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Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Yamanashi Central Hospital

The University of Tokyo

the university of tokyo

In both SHARP and Asia-Pacific Study, sorafenib was proved to improve the overall survival of the patients with hepatocellular carcinoma. However, factors contributing to the improvement of overall survival of the patients treated by sorafenib have not been fully evaluated. In this study, patient-derived, background liver disease-derived and tumor-derived factors before treatment were evaluated whether they have contributed to the improvement of the overall survival.

Factors contributing to the overall survival in patients with hepatocellular carcinoma treated by sorafenib.

Sarcomatoid cancer is defined by the World Health Organization as a category of non-small cell lung cancers with sarcoma or sarcoma-like differentiation. They are characterized by poor prognosis and resistance to conventional chemotherapy. However, the mutational profile of sarcomatoid cancer remains yet to be elucidated. Sarcomatoid cancers are usually biphasic tumors composed of carcinomatous and sarcomatous components, but the evolutional development of sarcomatoid cancer is controversial.

Stepwise addition of genetic changes correlated with histological change from "well-differentiated" to "sarcomatoid" phenotypes: a case report.

Bronchoendoscopic examination is not necessarily comfortable procedure and limited by its sensitivity, depending on the location and size of the tumor lesion. Patients with a non-diagnostic bronchoendoscopic examination often undergo further invasive examinations. Non-invasive diagnostic tool of lung cancer is desired. A 72-year-old man had a 3.0 cm × 2.5 cm mass lesion in the segment B1 of right lung. Cytological examination of sputum, bronchial washing and curetted samples were all "negative". We could confirm a diagnosis of lung cancer after right upper lung lobe resection pathologically, and also obtained concordant results by genomic analysis using cytological negative samples from airways collected before operation. Genetic analysis showed mutational profiles of both resected specimens and samples from airways were identical. These data clearly indicated the next generation sequencing (NGS) may yield a diagnostic tool to conduct "precision medicine".

Analysis of significantly mutated genes as a clinical tool for the diagnosis in a case of lung cancer.

In cases of multiple lung cancers, individual tumors may represent either a primary lung cancer or both primary and metastatic lung cancers. Treatment selection varies depending on such features, and this discrimination is critically important in predicting prognosis. The present study was undertaken to determine the efficacy and validity of mutation analysis as a means of determining whether multiple lung cancers are primary or metastatic in nature. The study involved 12 patients who underwent surgery in our department for multiple lung cancers between July 2014 and March 2016. Tumor cells were collected from formalin-fixed paraffin-embedded tissues of the primary lesions by using laser capture microdissection, and targeted sequencing of 53 lung cancer-related genes was performed. In surgically treated patients with multiple lung cancers, the driver mutation profile differed among the individual tumors. Meanwhile, in a case of a solitary lung tumor that appeared after surgery for double primary lung cancers, gene mutation analysis using a bronchoscopic biopsy sample revealed a gene mutation profile consistent with the surgically resected specimen, thus demonstrating that the tumor in this case was metastatic. In cases of multiple lung cancers, the comparison of driver mutation profiles clarifies the clonal origin of the tumors and enables discrimination between primary and metastatic tumors.

Mutational analysis of multiple lung cancers: Discrimination between primary and metastatic lung cancers by genomic profile.

Stereotactic radiotherapy (SRT) for inoperable stage I non-small cell lung cancer has shown promising results and is now an alternative therapy for this disease. Several reports have detailed changes in mutation profiles after treatment with chemotherapy; however, such changes after SRT for lung cancer have not been reported. A patient who received SRT for lung cancer developed local recurrence 9 months after treatment and underwent surgery in our department. Using bronchoscopically biopsied and surgically resected specimens, we performed targeted sequencing of 53 lung cancer-related genes and compared the tumor mutation profiles before and after SRT. Identical mutations were detected from tumor specimens collected before and after SRT, and the specimens were confirmed to be clonal. However, the number of mutations decreased after SRT, suggesting that it induced mutation selection. Analyses of the statistical inference of clonal population structure showed that this evolving heterogeneous genomic landscape may be caused by heterogeneous responsiveness to SRT.

Elucidation of radiation-resistant clones by a serial study of intratumor heterogeneity before and after stereotactic radiotherapy in lung cancer.

