Next generation sequencing demonstrates association between tumor suppressor gene aberrations and poor outcome in patients with cancer

Next generation sequencing is transforming patient care by allowing physicians to customize and match treatment to their patients’ tumor alterations. Our goal was to study the association between key molecular alterations and outcome parameters. We evaluated the characteristics and outcomes (overall survival (OS), time to metastasis/recurrence, and best progression-free survival (PFS)) of 392 patients for whom next generation sequencing (182 or 236 genes) had been performed. The Kaplan-Meier method and Cox regression models were used for our analysis, and results were subjected to internal validation using a resampling method (bootstrap analysis). In a multivariable analysis (Cox regression model), the parameters that were statistically associated with a poorer overall survival were the presence of metastases at diagnosis (P = 0.014), gastrointestinal histology (P < 0.0001), PTEN (P < 0.0001), and CDKN2A alterations (P = 0.0001). The variables associated with a shorter time to metastases/recurrence were gastrointestinal histology (P = 0.004), APC (P = 0.008), PTEN (P = 0.026) and TP53 (P = 0.044) alterations. TP53 (P = 0.003) and PTEN (P = 0.034) alterations were independent predictors of a shorter best PFS. A personalized treatment approach (matching the molecular aberration with a cognate targeted drug) also correlated with a longer best PFS (P = 0.046). Our study demonstrated that, across diverse cancers, anomalies in specific tumor suppressor genes (PTEN, CDKN2A, APC, and/or TP53) were independently associated with a worse outcome, as reflected by time to metastases/recurrence, best PFS on treatment, and/or overall survival. These observations suggest that molecular diagnostic tests may provide important prognostic information in patients with cancer.     

New reactive extraction systems for separation of bio-succinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses and to become an important feedstock for the chemical industry. In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production processes. In this context, high molecular weight amines are known to be effective extractants for organic acids. For this reason, as a first step of isolation and purification, the reactive extraction of succinic acid was studied by mixing aqueous succinic acid solutions with 448 different amine–solvent mixtures as extraction agents (mixer-settler studies). The extraction agents consist either of one amine and one solvent (208 reactive extraction systems) or two amines and two solvents (240 reactive extraction systems). Maximum extraction yields of succinic acid from an aqueous solution with 423 mM succinic acid at pH 2.0 were obtained with more than 95% yield with trihexylamine solved in 1-octanol or with dihexylamine and diisooctylamine solved in 1-octanol and 1-hexanol. Applying these optimized reactive extraction systems with Escherichia coli fermentation broth resulted in extraction yields of 78–85% due to the increased ionic strength of the fermentation supernatant and the co-extraction of other organic acids (e.g., lactic acid and acetic acid), which represent typical fermentation byproducts.     

Urothelial progenitor cells: regional differences in the rat bladder

OBJECTIVES: Because the trigone is a unique region in the caudal bladder with a higher risk of neoplasia, we hypothesized that this area would have a high proportion of progenitor cells. As yet there is no marker nor methodology to specifically isolate urothelial stem cells, and thus demonstrate multi-potential differentiation and self-renewal. Here, our goal was to evaluate the distribution of progenitor cells that carry two general major attributes of stem cells: clonogenicity and proliferative capacity. MATERIALS AND METHODS: The bladders of Fisher rats were divided into caudal and cephalic segments and primary cultures were established from the harvested urothelial cells. RESULTS: We found that colony-forming efficiency was almost 2-fold higher for cells from the caudal bladder compared to the cephalic bladder. Doubling time was significantly faster for cells harvested from the caudal bladder at initial plating. This suggested that the caudal bladder harbours a higher density of urothelial progenitor cells. With passage to p4, the differences between the upper and lower bladder were lost, suggesting selection of proliferative cells with serial passage. Based on Ki-67 staining, there was no geographical difference in cell proliferation under normal homeostatic in vivo conditions. CONCLUSIONS: These results demonstrate geographical sequestration of urothelial progenitor cells to the area of the bladder that encompasses the bladder neck and trigone, which may be a factor in pathological disparities between the trigone and remaining bladder.     

Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells

Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.     

Targeted morphoproteomic profiling of Ewing's sarcoma treated with insulin-like growth factor 1 receptor (IGF1R) inhibitors: response/resistance signatures

Background

Insulin-like growth factor 1 receptor (IGF1R) targeted therapies have resulted in responses in a small number of patients with advanced metastatic Ewing''s sarcoma. We performed morphoproteomic profiling to better understand response/resistance mechanisms of Ewing''s sarcoma to IGF1R inhibitor-based therapy.

Methodology/Principal Findings

This pilot study assessed two patients with advanced Ewing''s sarcoma treated with IGF1R antibody alone followed by combined IGF1R inhibitor plus mammalian target of rapamycin (mTOR) inhibitor treatment once resistance to single-agent IGF1R inhibitor developed. Immunohistochemical probes were applied to detect p-mTOR (Ser2448), p-Akt (Ser473), p-ERK1/2 (Thr202/Tyr204), nestin, and p-STAT3 (Tyr 705) in the original and recurrent tumor. The initial remarkable radiographic responses to IGF1R-antibody therapy was followed by resistance and then response to combined IGF1R plus mTOR inhibitor therapy in both patients, and then resistance to the combination regimen in one patient. In patient 1, upregulation of p-Akt and p-mTOR in the tumor that relapsed after initial response to IGF1R antibody might explain the resistance that developed, and the subsequent response to combined IGF1R plus mTOR inhibitor therapy. In patient 2, upregulation of mTOR was seen in the primary tumor, perhaps explaining the initial response to the IGF1R and mTOR inhibitor combination, while the resistant tumor that emerged showed activation of the ERK pathway as well.

