30 research outputs found
Multiplexed and Reiterative Detection of Protein Markers in Cells using Dynamic Nucleic Acid Complexes
The diagnosis, staging and clinical management of cancer and other diseases is
becoming increasingly reliant upon the identification and quantification of molecular
markers as well their spatial distribution in histological samples. Yet, due to spectral
overlap of dyes and the inability to remove probes without affecting marker integrity,
immunohistological methods are limited by the number of markers that can be examined
on a single specimen resulting in loss of information that could be vital to diagnosis or
treatment.
This dissertation describes the development and characterization of an erasable
multi-color imaging technology capable of detecting large numbers of molecular markers
on a single biological sample. The system consists of (1) 'targets', which are single or
partially hybridized DNA strands conjugated to a protein of interest for biomarker
recognition in cells, and (2) multi-strand, fluorophore-containing DNA 'probe
complexes' that react with the DNA portion of the target in a sequence dependent fashion
to create fluorescent reporting complexes. The addition of a quencher-bearing ssDNA
displaces the target's DNA strand to effectively remove the dye from the marker so that
the sample can be re-imaged for other markers with minimal interference from prior
iii
rounds of labeling. Orthogonal DNA sequences and spectrally-separated dyes can be used
to create multiple, unique target/probe pairs that associate specifically and can be imaged
in parallel.
The overall utility of this technology depends on high specificity of targets to
respective probe complexes, highly efficient labeling and erasing to ensure that
fluorescent signals can be used to fully quantify target abundance without the interference
of signals from previous rounds of labeling, and short reaction times to allow for multiple
rounds of processing on the same sample without loss of integrity. Based on the above
criteria, three classes of probes were designed and their structure-function relationships
elucidated to determine the contributions of complex size, free energy differences
between intermediate states, and strand displacement on labeling and erasing kinetics and
efficiencies on cells.
A comparison of the kinetics of the labeling and erasing reactions for the three
different constructs showed that reaction efficiencies depend less on calculated net free
energy change than on the engineered state of the complex during the strand
displacement reaction (i.e., the type of strand displacement reaction it participates in).
This new paradigm in probe design allowed the system to meet its design goals,
potentially increasing the diagnostic power of individual histological specimens and
opening the door to more sophisticated analyses of cell phenotype and its functional
relationship to disease
Detection of the MYD88 p.L265P Mutation in the CSF of a Patient With Secondary Central Nervous System Lymphoma
Primary Central Nervous System Lymphoma (PCNSL) and Metastatic (or Secondary) Central Nervous System Lymphoma (MCNSL) are rare central nervous system (CNS) malignancies that exhibit aggressive clinical behavior and have a poor prognosis. The majority of CNS lymphomas are histologically classified as diffuse large-B cell lymphoma (DLBCL). DLBCL harbors a high frequency of mutations in MYD88 and CD79b. The MYD88 p.L265P mutation occurs at high frequency in CNS lymphoma and is extremely rare in non-hematologic malignancies. Currently, brain biopsy is considered the gold standard for CNS lymphoma diagnosis. However, brain biopsy is invasive, carries a risk of complications, and can delay initiation of systemic therapy. Circulating tumor DNA (ctDNA) in the cerebrospinal fluid (CSF) can be utilized to detect tumor-derived mutations. Testing of CSF-ctDNA is a minimally-invasive methodology that can be used to assess the genomic alterations present in CNS malignancies. We present a case of an 82-year-old man with a history of testicular lymphoma who presented with speech difficulty and a multifocal enhancing left inferior frontal mass. Analysis for both CSF-cytology and flow cytometry did not show evidence of neoplastic cells. A brain biopsy was performed and microscopic examination showed DLBCL. We isolated CSF-ctDNA and used droplet digital PCR (ddPCR) to detect the most common lymphoma-associated mutations in MYD88, L265P, and V217F. In conjunction, we evaluated the patient-matched CNS lymphoma tissue for MYD88 mutations. We detected the MYD88 p.L265P mutation in formalin fixed paraffin embedded (FFPE) tissue from the brain biopsy and the CSF-ctDNA. In contrast, both the tumor tissue and the CSF ctDNA were negative for the MYD88 p.V217F mutation. This study shows that testing CSF ctDNA for MYD88 mutations is a potentially minimally-invasive approach to diagnosing patients with suspected CNS lymphomas
