Intratumor heterogeneity (ITH) contributes to cancer progression and chemoresistance. We sought to comprehensively describe ITH of somatic mutations, copy number, and transcriptomic alterations involving clinically and biologically relevant gene pathways in colorectal cancer (CRC). We performed multiregion, high-depth (384× on average) sequencing of 799 cancer-associated genes in 24 spatially separated primary tumor and nonmalignant tissues from four treatment-naïve CRC patients. We then used ultra-deep sequencing (17 075× on average) to accurately verify the presence or absence of identified somatic mutations in each sector. We also digitally measured gene expression and copy number alterations using NanoString assays. We identified the subclonal point mutations and determined the mutational timing and phylogenetic relationships among spatially separated sectors of each tumor. Truncal mutations, those shared by all sectors in the tumor, affected the well-described driver genes such as APC, TP53, and KRAS. With sequencing at 17 075×, we found that mutations first detected at a sequencing depth of 384× were in fact more widely shared among sectors than originally assessed. Interestingly, ultra-deep sequencing also revealed some mutations that were present in all spatially dispersed sectors, but at subclonal levels. Ultra-high-depth validation sequencing, copy number analysis, and gene expression profiling provided a comprehensive and accurate genomic landscape of spatial heterogeneity in CRC. Ultra-deep sequencing allowed more sensitive detection of somatic mutations and a more accurate assessment of ITH. By detecting the subclonal mutations with ultra-deep sequencing, we traced the genomic histories of each tumor and the relative timing of mutational events. We found evidence of early mixing, in which the subclonal ancestral mutations intermixed across the sectors before the acquisition of subsequent nontruncal mutations. Our findings also indicate that different CRC patients display markedly variable ITH, suggesting that each patient's tumor possesses a unique genomic history and spatial organization.

High-grade gliomas are the most aggressive and devastating brain neoplasms. Therefore much effort is put on understanding their background as well as development of new effective diagnostic and therapeutic methods. However, until now the genetic only approach has not provided a satisfactory answer. Recently, it has been shown that the epigenetic issue is important for high-grade gliomas' development and progression. Out of many epigenetic mechanisms, as DNA methylation, histone methylation and acetylation, especially microRNAs showed to be deeply involved in the carcinogenesis process. MicroRNAs are short non-coding RNAs. They are new candidates for human disease biomarkers due to their simple identification. MicroRNAs are stable in tissue and body fluids, what makes them very prospective non-invasive, blood-based biomarkers. There is a lot of data showing that various profiles of serum microRNAs are linked to numerous neoplastic processes, indicating that microRNAs can be really a new class of biomarkers for human diseases.

Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine disorder of premenopausal women. Given the phenotypic overlap between PCOS and type 2 diabetes mellitus (T2DM), this study was carried out to investigate whether genes implicated in T2DM were also involved in the susceptibility to PCOS among women from southern India.

The Arg>Pro polymorphism in codon 72 of p53 gene is known to affect the susceptibility of cervical cancer differently in different population worldwide although information regarding its role in determining survival status and disease outcome in patients is lacking. The present study was conducted to determine the genotype frequency and prognostic role of p53 codon 72 Arg>Pro polymorphism in patients with advanced stage cervical cancer in India.

To develop a NB animal model which makes possible studies related to tumor immunity.

The complexity by which cells regulate gene and protein expression is multifaceted and intricate. Regulation of 3' untranslated region (UTR) processing of mRNA has been shown to play a critical role in development and disease. However, the process by which cells select alternative mRNA forms is not well understood. We discovered that the Saccharomyces cerevisiae lysine demethylase, Jhd2 (also known as KDM5), recruits 3'UTR processing machinery and promotes alteration of 3'UTR length for some genes in a demethylase-dependent manner. Interaction of Jhd2 with both chromatin and RNA suggests that Jhd2 affects selection of polyadenylation sites through a transcription-coupled mechanism. Furthermore, its mammalian homolog KDM5B (also known as JARID1B or PLU1), but not KDM5A (also known as JARID1A or RBP2), promotes shortening of CCND1 transcript in breast cancer cells. Consistent with these results, KDM5B expression correlates with shortened CCND1 in human breast tumor tissues. In contrast, both KDM5A and KDM5B are involved in the lengthening of DICER1. Our findings suggest both a novel role for this family of demethylases and a novel targetable mechanism for 3'UTR processing.