Conclusion/Significance

Morphoproteomic analysis revealed that the mTOR pathway was activated in these two patients with advanced Ewing''s sarcoma who showed response to combined IGF1R and mTOR inhibition, and the ERK pathway in the patient in whom resistance to this combination emerged. Our pilot results suggests that morphoproteomic assessment of signaling pathway activation in Ewing''s sarcoma merits further investigation as a guide to understanding response and resistance signatures.     

Ontogeny of the male urethra: theory of endodermal differentiation

The most widely accepted mechanism of male urethral development proposes that the urethral plate is elevated by urethral folds which fuse ventrally in a proximal-to-distal sequence. Unlike its proximal counterpart, the urethra which forms within the glans is lined by a stratified squamous epithelium and has a more controversial development. One theory supports the idea that fusion of the urethral folds extends all the way to the tip of the glans. Another theory suggests that a solid ectodermal in-growth of epidermis canalizes the glandar urethra. We hypothesized that the use of immunohistochemical staining and tissue recombinant grafting would delineate the epithelia involved and lend clues to their origin. Thirty-six human fetal phallic specimens of gestational ages 5-22 weeks were sectioned and stained immunohistochemically with antibodies raised against different cytokeratins. Evaluation of the sections showed that the urethral plate, an extension of the urogenital sinus, extended to the tip of the phallus and maintained patency and continuity throughout the process of urethral development. The entire urethra, including the glans portion, was formed by dorsal extension and disintegration of the urethral plate combined with ventral growth and fusion of the urethral folds. Sections of the distal glandar urethra showed no evidence of a solid ectodermal ingrowth. Rather, immunostaining results at different ages suggested differentiation of the endodermal urethral plate into a stratified squamous epithelium. To determine whether urothelium could be induced to express a stratified squamous phenotype, mouse fetal bladder epithelium was combined with rat fetal genital tubercle mesenchyme and grown under the renal capsule of athymic mice. The bladder epithelium differentiated into a stratified squamous epithelium. Thus, proper mesenchymal signaling may induce differentiation of urothelium into a stratified squamous phenotype, such as during development of the urethra of the glans penis.     

Recovery of succinic acid from fermentation broth

Succinic acid is of high interest as bio-feedstock for the chemical industry. It is a precursor for a variety of many other chemicals, e.g. 1,4-butandiol, tetrahydrofuran, biodegradable polymers and fumaric acid. Besides optimized production strains and fermentation processes it is indispensable to develop cost-saving and energy-effective downstream processes to compete with the current petrochemical production process. Various methods such as precipitation, sorption and ion exchange, electrodialysis, and liquid–liquid extraction have been investigated for the recovery of succinic acid from fermentation broth and are reviewed critically here.     

Precision oncology for patients with advanced cancer: the challenges of malignant snowflakes

Precision oncology implies customizing treatment to the unique molecular and biologic characteristics of each individual and their cancer. Its implementation is being facilitated by remarkable technological advances in genomic sequencing, as well as the increasing availability of targeted and immunotherapeutic drugs. Yet, next generation sequencing may be a disruptive technology in that its results suggest that classic paradigms for clinical research and practice are a poor fit with the complex reality encountered in metastatic malignancies. Indeed, it is evident that advanced tumors have heterogeneous molecular landscapes that mostly differ between patients. Traditional modes of clinical research/practice are drug centered, with a strategy of finding commonalities between patients so that they can be grouped together and treated similarly. However, if each patient with metastatic cancer has a unique molecular portfolio, a new patient-centered, N-of-one approach that utilizes individually tailored treatment is needed.     

Integrated separation process for isolation and purification of biosuccinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses. Therefore, it is of high interest for the chemical, pharmaceutical, and food industry.In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production of succinic acid. Isolation and purification of succinic acid from an Escherichia coli fermentation broth were studied with two amine-based reactive extraction systems: (i) trihexylamine in 1-octanol and (ii) diisooctylamine and dihexylamine in a mixture of 1-octanol and 1-hexanol. Back extraction of succinic acid from the organic phase was carried out using an aqueous trimethylamine solution. The trimethylammonium succinate generated after back extraction was split with an evaporation-based crystallization.The focus was on process integration, for example, reuse of the applied amines for extraction and back extraction. It was shown that the maximum trimethylamine concentration for back extraction should not exceed the stoichiometric amount (2 mol trimethylamine/mol the succinic acid in the organic phase) to ensure maximal extraction yields with the reused organic phase in subsequent extractions. Moreover, mixer-settler extraction and back extraction of succinic acid were scaled up from the milliliter- to the liter-scale making use of liquid–liquid centrifuges. The overall yield was 83.5% of the succinic acid from thefermentation supernatant. The final purity of the succinic acid crystals was 99.5%. Organic phase and amines can easily be recycled and reused.