Multianalyte Conventional Reference Material (MacRM): A Useful Tool for Nationwide Standardization of Laboratory Measurements for Medical Care-A Model Study in Japan.

BACKGROUND: The Japanese Committee for Clinical Laboratory Standards (JCCLS) has developed a multianalyte conventional reference material (MacRM) for nationwide standardization of laboratory measurements. METHODS: To prepare the MacRM, pooled sera were obtained from healthy Japanese individuals. Target values of the pooled sera for 30 analytes were assigned on the basis of the measurement results of 45 certified clinical laboratories whose calibration was verified by measuring certified reference materials (CRMs) provided by the National Institute of Standards and Technology, the Institute for Reference Materials and Measurements, and JCCLS. Commutability of MacRM was assessed by comparison with results for 150 individual inpatients at Fukuoka University Chikushi Hospital. Survey samples were prepared by essentially the same method for MacRM but without target values. The survey samples were used to assess agreement among 165 laboratories that used various assay kits and platforms calibrated with the MacRM. RESULTS: The commutability of MacRM was confirmed for 30 analytes with sera from 150 individual patients. The imprecision (CV) of measurements of survey samples (high and low concentrations) among the 165 laboratories was 0.4%-10.0%. Twenty-six of 30 analytes were within the goals for interinstitutional allowable bias. An aliquot of MacRM stored frozen at -80 °C remained stable for ≥4 years. CONCLUSIONS: The MacRM was successfully applied as a calibrator to achieve nationwide standardization for 30 analytes measured by 165 laboratories that used various methods from different manufacturers.

Analysis to Estimate Genetic Variations in the Idarubicin-Resistant Derivative MOLT-3.

Gene alterations are a well-established mechanism leading to drug resistance in acute leukemia cells. A full understanding of the mechanisms of drug resistance in these cells will facilitate more effective chemotherapy. In this study, we investigated the mechanism(s) of drug resistance in the human acute leukemia cell line MOLT-3 and its idarubicin-resistant derivative MOLT-3/IDR through complete mitochondrial and nuclear DNA analyses. We identified genetic differences between these two cell lines. The ND3 mutation site (p.Thr61Ile) in the mitochondrial DNA sequence was unique to MOLT-3/IDR cells. Moreover, we identified five candidate genes harboring genetic alterations, including GALNT2, via CGH array analysis. Sequencing of the GALNT2 exon revealed a G1716K mutation present within the stop codon in MOLT-3/IDR cells but absent from MOLT-3 cells. This mutation led to an additional 18 amino acids in the protein encoded by GALNT2. Using real-time PCR, we determined an expression value for this gene of 0.35. Protein structure predictions confirmed a structural change in GALNT2 in MOLT-3/IDR cells that corresponded to the site of the mutation. We speculate that this mutation may be related to idarubicin resistance.

[Accreditation of Medical Laboratory under International Standards in Clinical Trial].

In Japan, a medical laboratory accreditation program has been implemented on the basis of the international standard ISO 15189 (Medical laboratories-requirements for quality and competence), under the cooperation of the Japanese Committee for Clinical Laboratory Standards (JCCLS) and the Japan Accreditation Body (JAB). It is carried out in accordance with the specific guidance document 'Guide RM300: 2014'. The subject in the accreditation program is currently limited to common laboratory tests, which are covered by medical insur- ance, with regulatory approval. The 2012 version of ISO 15189 (ISO 15189:2012) expanded its scope, cover- ing genetic testing. In order to achieve the purpose of a clinical research core hospital that has recently been established in Japan, there is a need for an accreditation program to cover the lack of insurance coverage, such as molecular-genetic testing based on emerging technologies. To this end, the development of guid- ance documents intended for genetic testing by which medical laboratories can implement a quality system to meet ISO 15189:2012 is to be considered. In addition, it is necessary to secure human resources who are familiar with oversight and inspection regarding genome-specific matters. [Review].

[Education and Career Development of Laboratory Personnel through Qualification System of Molecular Analysis Technologist and Specialist}.

Clinical applications of molecular-genetic testing are rapidly disseminating and expanding. A qualification system of technological professionals for molecular-genetic testing has been implemented and operating in Japan, aiming at educating and evaluating personnel who are to engage in molecular-genetic testing. It con- sists of two stages in career development with molecular analysis technologist and specialist, both of which need to be renewed every 5 years. The qualification is of significance across many fields. Human resource development of molecular-genetic testing is a cornerstone of quality assurance of measurement by medical and research laboratory personnel. In addition, expectations of the qualified personnel include the manage- ment of relevant guidelines, compliance regarding the use of personal genetic information (medical ethics) and bio-risk in the laboratories, as well as engagement in the biotechnology industry. [Review].

[Global Trends in the Use of Point-of-Care Testing Devices and Efforts toward Standardization.]

Advances in testing and information technologies have expanded the availability of Point-of-Care Testing (POCT) devices not only in healthcare but also in non-healthcare settings. POCT laboratory accreditation under ISO 22870 has been implemented in conjunction with ISO 15189 in European countries. A markedly increasing number of facilities are performing POCT in non-healthcare settings in the United States with a certificate of waiver and in European countries; concerns are being raised on the lack of oversight and re- quirements for personnel qualifications and training. Centers for Disease Control and Prevention have been making efforts to develop educational tools. In ISO Technical Committee 212 (ISO/TC212) on clinical labor- atory testing and in vitro diagnostic test systems, guidance for POCT operators is proposed, being designed as a document for operators in healthcare and non-healthcare settings who lack specific medical laboratory training. In Japan, on the basis of the international efforts on standardization and quality assurance, nation- wide efforts are necessary for increased reliability and confidence on performing POCT to promote the cost- effectiveness and quality of healthcare services.

