Feasibility of whole genome and transcriptome profiling in pediatric and young adult cancers.

The utility of cancer whole genome and transcriptome sequencing (cWGTS) in oncology is increasingly recognized. However, implementation of cWGTS is challenged by the need to deliver results within clinically relevant timeframes, concerns about assay sensitivity, reporting and prioritization of findings. In a prospective research study we develop a workflow that reports comprehensive cWGTS results in 9 days. Comparison of cWGTS to diagnostic panel assays demonstrates the potential of cWGTS to capture all clinically reported mutations with comparable sensitivity in a single workflow. Benchmarking identifies a minimum of 80× as optimal depth for clinical WGS sequencing. Integration of germline, somatic DNA and RNA-seq data enable data-driven variant prioritization and reporting, with oncogenic findings reported in 54% more patients than standard of care. These results establish key technical considerations for the implementation of cWGTS as an integrated test in clinical oncology.

Prostate cancer evolution from multilineage primary to single lineage metastases with implications for liquid biopsy.

The evolutionary progression from primary to metastatic prostate cancer is largely uncharted, and the implications for liquid biopsy are unexplored. We infer detailed reconstructions of tumor phylogenies in ten prostate cancer patients with fatal disease, and investigate them in conjunction with histopathology and tumor DNA extracted from blood and cerebrospinal fluid. Substantial evolution occurs within the prostate, resulting in branching into multiple spatially intermixed lineages. One dominant lineage emerges that initiates and drives systemic metastasis, where polyclonal seeding between sites is common. Routes to metastasis differ between patients, and likely genetic drivers of metastasis distinguish the metastatic lineage from the lineage that remains confined to the prostate within each patient. Body fluids capture features of the dominant lineage, and subclonal expansions that occur in the metastatic phase are non-uniformly represented. Cerebrospinal fluid analysis reveals lineages not detected in blood-borne DNA, suggesting possible clinical utility.

KLF2 mutation is the most frequent somatic change in splenic marginal zone lymphoma and identifies a subset with distinct genotype.

To characterise the genetics of splenic marginal zone lymphoma (SMZL), we performed whole exome sequencing of 16 cases and identified novel recurrent inactivating mutations in Kruppel-like factor 2 (KLF2), a gene whose deficiency was previously shown to cause splenic marginal zone hyperplasia in mice. KLF2 mutation was found in 40 (42%) of 96 SMZLs, but rarely in other B-cell lymphomas. The majority of KLF2 mutations were frameshift indels or nonsense changes, with missense mutations clustered in the C-terminal zinc finger domains. Functional assays showed that these mutations inactivated the ability of KLF2 to suppress NF-κB activation by TLR, BCR, BAFFR and TNFR signalling. Further extensive investigations revealed common and distinct genetic changes between SMZL with and without KLF2 mutation. IGHV1-2 rearrangement and 7q deletion were primarily seen in SMZL with KLF2 mutation, while MYD88 and TP53 mutations were nearly exclusively found in those without KLF2 mutation. NOTCH2, TRAF3, TNFAIP3 and CARD11 mutations were observed in SMZL both with and without KLF2 mutation. Taken together, KLF2 mutation is the most common genetic change in SMZL and identifies a subset with a distinct genotype characterised by multi-genetic changes. These different genetic changes may deregulate various signalling pathways and generate cooperative oncogenic properties, thereby contributing to lymphomagenesis.

Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.

BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

p27Kip1 represses transcription by direct interaction with p130/E2F4 at the promoters of target genes.

The cyclin-cdk (cyclin-dependent kinase) inhibitor p27Kip1 (p27) has a crucial negative role on cell cycle progression. In addition to its classical role as a cyclin-cdk inhibitor, it also performs cyclin-cdk-independent functions as the regulation of cytoskeleton rearrangements and cell motility. p27 deficiency has been associated with tumor aggressiveness and poor clinical outcome, although the mechanisms underlying this participation still remain elusive. We report here a new cellular function of p27 as a transcriptional regulator in association with p130/E2F4 complexes that could be relevant for tumorigenesis. We observed that p27 associates with specific promoters of genes involved in important cellular functions as processing and splicing of RNA, mitochondrial organization and respiration, translation and cell cycle. On these promoters p27 co-localizes with p130, E2F4 and co-repressors as histone deacetylases (HDACs) and mSIN3A. p27 co-immunoprecipitates with these proteins and by affinity chromatography, we demonstrated a direct interaction of p27 with p130 and E2F4 through its carboxyl-half. We have also shown that p130 recruits p27 on the promoters, and there p27 is needed for the subsequent recruitment of HDACs and mSIN3A. Expression microarrays and luciferase assays revealed that p27 behaves as transcriptional repressor of these p27-target genes (p27-TGs). Finally, in human tumors, we established a correlation with overexpression of p27-TGs and poor survival. Thus, this new function of p27 as a transcriptional repressor could have a role in the major aggressiveness of tumors with low levels of p27.

Clinical symptoms and histopathological findings in subjects with adenomyosis uteri.

OBJECTIVE: The purpose of this study was to compare the clinical symptoms and histopathological findings in subjects with adenomyosis uteri. METHOD: Infiltration depth and spread of adenomyotic foci together with clinical symptoms and findings were compared in a total of 103 subjects who had undergone hysterectomy and were diagnosed with adenomyosis uteri through histopathological examinations. RESULTS: The spread of adenomyotic foci in myometrial tissues was observed to significantly increase as the depth of myometrial infiltration increased in subjects with adenomyosis (p < 0.05). It was observed that there was significantly higher myometrial infiltration depth in subjects with dysmenorrhea and severe anemia, and diffuse adenomyotic foci in subjects with menometrorrhagia (p < 0.05). CONCLUSION: Increased infiltration depth and spread of adenomyotic foci in myometrial tissues in subjects with adenomyosis uteri were studied. When clinical symptoms and findings in subjects with adenomyosis, such as dysmenorrhea, anemia and menometrorrhagia are compared with these histopathological findings, infiltration depth and spread of adenomyotic foci appear to determine the clinical severity of adenomyosis.