Histone posttranslational modifications (PTMs) are key epigenetic marks involved in gene silencing or activation. Histone modifications impact chromatin organization and transcriptional processes through the changes in charge density between histones and DNA. They also serve as recognition and binding sites for specific binding proteins. Histone tails and globular cores contain many basic amino acid residues, which are subject to various dynamic modifications, making the modification repertoire extremely diverse. Consequently, determination of histone PTM identity and quantity has been a challenging task. In recent years, mass spectrometry-based methods have proven useful in histone PTM characterization. This chapter provides a brief overview of these methods and describes the approach to analyze the PTMs of the histone variant CENP-A, essential for the cell cycle progression, when present in minute amounts from tumor and mammalian tissues. Because this method does not rely on antibody-based immunopurification, we anticipate that these tools could be readily adaptable to the investigation to other histone variants in a range of mammalian tissues and solid tumors.

The aim of the study was to evaluate the relationship between patient's age and biochemical recurrence (BCR) after radical retropubic prostatectomy (RRP). Data from RRP applied to 305 patients with clinically localized prostate cancer were included in the study. Patients were divided into the three age groups, < 60 years, 60-70 years, and > 70 years. The groups were compared regarding adverse pathological findings on RRP specimen, BCR, and biochemical recurrence-free survival (bRFS) rates. The rates of positive surgical margin, seminal vesicle invasion, lymph node involvement, RRP specimens' Gleason score, and BCR were not significantly different among the three age groups. bRFS rates were not different either. Nonorgan-confined disease and extracapsular extension (ECE) rates were significantly higher in the group of 60-70 years group than in the other two age groups. Factors associated with BCR in multivariate Cox regression analysis were ECE, seminal vesicle invasion, positive surgical margin, and RRP specimens' Gleason score of ≥ 4+3. Patient age and preoperative prostate specific antigen levels were not identified to be associated with BCR. Post-RRP nonorgan-confined disease and ECE are more frequently seen in patients of 60-70 years of age group than in other age groups. However, patient age is not an independent prognostic factor associated with bRFS.

The mouse lymphoma L5178Y-3.7.2C (TK+/-) cell line is extensively used in genetic toxicology to conduct the mouse lymphoma assay (MLA). The MLA is used to establish the mutagenic and clastogenic effects of chemicals and pharmaceuticals, and is one of the few genetic tests widely accepted by regulatory agencies throughout the world. Despite the extensive use and regulatory impact of L5178Y-3.7.2C (TK+/-) cells, little is known about their genetic composition or how it affects the outcome of the MLA. To determine the genetic background of this cell line, we sequenced and analyzed its entire genome. Our results confirm the existence of previously described mutations in the Tk1 and Trp53 genes and catalog millions of other mutations, many of which impair the function of genes with key roles in cell physiology and genetic toxicology.

We report a case of a gastrointestinal stromal tumor(GIST)that originated in the anal canal. A 70's woman with a subcutaneous tumor reaching from the anal canal was referred to our hospital. After a thorough examination, the tumor was resected percutaneously in the jackknife position. Histopathological examination showed proliferation of spindle-shaped tumor cells arranged in irregular bundles. Immunohistochemical staining showed that the tumor was positive for c-kit and CD34, and negative for a-SMA and S-100, so the tumor was diagnosed as GIST. As a-SMA-positive smooth muscle cells were seen around the tumor, we suspected that this tumor originated from the internal sphincter muscle.

A 59-year-old man underwent total gastrectomy(with D2 dissection)and cholecystectomy for gastric cancer and a submucosal tumor of the stomach. The specimen was immunohistochemically positive for c-kit, the Ki-67 label index was 10%, and the mitotic count was 20/HPF. Finally, the patient was diagnosed with high-risk gastrointestinal stromal cancer with normal type gastric cancer. After discharge from hospital, we started administration of TS-1 as adjuvant therapy for the gastric cancer. As multiple recurrences of the GIST in the abdomen developed, the patient underwent 3 radical local resections. Mutational analysis revealed a PDGFRA mutation in exon 18, which causes resistance to both imatinib and sunitinib. As he was refractory to imatinib, the patient received regorafenib. After a while, it caused liver failure, which required 7 rounds of plasmapheresis. The patient died from multiple organ failure resulting from multiple recurrences 4 years after the first surgery.