Sonographic evaluation of the treatment response in patients with immunoglobulin G4-related disease of the submandibular glands.

OBJECTIVES: To evaluate the usefulness of sonography for monitoring the response to glucocorticoid treatment in patients with immunoglobulin G4 (IgG4)-related disease. METHODS: We conducted a retrospective study using sonography in 12 patients with bilateral swollen submandibular glands who had a diagnosis of IgG4-related disease based on an elevated serum IgG4 level (>135 mg/dL) and histopathologic findings between January 2010 and December 2012. Among these patients, 6 were treated with prednisolone, and the other 6 were placed under observation. B-mode sonographic examinations of the submandibular glands were performed with or without color Doppler imaging at the initial examination and 6 months later. Findings were compared between the groups (treated and untreated), and their relationship with the treatment response of the primarily involved organs was investigated. RESULTS: In the treated group, the submandibular glands of all 6 patients decreased in both size and volume after treatment (average volume ± SD, 27,449.7 ± 24,227.6 to 4609.7 ± 1911.4 mm(3); P = .004). The internal echo texture, characterized by multiple hypoechoic foci scattered against a heterogeneous hyperechoic background of submandibular tissue with demarcated hyperechoic lines, with or without hypoechoic tumor formation, disappeared or was obscured in all cases. In addition, the blood flow signals were reduced in all 3 patients who underwent color Doppler sonography, and the response observed on sonography was found to correlate with the IgG4 level and recovery of specific organ involvement. In contrast, in the untreated group, the submandibular glands showed a tendency to increase in both size and volume (average volume, 9326.3 ± 3054.8 to 12,217.4 ± 4605.5 mm(3); P= .2) without a decrease in the blood flow signals. CONCLUSIONS: Sonography is considered useful for evaluating the response to glucocorticoid therapy in patients with IgG4-related disease of the submandibular glands.

[Molecular-Genetic Diagnosis and Molecular-Targeted Therapy in Cancer: Challenges in the Era of Precision Medicine].

Elucidation of the molecular pathogenesis of neoplasms and application of emerging technologies for testing and therapy have resulted in a series of paradigm shifts in patient care, from conventional to personalized medicine. This has been promoted by companion diagnostics and molecular targeted therapy, tailoring the treatment to the individual characteristics of each patient. Precision oncology has been accelerated by integrating the enhanced resolution of molecular analysis, mechanism clarity, and therapeutic relevance through genomic knowledge. In its clinical implementation, there are laboratory challenges concerning accurate measurement using stored samples, differentiation between driver and passenger mutations as well as between germline and somatic mutations, bioinformatics availability, practical decision-making algorithms, and ethical issues regarding incidental findings. The medical laboratory has a new role in providing not only testing services but also an instructive approach to users to ensure the sample quality and privacy protection of personal genome information, supporting the quality of patient practice based on laboratory diagnosis.

[Development of Standards for Baseline Quality in Quality Management of Molecular-Diagnostic Testing].

As molecular-diagnostic testing is expanding in clinical use, the demand for its quality assurance is increasing. To this end, efforts towards quality management have been made regionally and globally. An entire testing procedure needs to be properly performed from the preanalytic, analytic, and postanalytic processes. Particularly, the preanalytic process largely affects the measurement and, thus, the result. The Japanese Committee for Clinical Laboratory and Standard developed the standard documents, such as that for the quality management of clinical specimens and best-practice guideline for quality assurance of molecular-genetic testing. These standard documents would provide not only the requirements as the best practice for testing, but also the basis of baseline quality and reliability. They can be used as the basis for assessment of the quality of practice in reimbursement coverage by payers and in certification or accreditation by a third party.

[Overview of Proficiency Testing and External Quality Assessment Programs of Laboratory Tests from the Standpoint of International Standardization].

In Japan, laboratory comparison, by means of proficiency testing or external quality assessment, has been conducted with separate programs on a nationwide basis, markedly contributing to quality improvement of clinical laboratory testing. However, from the standpoint of international standardization, there are issues to be addressed. Performance is evaluated once a year in each program, with time-consuming studies and different methods for data collection, analysis, and its assessment among programs, thus not leading to real-time monitoring and correction of the performance. Only the examination procedure of routine laboratory testing is focused on, while the entire process of the examination needs to be properly performed from the pre-examination down to the post-examination procedures. The development of proficiency testing for esoteric tests is needed, since both routine and esoteric ones have been provided by most medical laboratories, and a combination of them is used for decision-making in patient care. As molecular-genetic tests have been widely used, demand for the development of laboratory comparison programs has increased. In order to comprehensively evaluate the performance of laboratory, medical imaging and biophysical examination are also necessary. The overall performance of the medical laboratory must be assured along with the availability of proficiency testing or external quality assessment programs for each test to be performed, in order to contribute to optimal patient care.