Circulating tumor DNA (ctDNA), extracted from plasma, is a non-invasive surrogate biomarker. However, the distribution of ctDNA in the body still remains to be elucidated. In this study, resected lung tumors, with simultaneous blood and bone marrow samples, were analyzed to elucidate the distribution of ctDNA. Rib bone marrow, pulmonary venous blood (Pul.V) and peripheral blood (Peri.B) were obtained from 30 patients. The liquid samples were divided into cell pellets and supernatant by centrifugation; a total of 212 DNA samples were subjected to massively parallel sequencing. ctDNA was detected in 5 patients. Given that the frequency of mutations in the primary tumor was considered to be 100%, those in the other specimens were as follows; Pul.V plasma 20%, Peri.B plasma 11%, and the other samples 0%. Furthermore, ctDNA reflected the predominant mutations in the primary lesion. Clinically, the presence of ctDNA was associated with significantly poorer survival. These results suggest ctDNA "spill over" into an immediate outflow tract (Pul.V), and from there is disseminated to the entire body. Thus, it can be inferred that ctDNA reflects the cancer progression and could function as a prognostic marker.

Distribution of circulating tumor DNA in lung cancer: analysis of the primary lung and bone marrow along with the pulmonary venous and peripheral blood.

Immunotherapy has become a treatment option for lung cancer. The utility of nivolumab as second-line treatment for non-small cell lung cancer has been proven, but predictive biomarkers influencing its efficacy remain unknown.

Very early response of circulating tumour-derived DNA in plasma predicts efficacy of nivolumab treatment in patients with non-small cell lung cancer.

Pancreatic cancer is classified as ductal, acinar, neuroendocrine carcinoma or pancreatoblastoma. Ductal and acinar cells derive from exocrine glands and neuroendocrine cells from endocrine glands; however, mixed acinar-neuroendocrine-ductal carcinoma has different histological carcinomas coexisting within a nodule. The mixed pancreatic carcinoma forms from different developmental origins and therefore requires investigation. The current case report presents a 50-year-old male who had a tumor within the body of the pancreas. Pathological examination clarified the tumor as a mixed acinar-neuroendocrine-ductal carcinoma. The ductal and acinar/neuroendocrine tumor components were isolated using laser-capture microdissection, and next-generation sequencing analysis was performed. Consequently,frameshift (p.N210fs) andmissense (p.G12R) mutations were identified in both ductal and acinar/neuroendocrine tumors. These results suggested a pancreatic mixed acinar-neuroendocrine-ductal carcinoma was derived from a founder tumor clone, and supports the notion that a founder tumor clone may differentiate and transform into a diverse histological type and form a pancreatic mixed carcinoma.

Genetic basis of a common tumor origin in the development of pancreatic mixed acinar-neuroendocrine-ductal carcinoma: A case report.

The diagnostic accuracy of bronchoscopy for detecting lung cancer, especially peripheral lung cancer with lesions outside the endoscopically visible range, remains unsatisfactory. The aim of this study was to perform next-generation sequencing on bronchoscopic specimens to determine whether this improves the accuracy of bronchoscopy for diagnosing lung cancer and to identify factors influencing sensitivity. The bronchoscopic sensitivity for diagnosing lung cancer was initially evaluated in 191 patients who underwent lobectomy after bronchoscopy at our hospital. Sputum, bronchial wash fluid, and resected lung cancer specimens were subsequently collected from 18 patients with peripheral small cell lung cancer for genomic analysis. DNA was extracted from formalin-fixed, paraffin-embedded surgical tissue specimens and the supernatant and cell fractions of sputum and bronchial wash fluid. Deep sequencing was performed using a lung cancer panel covering all exons of 53 lung cancer-related genes. The bronchoscopic sensitivity for diagnosing lung cancer at our hospital was 60.7%. Multivariate analysis revealed that this was influenced by tumor size and location, but not histological type or lymph node metastasis. The sensitivity was the highest for biopsy followed by curettage and bronchial wash specimens. DNA mutations homologous to those identified in the primary lesions were detected in the bronchial wash fluid of 10 patients (55.6%), while only 2 patients (11.1%) were diagnosed with lung cancer based on conventional cytological examinations. In conclusion, the addition of genomic analysis to routine pathological examinations improves the diagnostic accuracy of bronchoscopy.

Detection of tumor-derived DNA dispersed in the airway improves the diagnostic accuracy of bronchoscopy for lung cancer.