A 44-year-old man with familial adenomatous polyposis underwent laparoscopic-assistedtotal proctocolectomy with ilealpouch anal anastomosis(IPAA). Computed tomography conducted 21 months after IPAA demonstrated bilateral hydronephrosis andan intra-abdominal mass with a maximal diameter of 22 cm, leading to a diagnosis of stage IV desmoid disease, according to the classification by Church and associates. Six courses of combination chemotherapy with doxorubicin plus dacarbazine were administered. Computed tomography after chemotherapy demonstrated marked shrinkage of the desmoidtumor with intraabdominal air andfluidcollection extending just below the skin of the ileostomy closure site. Stoollike fluidoverflowedspontaneously through the site of the ileostomy closure andthe abscess cavity was successfully drained. The patient was discharged 30 days after the start of drainage. The patient is doing well 10 months after the drainage without regrowth of the desmoid tumor, even though a cavity-like lesion encapsulatedby a thick wall remains.

We report a case of panitumumab-resistant rectal cancer with HER2 gene amplification detected by CancerPlex®. A 51- year-old man was diagnosed with an obstructive rectal cancer having lung and adrenal metastases. He underwent the Hartmann 's operation, and KRAS mutations were not detected. After the surgery, 3 courses of CapeOx plus bevacizumab were administered as first-line chemotherapy; however, the lung and adrenal metastases progressed. Subsequently, 24 courses of IRIS/panitumumab was administered as second-line chemotherapy, and the metastases slowly progressed. Six courses of regorafenib were administered as third-line chemotherapy followed by a course of TAS-102 as fourth-line chemotherapy. Subsequently, a left femoral head metastasis and cerebellar metastases were detected. The patient received best supportive care including palliative femoral head replacement and stereotactic irradiation for the cerebellar metastases, and he died of cancer 3 years 5 months after the primary surgery. The comprehensive genomic analysis focusing on 413 cancer-related genes with CancerPlex®revealed that EGFR, BRAF, KRAS, NRAS, and HRAS had no mutations; however, ERBB2 amplification was detected. Furthermore, immunohistochemical staining revealed overexpression of HER2 protein in both the primary and bone metastatictumor. HER2 and EGFR independently promote the RAS-RAF-MAPK pathway. In the present case, the efficacy of anti-EGFR therapy may be attenuated because of ERBB2 amplification in the metastatic tumor.

A 58-year-old man was admitted with the complaint of bloody stools. Colonoscopy and computed tomography revealed a rectal cancer with a liver metastasis and multiple lung metastases. After administering a regimen comprising 3 courses of XELOX plus bevacizumab chemotherapy, the sizes of the primary and metastatic lesions decreased remarkably. Abdominoperineal resection was performed for local control of the cancer; the specimen from the initial tumor was found to be KRAS wild type. After 14 courses of XELOX chemotherapy, brain metastases were detected. Although 3 courses of IRIS plus panitumumab were administered, the liver, lung, and brain metastases spread rapidly. A comprehensive genomic analysis focused on cancer-related genes with CancerPlex®found a mutation of the BRAF gene(I326V). BRAF is a downstream molecule of KRAS in the RAS-RAF-MAPK pathway. Therefore, this mutation of the BRAF gene has the possibility of causing resistance against panitumumab that was found in this case. Furthermore, we expect that the systematic analysis of oncogene and suppressor oncogenes will enable us to choose the optimal regimen of chemotherapy or molecular targeting therapy for each patient with colorectal cancer.

We report a rare case of male hereditary breast cancer in which a sentinel lymph node biopsy was performed. A 62-yearold man was admitted to our hospital because of a palpable tumor in his right breast. Both his younger sister and daughter had had breast cancer. Genetic testing revealed a morbid mutation in the BRCA2 gene. The tumor was palpated to an elastic hard mass and had a clear border in the right DCE area. We performed a core needle biopsy and diagnosed invasive ductal carcinoma, specifically, cT1cN0cM0, cStage I hereditary breast cancer. The patient underwent mastectomy and a sentinel lymph node biopsy. Nine days later, tamoxifen therapy was initiated. There has been no sign of recurrence during the 9 months after the operation.

A 49-year-old man visited our hospital with a chief complaint of abdominal pain that began 1 day before his visit.An approximately 30 cm tumor that was extensively in contact with the gastric wall in the abdominal cavity was detected on computed tomography(CT).An elevated lesion covered with normal mucosa on the posterior wall of the greater curvature was detected on upper endoscopy.He was diagnosed with a submucosal tumor of the stomach, and he underwent surgery. Surgical findings revealed an elastic soft tumor with a maximal dimension of 38 cm that projected from the posterior wall of the stomach beyond the gastric wall.No invasion and metastasis to other organs were detected.Partial gastrectomy was performed.On histopathological examination, proliferation of atypical round and spindle cells was found, and immunostaining was negative for KIT but positive for CD34.In the gene search, an Asp842Val mutation was detected in exon 18 of the PDGFRA gene.Currently, the patient has survived for 7 months after surgery without recurrence.