Adenomyosis: prevalence, risk factors, symptoms and clinical findings.

OBJECTIVE: This prospective study investigated the prevalence of adenomyosis in histopathological examinations of patients who had undergone hysterectomy due to various indications in our clinic. Epidemiological characteristics, predisposing risk factors, symptoms and clinical findings of adenomyosis were evaluated. METHOD: A total of 298 subjects who had undergone abdominal, vaginal or laparoscopic hysterectomy with/without salpingooophorectomy between October 2003 and April 2004 in our clinic were included. Uterine specimens obtained through hysterectomy were weighed and histopathologically examined in the Pathology Department of Ege University. The study group (n = 103), cases with adenomyosis, was compared with the control group (n=195), cases without adenomyosis, with respect to the epidemiological, clinical and histopathological characteristics. RESULTS: The prevalence of adenomyosis in 298 subjects was 36.2% (103). Duration of the reproductive period in patients with adenomyosis was found to be significantly longer than for those in the control group (p < 0.05). Prevalence of pelvic pain, dysmenorrhea and dyspareunia was also significantly higher in the study group (p < 0.05). Moreover, the number of cases requiring blood transfusion before the operation was significantly higher in the study group (p < 0.05) as were the rates of smoking, previous uterine surgery and nulliparity (p < 0.05). The most common gynecological condition accompanying adenomyosis was found to be uterine myoma in both groups, but the difference was not significant. CONCLUSION: Adenomyosis is not a rare histopathological finding. Due to diagnostic and therapeutic methods which are being developed as an alternative to hysterectomy, the clinical effects of adenomyosis and its association with other gynecological conditions, adenomyosis appears to be an issue which will be more intensively investigated in the future.

Does raloxifene therapy affect mammographic breast cancer screening in postmenopausal patients?

OBJECTIVE: The aim of the study was to determine mammographic breast density changes during raloxifene therapy in postmenopausal patients MATERIALS AND METHODS: Fifty-five cases who were using raloxifen therapy were included in this retrospective analysis. Raloxifene was given for osteopenia and osteoporosis according to low bone mineral density measured by dual-energy X-ray absorptiometry (DEXA). None of the patients were using hormone replacement therapy 12 months before the initiation of raloxifene treatment or during the study. Mammographic breast density was determined by mammography before the initiation of raloxifene treatment (baseline) and after 12 to 16 months of therapy. The Breast Imaging Reporting and Data System (BI-RADS) breast density score was used for the evaluation of mammographic density. RESULTS: There was no change in mammographic breast density when the baseline and the first mammography taken after the initiation of therapy were compared (p = 0.32). There was no significant correlation between the duration of raloxifene treatment and mammographic density measured after raloxifene treatment (r = -0.158, p = 0.25). Only in one patient did the BI-RADS classification of 2 change to 3 after 12 months of therapy. CONCLUSIONS: In conclusion, raloxifene therapy for 12 to 16 months does not increase mammographic breast density in postmenopausal women with low bone mass.

Preoperative and postoperative correlation of histopathological findings in cases of endometrial hyperplasia.

OBJECTIVES: To determine the preoperative and postoperative correlation of histopathological findings in cases of endometrial hyperplasia. MATERIAL AND METHODS: One hundred and three patients with endometrial hyperplasia detected by surgical curettage performed due to various gynecologic pathologies were treated by hysterectomy. We compared retrospectively the histopathological diagnoses found on curettage with those found on hysterectomy specimens. The classification scheme endorsed by the International Society of Gynecological Pathologists was used to classify the endometrial hyperplasia. The histologic findings found on the endometrial tissue of curettage specimens were correlated with those from hysterectomy specimens. Histopathologic evaluation was performed by a single skilled gynecologic pathologist. RESULTS: A total number of 103 women--76 (73.8%) premenopausal and 27 (26.2%) postmenopausal--were determined to have endometrial hyperplasia on histopathological evaluation of endometrial tissues obtained by endometrial curettage performed for evaluation of various bleeding abnormalities. These included 94 patients with simple hyperplasia without atypia (91.3%), two patients with simple hyperplasia with atypia (1.9%), five patients with complex hyperplasia without atypia (4.9%), and two patients with complex hyperplasia with atypia (1.9%). Histopathological evaluation of endometrial tissue obtained from hysterectomy specimens (of patients diagnosed with hyperplasia on curettage) revealed a total number of 65 cases (63.1%) with endometrial hyperplasia, and 38 cases (36.9%) with various histopathological findings. The correlation between preoperative and postoperative endometrial histologic findings was found to be statistically insignificant (r = 0.105, p = 0.29). Among 94 patients who were found to have simple hyperplasia without atypia on curettage specimens, 55.3%, were found to have simple hyperplasia without atypia, 1.1% simple hyperplasia with atypia, 5.3% complex hyperplasia without atypia, 9.6% secretory endometrium, 4.3% proliferative endometrium, 21.3% disorganized proliferative endometrium, 1.1% corpus luteum persistency, 1.1% basal endometrium, and 1.1% endometrium cancer on final hysterectomy specimens. CONCLUSION: Postoperative diagnosis of endometrial pathology might be different from that of preoperative especially in cases with simple endometrial hyperplasia without atypia.