Utility of combined annotation-dependent depletion (CADD) score was recently reported to rank pathogenicity as C-scores ranging 1-99 for both confirmed deleterious mutation. Using C-scores for BRCA1/2 variants, we tried to constitute the classification system for variant of uncertain significance (VUS), which had been a major problem of genetic testing for hereditary breast and/or ovarian cancer. We analyzed BRCA1/2 genes for 283 patients with breast and/or ovarian cancer. The deleterious mutation and missesne mutations, minor variant, and wild type of BRCA1 and -2 were 5, 27, 251 and 15, 85, 183, respectively. Meanwhile, the variants with C-score ≥10 were involved in 19/283 (6.7%) in BRCA1 and 34/283 (12%) in BRCA2. All deleterious mutations were included in this group. Frequency of personal history and family history of ovarian cancer were significantly high, and frequency of serous adenocarcinoma of ovary and triple negative breast cancer was relatively high in the group with deleterious mutations. Similar findings were seen in patients with variants of C-score ≥10. According to the C-score and population frequency, we could define VUS for 11 patients out of 283 patients (3.9 CADD is useful to classify the variant of BRCA1/2 and selecting the patient who needs further segregation studies.

Combined annotation-dependent depletion score for BRCA1/2 variants in patients with breast and/or ovarian cancer.

Germline mutations inandgenes () predispose to hereditary breast and ovarian cancer syndrome (HBOC), and their dysregulation increases the risk of cancers. The detection of pathogenicvariants is essential for the diagnosis and prevention of HBOC, and for offering treatment decisions for patients. Therefore, there is a growing demand for the development of accurate, rapid assay systems that simultaneously detect pathogenic variants and copy number alterations. Here, we tested Thermo Fisher Scientific's newly developed OncominePanel. We showed that all mutations in standard reference DNA were detected with high accuracy, and that values of allelic fractions were detected with high concordance (= 0.9986). The OncominePanel detected 21 pathogenic germline variants in 147 patients with breast and/or ovarian cancer, of which 20 were detected by the previously-launched Ion AmpliSeq™Panel, except for one frameshift mutation. The OncominePanel precisely captured one additional frameshift mutation, which is difficult to detect because of the homopolymer site. Large genomic deletion was identified in one sample, which was previously detected by multiplex ligation-dependent probe amplification. OncominePanel could accurately detect pathogenic variant and copy number alteration, and be an alternative assay to investigategermline and somatic mutations.

Simultaneous detection of genetic and copy number alterations ingenes.

Pulmonary sarcomatoid carcinomas are rare and generally aggressive tumors composed of carcinomatous and sarcomatous components; however, the evolution of sarcomatoid cancer has not been elucidated. Here, we aimed to evaluate the mutational profiles and phylogeny of sarcomatoid carcinomas using next generation sequencing andanalysis to facilitate the development of novel therapies.

Article	Year
Factors contributing to the overall survival in patients with hepatocellular carcinoma treated by sorafenib. Hepato-gastroenterology\| PubMed ID: 22497953	2012
Stepwise addition of genetic changes correlated with histological change from "well-differentiated" to "sarcomatoid" phenotypes: a case report. BMC cancer\| PubMed ID: 28103823	2017
Analysis of significantly mutated genes as a clinical tool for the diagnosis in a case of lung cancer. Respiratory medicine case reports\| PubMed ID: 28275538	2017
Mutational analysis of multiple lung cancers: Discrimination between primary and metastatic lung cancers by genomic profile. Oncotarget\| PubMed ID: 28415711	2017
Elucidation of radiation-resistant clones by a serial study of intratumor heterogeneity before and after stereotactic radiotherapy in lung cancer. Journal of thoracic disease\| PubMed ID: 28840024	2017
Distribution of circulating tumor DNA in lung cancer: analysis of the primary lung and bone marrow along with the pulmonary venous and peripheral blood. Oncotarget\| PubMed ID: 28938635	2017
Very early response of circulating tumour-derived DNA in plasma predicts efficacy of nivolumab treatment in patients with non-small cell lung cancer. European journal of cancer (Oxford, England : 1990)\| PubMed ID: 29078173	2017
Genetic basis of a common tumor origin in the development of pancreatic mixed acinar-neuroendocrine-ductal carcinoma: A case report. Oncology letters\| PubMed ID: 29085438	2017
Detection of tumor-derived DNA dispersed in the airway improves the diagnostic accuracy of bronchoscopy for lung cancer. Oncotarget\| PubMed ID: 29108319	2017
Combined annotation-dependent depletion score for BRCA1/2 variants in patients with breast and/or ovarian cancer. Cancer science\| PubMed ID: 29215753	2018
Simultaneous detection of genetic and copy number alterations ingenes. Oncotarget\| PubMed ID: 29383094	2017
New therapeutic targets for pulmonary sarcomatoid carcinomas based on their genomic and phylogenetic profiles. Oncotarget\| PubMed ID: 29535832	2018

Masao Omata

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