Use of WGS in M. tuberculosis routine diagnosis

AbstractWhole Genome Sequencing (WGS) is becoming affordable with overall costs comparable to other tests currently in use to perform the diagnosis of drug resistant tuberculosis and cluster analysis. The WGS approach allows an “all-in one” approach providing results on expected sensitivity of the strains, genetic background, epidemiological data and indication of risk of laboratory cross-contamination.Although ideal, WGS from the direct diagnostic specimen is not yet standardized and up today the two most promising approaches are WGS from early positive liquid culture and targeted sequencing from diagnostic specimens using Next Generation Technology. Both have advantages and disadvantages. Sequencing from early MGIT requires positive cultures while targeted sequencing can be performed from a specimen positive for M. tuberculosis with a consistent gain in time to information. Aim of this study is to evaluate the feasibility and cost to use WGS with a centralized approach to speed up diagnosis of tuberculosis in a low incidence country.From March to September 2016 we collected and processed by WGS 89 early positive routine MGIT960 tubes. Time to diagnosis and accuracy of this technique were compared with the standard testing performed in the routine laboratory.An aliquot of 2ml of early positive MGIT was processed, starting with heat inactivation. DNA was then isolated by using the Maxwell 16 Cell DNA Purification Kit and Maxwell 16 MDx for automated extraction. Paired-end libraries of read-length 75–151bp were prepared using the Nextera XT DNA Sample Preparation kit, and sequenced on Illumina Miseq/Miniseq platform (based on the first available run). Total variant calling was performed according to the pipeline of the Phyresse web-tool.The DNA isolation step required 30′ for inactivation plus 30′ for extraction. The concentration obtained ranged from 0.1 to 1ng/μL, suitable for library preparation. Samples were sequenced with a turn around time of 24–48h. The percentage of reads mapped to H37Rv reference genome was 83% on average. Mean read coverage was 65×. Main challenge was the presence of non–mycobacterial DNA contamination in a variable amount. Lineage detection was possible for all cases, and mutations associated to drug resistance to antitubercular drugs were examined. We observed high diagnostic accuracy for species identification and detection of full drug resistance profile compared to standard DST testing performed in MGIT.Two events of recent transmissions including respectively three and two patients were identified and two laboratory cross-contamination were investigated and confirmed based on the analysis. Time to availability of report was around 72h from MGIT positivity compared to up to 6–9weeks for XDR-TB diagnosis with standard testing.In addition to speed, main advantages were the availability of a full prediction of resistance determinants for rifampicin resistant cases, the fast detection of potential cross-contaminations and clusters to guide epidemiological investigation and cross border tracing.Cost analysis showed that the cost per strain was approximately 150 Euro inclusive of staff cost, reagents and machine cost.WGS is a rapid, cost-effective technique that promises to integrate and replace the other tests in routine laboratories for an accurate diagnosis of DR-TB, although suitable nowadays for cultured samples only

Patient-derived xenografts and organoids model therapy response in prostate cancer

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe an androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naive, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harbors BRCA2 and CHD1 somatic mutations, shows an SPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modeled in vivo. Comparison of the treatment-naive PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds. To date, patients still succumb to cancer, due to tumors not responding to therapy or ultimately acquiring resistance. Here the authors show that by exploiting patient derived organoids and a treatment-naive patient derived xenograft, patient therapy can be personalized.Prostatic carcinom

Genetic heterogeneity and actionable mutations in HER2-positive primary breast cancers and their brain metastases