Configuring robust DNA strand displacement reactions for in situ molecular analyses
The number of distinct biomolecules that can be visualized within individual cells and tissue sections via fluorescence microscopy is limited by the spectral overlap of the fluorescent dye molecules that are coupled permanently to their targets. This issue prohibits characterization of important functional relationships between different molecular pathway components in cells. Yet, recent improved understandings of DNA strand displacement reactions now provides opportunities to create programmable labeling and detection approaches that operate through controlled transient interactions between different dynamic DNA complexes. We examined whether erasable molecular imaging probes could be created that harness this mechanism to couple and then remove fluorophore-bearing oligonucleotides to and from DNA-tagged protein markers within fixed cell samples. We show that the efficiency of marker erasing via strand displacement can be limited by non-toehold mediated stand exchange processes that lower the rates that fluorophore-bearing strands diffuse out of cells. Two probe constructions are described that avoid this problem and allow efficient fluorophore removal from their targets. With these modifications, we show one can at least double the number of proteins that can be visualized on the same cells via reiterative in situ labeling and erasing of markers on cells
A preexisting rare PIK3CA e545k subpopulation confers clinical resistance to MEK plus CDK4/6 inhibition in NRAS melanoma and is dependent on S6K1 signaling
Combined MEK and CDK4/6 inhibition (MEKi + CDK4i) has shown promising clinical outcomes in patients with NRAS- mutant melanoma. Here, we interrogated longitudinal biopsies from a patient who initially responded to MEKi + CDK4i therapy but subsequently developed resistance. Whole-exome sequencing and functional validation identified an acquired PIK3CA E545K mutation as conferring drug resistance. We demonstrate that PIK3CA E545K preexisted in a rare subpopulation that was missed by both clinical and research testing, but was revealed upon multiregion sampling due to PIK3CA E545K being nonuniformly distributed. This resistant population rapidly expanded after the initiation of MEKi + CDK4i therapy and persisted in all successive samples even after immune checkpoint therapy and distant metastasis. Functional studies identified activated S6K1 as both a key marker and specific therapeutic vulnerability downstream of PIK3CA E545K -induced resistance. These results demonstrate that difficult-to-detect preexisting resistance mutations may exist more often than previously appreciated and also posit S6K1 as a common downstream therapeutic nexus for the MAPK, CDK4/6, and PI3K pathways. SIGNIFICANCE: We report the first characterization of clinical acquired resistance to MEKi + CDK4i, identifying a rare preexisting PIK3CA E545K subpopulation that expands upon therapy and exhibits drug resistance. We suggest that single-region pretreatment biopsy is insufficient to detect rare, spatially segregated drug-resistant subclones. Inhibition of S6K1 is able to resensitize PIK3CA E545K -expressing NRAS-mutant melanoma cells to MEKi + CDK4i. Ā© 2018 AAC
Immunohistochemical and Molecular Features of Melanomas Exhibiting Intratumor and Intertumor Histomorphologic Heterogeneity