A 39-year-old woman underwent right colectomy for type-3 transverse colon cancer, which was histologically identified as well-differentiated stage II A adenocarcinoma with a mucinous component and tumor-infiltrating lymphocytes. The patient was suspected of having Lynch syndrome(LS)since she fulfilled 2 of the revised Bethesda criteria, even though there was no family history of LS. Twelve months after colectomy, abdominal CT revealed thickening of the uterine endometrium. Histopathological examination of biopsy specimens revealed well-differentiated endometrioid carcinoma. Extended hysterectomy with bilateral oophorectomy was performed. Histological examination of the resected specimen revealed well-differentiated endometrioid carcinoma of stage I . Immunohistochemistry analysis of mismatch repair proteins demonstrated loss of MLH1/ PMS2 expression in the colon cancer, but normal expression in the uterine cancer. Genetic testing identified duplication of exons 10-15 of the MLH1 gene, leading to a definitive diagnosis of LS. The patient has not shown any evidence of recurrence or new LS-associated tumors in the 12 years since the last surgery. There is an ongoing debate regarding the pathogenesis of endometrioid cancer, and this case emphasizes the importance of surveillance for gynecological malignancies after colon cancer surgery in female LS patients.

RAS mutation is an established predictive biomarker of resistance to anti-epidermal growth factor receptor(EGFR)therapy in metastatic colorectal cancer. In addition, previous studies identified mutations in ERBB2, FGFR1, PDGFRA, BRAF, MAP2K1, PTEN, and PIK3CA as potential mechanisms of resistance to anti-EGFR therapy. Testing for these mutations might be necessary to determine eligibility for anti-EGFR therapy in patients with metastatic colorectal cancer. CancerPlex®is a nextgeneration sequencer for 413 cancer genes. An analysis panel includes genes that may be associated with resistance to anti- EGFR therapy. A 65-year-old man with unresectable rectal cancer, multiple lung metastases, and a bulky liver metastasis was evaluated for expression of genes associated with resistance to anti-EGFR. The analysis found that all genes indicating resistance were wild-type genes. Cetuximab monotherapy was administered after rectal resection, with dramatic shrinkage of the metastatic tumors. A more accurate selection of patients according to tumor genetic status using CancerPlex®might improve the risk-benefit profile of anti-EGFR therapy.

Mismatch repair(MMR)protein deficiency in colorectal cancer is well correlated with high-level microsatellite instability (MSI-H). There are little data on mismatch repair deficiency(dMMR)colorectal cancers in Japan. In addition, we have no available data on the therapeutic efficacy of oxaliplatin(oxa)-based chemotherapy, one of the standard treatment regimens for metastatic colorectal cancer, for patients with dMMR colorectal cancer. The subjects were 254 patients with Stage IV colorectal cancer whose tumors were immunohistochemically stained for MMR proteins, MLH1, MSH2, MSH6, and PMS2. Patients who underwent R0 resection were excluded. Clinicopathologic factors and the efficacy of oxa-based chemotherapy were compared between patients with dMMR colorectal cancer and those with mismatch repair proficient(pMMR)colorectal cancer. There were 7(2.8%)patients with dMMR. Four patients demonstrated both MLH1 and PMS2 loss, while 3 patients demonstrated both MSH2 and MSH6 loss. Though the dMMR had a higher frequency in female patients(p=0.02) and a lower frequency in those with liver metastasis(p<0.01), the other clinicopathologic factors evaluated did not significantly differ between the dMMR group and the pMMR group. One hundred and fifty patients with unresectable disease or R1/2 resection received first-line oxa-based chemotherapy. The median overall survival was 23.2 months and 16.2 months in patients with dMMR(n=4)and those with pMMR, respectively(n=146)(p=0.33). The frequency of dMMR amongStag e IV colorectal cancers was lower than those(4-11%)reported in Western countries. Therefore, the clinical significance of universal screeningfor dMMR in all colorectal cancer samples may not be valid. Concerningsurvival benefit, oxa-based chemotherapy seems to be an effective alternative in clinical practice for metastatic colorectal cancer patients with dMMR.