Brain metastases constitute a challenge in the management of patients with HER2- positive breast cancer treated with anti-HER2 systemic therapies. Here we sought to define the repertoire of mutations private to or enriched for in HER2-positive brain metastases. Massively parallel sequencing targeting all exons of 254 genes frequently mutated in breast cancers and/or related to DNA repair was used to characterize the spatial and temporal heterogeneity of HER2-positive breast cancers and their brain metastases in six patients. Data were analyzed with state-of-the-art bioinformatics algorithms and selected mutations were validated with orthogonal methods. Spatial and temporal inter-lesion genetic heterogeneity was observed in the HER2-positive brain metastases from an index patient subjected to a rapid autopsy. Genetic alterations restricted to the brain metastases included mutations in cancer genes FGFR2, PIK3CA and ATR, homozygous deletion in CDKN2A and amplification in KRAS. Shifts in clonal composition and the acquisition of additional mutations in the progression from primary HER2-positive breast cancer to brain metastases following anti-HER2 therapy were investigated in additional five patients. Likely pathogenic mutations private to or enriched in the brain lesions affected cancer and clinically actionable genes, including ATR, BRAF, FGFR2, MAP2K4, PIK3CA, RAF1 and TP53. Changes in clonal composition and the acquisition of additional mutations in brain metastases may affect potentially actionable genes in HER2-positive breast cancers. Our observations have potential clinical implications, given that treatment decisions for patients with brain metastatic disease are still mainly based on biomarkers assessed in the primary tumor

Predicting enteral nutrition-associated diarrhea with easily available clinical and biochemical parameters

Background: Artificial nutrition is now widely recognized as a medical treatment for patients unable to spontaneously meet their energy and protein requirements. Its efficacy may be limited, however, by the development of complications that may worsen the clinical status of the patients. During enteral nutrition, diarrhea frequently occurs, even if its pathogenic mechanisms are now better understood and enhanced delivery protocols have been implemented. Aim: We aimed at assessing whether the risk of developing enteral nutrition-associated diarrhea could be predicted by means of easily available and retrievable clinical and functional factors. Methods: The clinical records of 221 patients admitted to a rehabilitation center between 1999 and 2005 and receiving enteral nutrition or combined enteral+parenteral nutrition were retrospectively examined. Biographical (age, sex), clinical (comorbidity, quality of life, frailty) and nutritional parameters (anthropometry, biochemical markers, type of malnutrition) were collected if registered before the start of enteral nutrition/enteral+parenteral nutrition, and correlated with the development of diarrhea within the first 30 days of artificial nutrition. Results: Severe frailty and low circulating levels of cholinesterase at baseline were positively correlated with increased incidence of diarrhea during enteral nutrition/enteral+parenteral nutrition. Hypoalbuminemia did not correlate with the presence of diarrhea. Conclusions: Frailty class and circulating cholinesterase levels may represent useful markers to predict diarrhea when prescribing enteral nutrition or enteral+parenteral nutrition. Prospective clinical trials are needed to strengthen these results and translate them into clinical practice. © 2011 SINPE-GASAPE

The Microfluidic Environment Reveals a Hidden Role of Self-Organizing Extracellular Matrix in Hepatic Commitment and Organoid Formation of hiPSCs

The specification of the hepatic identity during human liver development is strictly controlled by extrinsic signals, yet it is still not clear how cells respond to these exogenous signals by activating secretory cascades, which are extremely relevant, especially in 3D self-organizing systems. Here, we investigate how the proteins secreted by human pluripotent stem cells (hPSCs) in response to developmental exogenous signals affect the progression from endoderm to the hepatic lineage, including their competence to generate nascent hepatic organoids. By using microfluidic confined environment and stable isotope labeling with amino acids in cell culture-coupled mass spectrometry (SILAC-MS) quantitative proteomic analysis, we find high abundancy of extracellular matrix (ECM)-associated proteins. Hepatic progenitor cells either derived in microfluidics or exposed to exogenous ECM stimuli show a significantly higher potential of forming hepatic organoids that can be rapidly expanded for several passages and further differentiated into functional hepatocytes. These results prove an additional control over the efficiency of hepatic organoid formation and differentiation for downstream applications

The repertoire of somatic genetic alterations of acinic cell carcinomas of the breast : an exploratory, hypothesis-generating study