Melanoma is a heterogeneous neoplasm at the histomorphologic, immunophenotypic, and molecular levels. Melanoma with extreme histomorphologic heterogeneity can pose a diagnostic challenge in which the diagnosis may predominantly rely on its immunophenotypic profile. However, tumor survival and response to therapy are linked to tumor genetic heterogeneity rather than tumor morphology. Therefore, understating the molecular characteristics of such melanomas become indispensable. In this study, DNA was extracted from 11 morphologically distinct regions in eight formalin-fixed, paraffin-embedded melanomas. In each region, mutations in 50 cancer-related genes were tested using next-generation sequencing (NGS). A tumor was considered genetically heterogeneous if at least one non-overlapping mutation was identified either between the histologically distinct regions of the same tumor (intratumor heterogeneity) or among the histologically distinct regions of the paired primary and metastatic tumors within the same patient (intertumor heterogeneity). Our results revealed that genetic heterogeneity existed in all tumors as non-overlapping mutations were detected in every tested tumor (n = 5, 100%; intratumor: n = 2, 40%; intertumor: n = 3, 60%). Conversely, overlapping mutations were also detected in all the tested regions (n = 11, 100%). Melanomas exhibiting histomorphologic heterogeneity are often associated with genetic heterogeneity, which might contribute to tumor survival and poor response to therapy
T-Cell Receptor Beta Variable Gene Polymorphism Predicts Immune-Related adverse Events During Checkpoint Blockade Immunotherapy
BACKGROUND: Immune checkpoint inhibitors have revolutionized cancer treatment. However, they are associated with a unique spectrum of side effects, called immune-related adverse events (irAEs), which can cause significant morbidity and quickly progress to severe or life-threatening events if not treated promptly. Identifying predictive biomarkers for irAEs before immunotherapy initiation is therefore a critical area of research. Polymorphisms within the T-cell receptor beta (TCRB) variable (TRBV) gene have been implicated in autoimmune disease and may be mechanistically linked to irAEs. However, the repetitive nature of the TCRB locus and incomplete genome assembly has hampered the evaluation of TRBV polymorphisms in the past.
PATIENTS AND METHODS: We used a novel method for long-amplicon next generation sequencing of rearranged TCRB chains from peripheral blood total RNA to evaluate the link between TRBV polymorphisms and irAEs in patients treated with immunotherapy for cancer. We employed multiplex PCR to create amplicons spanning the three beta chain complementarity-determining regions (CDR) regions to enable detection of polymorphism within the germline-encoded framework and CDR1 and CDR2 regions in addition to CDR3 profiling. Resultant amplicons were sequenced via the Ion torrent and TRBV allele profiles constructed for each individual was correlated with irAE annotations to identify haplotypes associated with severe irAEs (ā„ grade 3).
RESULTS: Our study included 81 patients who had irAEs when treated with immunotherapy for cancer. By using principal component analysis of the 81 TRBV allele profiles followed by k-means clustering, we identified six major TRBV haplotypes. Strikingly, we found that one-third of this cohort possessed a TRBV allele haplotype that appeared to be protective against severe irAEs.
CONCLUSION: The data suggest that long-amplicon TCRB repertoire sequencing can potentially identify TRBV haplotype groups that correlate with the risk of severe irAEs. Germline-encoded TRBV polymorphisms may serve as a predictive biomarker of severe irAEs
Immune evolution from preneoplasia to invasive lung adenocarcinomas and underlying molecular features
The mechanism by which anti-cancer immunity shapes early carcinogenesis of lung adenocarcinoma (ADC) is unknown. In this study, we characterize the immune contexture of invasive lung ADC and its precursors by transcriptomic immune profiling, T cell receptor (TCR) sequencing and multiplex immunofluorescenceĀ (mIF). Our results demonstrate that anti-tumor immunity evolved as a continuum from lung preneoplasia, to preinvasive ADC, minimally-invasive ADC and frankly invasive lung ADC with a gradually less effective and more intensively regulated immune response including down-regulation of immune-activation pathways, up-regulation of immunosuppressive pathways, lower infiltration of cytotoxic T cells (CTLs)Ā and anti-tumor helper T cellsĀ (Th), higher infiltration of regulatory T cellsĀ (Tregs), decreased T cell clonality, and lower frequencies of top T cell clones in later-stages. Driver mutations, chromosomal copy number aberrations (CNAs)Ā and aberrant DNA methylation may collectively impinge host immune responses and facilitate immune evasion, promoting the outgrowth of fit subclones in preneoplasia into dominant clones in invasive ADC