miRNAs in Newt Lens Regeneration: Specific Control of Proliferation and Evidence for miRNA Networking

Background: Lens regeneration in adult newts occurs via transdifferentiation of the pigment epithelial cells (PECs) of the dorsal iris. The same source of cells from the ventral iris is not able to undergo this process. In an attempt to understand this restriction we have studied in the past expression patterns of miRNAs. Among several miRNAs we have found that mir-148 shows an up-regulation in the ventral iris, while members of the let-7 family showed down-regulation in dorsal iris during dedifferentiation. Methodology/Principal Findings: We have performed gain- and loss-of–function experiments of mir-148 and let-7b in an attempt to delineate their function. We find that up-regulation of mir-148 caused significant decrease in the proliferation rates of ventral PECs only, while up-regulation of let-7b affected proliferation of both dorsal and ventral PECs. Neither miRNA was able to affect lens morphogenesis or induction. To further understand how this effect of miRNA up-regulation is mediated we examined global expression of miRNAs after up-regulation of mir148 and let-7b. Interestingly, we identified a novel level of mirRNA regulation, which might indicate that miRNAs are regulated as a network. Conclusion/Significance: The major conclusion is that different miRNAs can control proliferation in the dorsal or ventral iris possibly by a different mechanism. Of interest is that down-regulation of the let-7 family members has also been documented in other systems undergoing reprogramming, such as in stem cells or oocytes. This might indicate tha

Are one or two simple questions sufficient to detect depression in cancer and palliative care? A Bayesian meta-analysis

The purpose of this study is to examine the value of one or two simple verbal questions in the detection of depression in cancer settings. This study is a systematic literature search of abstract and full text databases to January 2008. Key authors were contacted for unpublished studies. Seventeen analyses were found. Of these, 13 were conducted in late stage palliative settings. (1) Single depression question: across nine studies, the prevalence of depression was 16%. A single ‘depression' question enabled the detection of depression in 160 out of 223 true cases, a sensitivity of 72%, and correctly reassured 964 out of 1166 non-depressed cancer sufferers, a specificity of 83%. The positive predictive value (PPV) was 44% and the negative predictive value (NPV) 94%. (2) Single interest question: there were only three studies examining the ‘loss-of-interest' question, with a combined prevalence of 14%. This question allowed the detection of 60 out of 72 cases (sensitivity 83%) and excluded 394 from 459 non-depressed cases (specificity of 86%). The PPV was 48% and the NPV 97%. (3) Two questions (low mood and low interest): five studies examined two questions with a combined prevalence of 17%. The two-question combination facilitated a diagnosis of depression in 138 of 151 true cases (sensitivity 91%) and gave correct reassurance to 645 of 749 non-cases (specificity 86%). The PPV was 57% and the NPV 98%. Simple verbal methods perform well at excluding depression in the non-depressed but perform poorly at confirming depression. The ‘two question' method is significantly more accurate than either single question but clinicians should not rely on these simple questions alone and should be prepared to assess the patient more thoroughly

Abstract P6-07-03: Broken promise of liquid biopsy: Plasma DNA does not accurately reflect tumor DNA in metastatic breast cancer

