Purpose
The enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular ...profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell‐free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection.
Methods
Tumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra‐ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA‐cleared tumor sequencing assay, MSK‐IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1.
Results
Tumor‐guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence.
Conclusion
Plasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow‐up schemes, and risk of metastasis.
The improved treatment strategy for retinoblastoma has led to increased ocular survival and less availability of tissue for molecular profiling. This study demonstrates that the identification of RB1 mutations by plasma cell‐free DNA and its absence in the buffy coat can conclusively determine whether or not the patient has a somatic RB1 mutation driving the disease.
Cell-free DNA (cfDNA) profiling is increasingly used to guide cancer care, yet mutations are not always identified. The ability to detect somatic mutations in plasma depends on both assay sensitivity ...and the fraction of circulating DNA in plasma that is tumor-derived (i.e., cfDNA tumor fraction). We hypothesized that cfDNA tumor fraction could inform the interpretation of negative cfDNA results and guide the choice of subsequent assays of greater genomic breadth or depth.
Plasma samples collected from 118 metastatic cancer patients were analyzed with cf-IMPACT, a modified version of the FDA-authorized MSK-IMPACT tumor test that can detect genomic alterations in 410 cancer-associated genes. Shallow whole genome sequencing (sWGS) was also performed in the same samples to estimate cfDNA tumor fraction based on genome-wide copy number alterations using z-score statistics. Plasma samples with no somatic alterations detected by cf-IMPACT were triaged based on sWGS-estimated tumor fraction for analysis with either a less comprehensive but more sensitive assay (MSK-ACCESS) or broader whole exome sequencing (WES).
cfDNA profiling using cf-IMPACT identified somatic mutations in 55/76 (72%) patients for whom MSK-IMPACT tumor profiling data were available. A significantly higher concordance of mutational profiles and tumor mutational burden (TMB) was observed between plasma and tumor profiling for plasma samples with a high tumor fraction (z-score≥5). In the 42 patients from whom tumor data was not available, cf-IMPACT identified mutations in 16/42 (38%). In total, cf-IMPACT analysis of plasma revealed mutations in 71/118 (60%) patients, with clinically actionable alterations identified in 30 (25%), including therapeutic targets of FDA-approved drugs. Of the 47 samples without alterations detected and low tumor fraction (z-score<5), 29 had sufficient material to be re-analyzed using a less comprehensive but more sensitive assay, MSK-ACCESS, which revealed somatic mutations in 14/29 (48%). Conversely, 5 patients without alterations detected by cf-IMPACT and with high tumor fraction (z-score≥5) were analyzed by WES, which identified mutational signatures and alterations in potential oncogenic drivers not covered by the cf-IMPACT panel. Overall, we identified mutations in 90/118 (76%) patients in the entire cohort using the three complementary plasma profiling approaches.
cfDNA tumor fraction can inform the interpretation of negative cfDNA results and guide the selection of subsequent sequencing platforms that are most likely to identify clinically-relevant genomic alterations.
We have developed a system that could potentially be used to identify the site of origin of ventricular tachycardia (VT) and to guide a catheter to that site to deliver radio-frequency ablation ...therapy. This system employs the Inverse Solution Guidance Algorithm based upon Single Equivalent Moving Dipole (SEMD) localization method. The system was evaluated in
in vivo
swine experiments. Arrays consisting of 9 or 16 bipolar epicardial electrodes and an additional mid-myocardial pacing lead were sutured to each ventricle. Focal tachycardia was simulated by applying pacing pulses to each epicardial electrode at multiple pacing rates during breath hold at the end-expiration phase. Surface potentials were recorded from 64 surface electrodes and then analyzed using the SEMD method to localize the position of the pacing electrodes. We found a close correlation between the locations of the pacing electrodes as measured in
computational
and
real spaces
. The reproducibility error of the SEMD estimation of electrode location was 0.21 ± 0.07 cm. The vectors between every pair of bipolar electrodes were computed in
computational and real spaces
. At 120 bpm, the lengths of the vectors in the
computational
and
real space
had a 95% correlation.
Computational space
vectors were used in catheter guidance simulations which showed that this method could reduce the distance between the
real space
locations of the emulated catheter tip and the emulated arrhythmia origin site by approximately 72% with each movement. We have demonstrated the feasibility of using our system to guide a catheter to the site of the emulated VT origin.
Lineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, facilitates therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon includes ...small cell and squamous cell (LUSC) histologic transformation in the context of acquired resistance to targeted inhibition of driver mutations. LUAD-to-LUSC transdifferentiation, occurring in up to 9% of EGFR-mutant patients relapsed on osimertinib, is associated with notably poor prognosis. We hypothesized that multi-parameter profiling of the components of mixed histology (LUAD/LUSC) tumors could provide insight into factors licensing lineage plasticity between these histologies.
We performed genomic, epigenomics, transcriptomics and protein analyses of microdissected LUAD and LUSC components from mixed histology tumors, pre-/post-transformation tumors and reference non-transformed LUAD and LUSC samples. We validated our findings through genetic manipulation of preclinical models in vitro and in vivo and performed patient-derived xenograft (PDX) treatments to validate potential therapeutic targets in a LUAD PDX model acquiring LUSC features after osimertinib treatment.
Our data suggest that LUSC transdifferentiation is primarily driven by transcriptional reprogramming rather than mutational events. We observed consistent relative upregulation of PI3K/AKT, MYC and PRC2 pathway genes. Concurrent activation of PI3K/AKT and MYC induced squamous features in EGFR-mutant LUAD preclinical models. Pharmacologic inhibition of EZH1/2 in combination with osimertinib prevented relapse with squamous-features in an EGFR-mutant patient-derived xenograft model, and inhibition of EZH1/2 or PI3K/AKT signaling re-sensitized resistant squamous-like tumors to osimertinib.
Our findings provide the first comprehensive molecular characterization of LUSC transdifferentiation, suggesting putative drivers and potential therapeutic targets to constrain or prevent lineage plasticity.
Lineage plasticity is implicated in treatment resistance in multiple cancers. In lung adenocarcinomas (LUAD) amenable to targeted therapy, transformation to small cell lung cancer (SCLC) is a ...recognized resistance mechanism. Defining molecular mechanisms of neuroendocrine (NE) transformation in lung cancer has been limited by a paucity of pre/posttransformation clinical samples. Detailed genomic, epigenomic, transcriptomic, and protein characterization of combined LUAD/SCLC tumors, as well as pre/posttransformation samples, supports that NE transformation is primarily driven by transcriptional reprogramming rather than mutational events. We identify genomic contexts in which NE transformation is favored, including frequent loss of the 3p chromosome arm. We observed enhanced expression of genes involved in the PRC2 complex and PI3K/AKT and NOTCH pathways. Pharmacologic inhibition of the PI3K/AKT pathway delayed tumor growth and NE transformation in an EGFR-mutant patient-derived xenograft model. Our findings define a novel landscape of potential drivers and therapeutic vulnerabilities of NE transformation in lung cancer.
The difficulty in collection of transformation samples has precluded the performance of molecular analyses, and thus little is known about the lineage plasticity mechanisms leading to LUAD-to-SCLC transformation. Here, we describe biological pathways dysregulated upon transformation and identify potential predictors and potential therapeutic vulnerabilities of NE transformation in the lung. See related commentary by Meador and Lovly, p. 2962. This article is highlighted in the In This Issue feature, p. 2945.
Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. ...48-49).
HMAX ("Hierarchical Model and X") system is among the best machine vision approaches developed today, in many object recognition tasks 1. HMAX decomposes an image into features which are passed to a classifier. These features each capture information about a small section of the input image but might not have information about the overall structure of the image if there is not a significant number of overlapping features. Therefore it can produce a false-positive if two images from two different classes having sufficiently similar features profile but completely different structures. To demonstrate the problem this thesis aimed to show that the features of a given subject are not unique because they lack geometric information. Genetic algorithm (GA) was used to create an image with a similar feature profile as a subject but which clearly does not belong to the subject. Using GA, random pixel images converged to an image whose feature profile has a small Euclidian distance from a target profile. This generated GA image does not resemble the target image but has a similar profile which successfully fooled the classifier in most cases. This implies that the "binding problem" is a major issue in a HMAX model of the size tested. Furthermore, methods of improving the system were investigated.
by Maysun Mazhar Hasan.
M.Eng.
PDF