Acinic cell carcinoma (ACC) of the breast is a rare form of triple-negative (that is, oestrogen receptor-negative, progesterone receptor-negative, HER2-negative) salivary gland-type tumour displaying serous acinar differentiation. Despite its triple-negative phenotype, breast ACCs are reported to have an indolent clinical behaviour. Here, we sought to define whether ACCs have a mutational repertoire distinct from that of other triple-negative breast cancers (TNBCs). DNA was extracted from microdissected formalin-fixed, paraffin-embedded sections of tumour and normal tissue from two pure and six mixed breast ACCs. Each tumour component of the mixed cases was microdissected separately. Tumour and normal samples were subjected to targeted capture massively parallel sequencing targeting all exons of 254 genes, including genes most frequently mutated in breast cancer and related to DNA repair. Selected somatic mutations were validated by targeted amplicon resequencing and Sanger sequencing. Akin to other forms of TNBC, the most frequently mutated gene found in breast ACCs was TP53 (one pure and six mixed cases). Additional somatic mutations affecting breast cancer-related genes found in ACCs included PIK3CA, MTOR, CTNNB1, BRCA1, ERBB4, ERBB3, INPP4B, and FGFR2. Copy number alteration analysis revealed complex patterns of gains and losses similar to those of common forms of TNBCs. Of the mixed cases analysed, identical somatic mutations were found in the acinic and the high-grade non-acinic components in two out of four cases analysed, providing evidence of their clonal relatedness. In conclusion, breast ACCs display the hallmark somatic genetic alterations found in high-grade forms of TNBC, including complex patterns of gene copy number alterations and recurrent TP53 mutations. Furthermore, we provide circumstantial genetic evidence to suggest that ACCs may constitute the substrate for the development of more aggressive forms of triple-negative disease

Microglandular adenosis associated with triple-negative breast cancer is a neoplastic lesion of triple-negative phenotype harbouring TP53 somatic mutations

Microglandular adenosis (MGA) is a rare proliferative lesion of the breast composed of small glands lacking myoepithelial cells and lined by S100-positive, oestrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative epithelial cells. There is evidence to suggest that MGA may constitute a non-obligate precursor of triple-negative breast cancer (TNBC). We sought to define the genomic landscape of pure MGA and of MGA, atypical MGA (AMGA) and associated TNBCs, and to determine whether synchronous MGA, AMGA, and TNBCs would be clonally related. Two pure MGAs and eight cases of MGA and/or AMGA associated with in situ or invasive TNBC were collected, microdissected, and subjected to massively parallel sequencing targeting all coding regions of 236 genes recurrently mutated in breast cancer or related to DNA repair. Pure MGAs lacked clonal non-synonymous somatic mutations and displayed limited copy number alterations (CNAs); conversely, all MGAs (n = 7) and AMGAs (n = 3) associated with TNBC harboured at least one somatic non-synonymous mutation (range 3-14 and 1-10, respectively). In all cases where TNBCs were analyzed, identical TP53 mutations and similar patterns of gene CNAs were found in the MGA and/or AMGA and in the associated TNBC. In the MGA/AMGA associated with TNBC lacking TP53 mutations, somatic mutations affecting PI3K pathway-related genes (eg PTEN, PIK3CA, and INPP4B) and tyrosine kinase receptor signalling-related genes (eg ERBB3 and FGFR2) were identified. At diagnosis, MGAs associated with TNBC were found to display subclonal populations, and clonal shifts in the progression from MGA to AMGA and/or to TNBC were observed. Our results demonstrate the heterogeneity of MGAs, and that MGAs associated with TNBC, but not necessarily pure MGAs, are genetically advanced, clonal, and neoplastic lesions harbouring recurrent mutations in TP53 and/or other cancer genes, supporting the notion that a subset of MGAs and AMGAs may constitute non-obligate precursors of TNBCs