Abstract Background: Circulating tumor DNA in plasma may present a minimally invasive approach to identify tumor-derived mutations that could be used to inform the selection of targeted therapies for individual patients, particularly in cases of metastatic disease where biopsy is often difficult. We hypothesized that plasma DNA will genetically reflect DNA derived from multiple tumors in patients with metastatic breast cancer. To test this hypothesis and assess the utility of plasma DNA obtained as a “liquid biopsy” for precision medicine, we sought to determine whether massively parallel sequencing of plasma DNA is a reliable surrogate for sequencing of DNA from tissue biopsies in patients with metastatic breast cancer. Methods: Blood samples were obtained from 7 patients with multiple advanced breast cancer lesions (recurrent breast and metastatic tumors), and tumor specimens were obtained thereafter by biopsy or surgical excision. DNA extracted from plasma, buffy coat of blood, and tumor tissues was used for probe-directed capture of all exons in 196 genes followed by massively parallel sequencing with an average coverage of 3000x for plasma DNA. Tumor and plasma DNA sequences were bioinformatically compared to buffy coat controls, and high-confidence somatic mutations were called. One patient with extensive metastatic disease was evaluated in further detail to study the contribution of different tumors to the overall plasma DNA pool. In this patient, 9 metastatic tumors were sampled in an axillary lymph node, heart, kidney (2), liver, omentum (3), and ovary by biopsy or at autopsy. Results: Mutations were found in plasma that were represented in one or more tumors in each patient. Three classes of mutations were discovered: 1) mutations overlapping between both plasma and tumors (e.g., TP53 p.R273C and SRC p.E527K); 2) mutations found in plasma but not tumors (e.g., AKT p.E17K and multiple known and novel ESR1 mutations); 3) mutations found in tumors but not plasma (e.g., PIK3CA p.H1047R, p.D350G, and p.N345K). The presence of mutations in each of these classes was validated in plasma and/or tumors using mutation-specific droplet digital PCR (ddPCR). In the patient with extensive metastatic disease, DNA sequencing revealed heterogeneity of tumor contribution to plasma DNA, with some tumors better represented than others. No correlation was found between tumor size (measured by CT scan) and mutational burden in plasma. Interestingly, a significant correlation was found between blood perfusion to the organ where the tumor resides and mutational burden in plasma, with the greatest tumor contribution coming from the heart metastasis (Pearson's r = 0.835, p=0.039). Conclusions: Plasma DNA sequencing adds a layer of depth to sequencing analysis of tumor biopsy samples, and serves to both confirm tumor-derived mutations as well as detect new mutations. However, plasma DNA profiling does not comprehensively reflect the mutational profiles of tumors in patients with metastatic breast cancer, and thus is unlikely to serve as a surrogate for tumor biopsy as a source of DNA for genetic profiling. Furthermore, plasma DNA contains many mutations not found in tumors, which will confound treatment decision-making and precision medicine.Background: Circulating tumor DNA in plasma may present a minimally invasive approach to identify tumor-derived mutations that could be used to inform the selection of targeted therapies for individual patients, particularly in cases of metastatic disease where biopsy is often difficult. We hypothesized that plasma DNA will genetically reflect DNA derived from multiple tumors in patients with metastatic breast cancer. To test this hypothesis and assess the utility of plasma DNA obtained as a “liquid biopsy” for precision medicine, we sought to determine whether massively parallel sequencing of plasma DNA is a reliable surrogate for sequencing of DNA from tissue biopsies in patients with metastatic breast cancer. Methods: Blood samples were obtained from 7 patients with multiple advanced breast cancer lesions (recurrent breast and metastatic tumors), and tumor specimens were obtained thereafter by biopsy or surgical excision. DNA extracted from plasma, buffy coat of blood, and tumor tissues was used for probe-directed capture of all exons in 196 genes followed by massively parallel sequencing with an average coverage of 3000x for plasma DNA. Tumor and plasma DNA sequences were bioinformatically compared to buffy coat controls, and high-confidence somatic mutations were called. One patient with extensive metastatic disease was evaluated in further detail to study the contribution of different tumors to the overall plasma DNA pool. In this patient, 9 metastatic tumors were sampled in an axillary lymph node, heart, kidney (2), liver, omentum (3), and ovary by biopsy or at autopsy. Results: Mutations were found in plasma that were represented in one or more tumors in each patient. Three classes of mutations were discovered: 1) mutations overlapping between both plasma and tumors (e.g., TP53 p.R273C and SRC p.E527K); 2) mutations found in plasma but not tumors (e.g., AKT p.E17K and multiple known and novel ESR1 mutations); 3) mutations found in tumors but not plasma (e.g., PIK3CA p.H1047R, p.D350G, and p.N345K). The presence of mutations in each of these classes was validated in plasma and/or tumors using mutation-specific droplet digital PCR (ddPCR). In the patient with extensive metastatic disease, DNA sequencing revealed heterogeneity of tumor contribution to plasma DNA, with some tumors better represented than others. No correlation was found between tumor size (measured by CT scan) and mutational burden in plasma. Interestingly, a significant correlation was found between blood perfusion to the organ where the tumor resides and mutational burden in plasma, with the greatest tumor contribution coming from the heart metastasis (Pearson's r = 0.835, p=0.039). Conclusions: Plasma DNA sequencing adds a layer of depth to sequencing analysis of tumor biopsy samples, and serves to both confirm tumor-derived mutations as well as detect new mutations. However, plasma DNA profiling does not comprehensively reflect the mutational profiles of tumors in patients with metastatic breast cancer, and thus is unlikely to serve as a surrogate for tumor biopsy as a source of DNA for genetic profiling. Furthermore, plasma DNA contains many mutations not found in tumors, which will confound treatment decision-making and precision medicine. Citation Format: Shee K, Chamberlin MD, Varn FS, Bean JR, Marotti JD, Wells WA, Trask HW, Hamilton JS, West RJ, Kaufman PA, Schwartz GN, Gemery JM, McNulty NJ, Tsapakos MJ, Barth RJ, Arrick BA, Gui J, Cheng C, Miller TW. Broken promise of liquid biopsy: Plasma DNA does not accurately reflect tumor DNA in metastatic breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-03.</jats:p