ABSTRACT
To investigate the cellular and molecular mechanisms associated with targeting CD30-expressing Hodgkin lymphoma (HL) and immune checkpoint modulation induced by combination therapies of CTLA4 and PD1, we leveraged Phase 1/2 multicenter open-label trial NCT01896999 that enrolled patients with refractory or relapsed HL (R/R HL). Using peripheral blood, we assessed soluble proteins, cell composition, T-cell clonality, and tumor antigen-specific antibodies in 54 patients enrolled in the phase 1 component of the trial. NCT01896999 reported high (>75%) overall objective response rates with brentuximab vedotin (BV) in combination with ipilimumab (I) and/or nivolumab (N) in patients with R/R HL. We observed a durable increase in soluble PD1 and plasmacytoid dendritic cells as well as decreases in plasma CCL17, ANGPT2, MMP12, IL13, and CXCL13 in N-containing regimens (BV + N and BV + I + N) compared with BV + I (P < 0.05). Nonresponders and patients with short progression-free survival showed elevated CXCL9, CXCL13, CD5, CCL17, adenosine-deaminase, and MUC16 at baseline or after one treatment cycle and a higher prevalence of NY-ESO-1-specific autoantibodies (P < 0.05). The results suggest a circulating tumor-immune-derived signature of BV ± I ± N treatment resistance that may be useful for patient stratification in combination checkpoint therapy. SIGNIFICANCE: Identification of multi-omic immune markers from peripheral blood may help elucidate resistance mechanisms to checkpoint inhibitor and antibody-drug conjugate combinations with potential implications for treatment decisions in relapsed HL.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Brentuximab Vedotin , Drug Resistance, Neoplasm , Hodgkin Disease , Ipilimumab , Nivolumab , Humans , Brentuximab Vedotin/therapeutic use , Hodgkin Disease/drug therapy , Hodgkin Disease/immunology , Hodgkin Disease/blood , Nivolumab/therapeutic use , Nivolumab/administration & dosage , Ipilimumab/therapeutic use , Ipilimumab/administration & dosage , Ipilimumab/pharmacology , Female , Male , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Adult , Middle Aged , Aged , Young AdultABSTRACT
PURPOSE: We initiated a biomarker-informed preoperative study of infigratinib, a fibroblast growth factor receptor (FGFR) inhibitor, in patients with localized upper tract urothelial carcinoma (UTUC), a population with high unmet needs and tumor with a high frequency of FGFR3 alterations. MATERIALS AND METHODS: Patients with localized UTUC undergoing ureteroscopy or nephroureterectomy/ureterectomy were enrolled on a phase 1b trial (NCT04228042). Once-daily infigratinib 125 mg by mouth × 21 days (28-day cycle) was given for 2 cycles. Tolerability was monitored by Bayesian design and predefined stopping boundaries. The primary endpoint was tolerability, and the secondary endpoint was objective response based on tumor mapping, done after endoscopic biopsy and post-trial surgery. Total planned enrollment: 20 patients. Targeted sequencing performed using a NovaSeq 6000 solid tumor panel. RESULTS: From May 2021 to November 2022, 14 patients were enrolled, at which point the trial was closed due to termination of all infigratinib oncology trials. Two patients (14.3%) had treatment-terminating toxicities, well below the stopping threshold. Responses occurred in 6 (66.7%) of 9 patients with FGFR3 alterations. Responders had median tumor size reduction of 67%, with 3 of 5 patients initially planned for nephroureterectomy/ureterectomy converted to ureteroscopy. Median follow-up in responders was 24.7 months (14.9-28.9). CONCLUSIONS: In this first trial of targeted therapy for localized UTUC, FGFR inhibition was well tolerated and had significant activity in FGFR3 altered tumors. Renal preservation was enabled in a substantial proportion of participants. These data support the design of a biomarker-driven phase 2 trial of FGFR3 inhibition in this population with significant unmet clinical needs.
Subject(s)
Carcinoma, Transitional Cell , Ureteral Neoplasms , Humans , Male , Female , Aged , Carcinoma, Transitional Cell/drug therapy , Carcinoma, Transitional Cell/surgery , Carcinoma, Transitional Cell/pathology , Carcinoma, Transitional Cell/genetics , Middle Aged , Ureteral Neoplasms/drug therapy , Ureteral Neoplasms/surgery , Ureteral Neoplasms/pathology , Receptor, Fibroblast Growth Factor, Type 3/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 3/genetics , Kidney Neoplasms/drug therapy , Kidney Neoplasms/surgery , Kidney Neoplasms/pathology , Ureteroscopy/adverse effects , Nephroureterectomy , Aged, 80 and over , Treatment Outcome , Phenylurea Compounds , PyrimidinesABSTRACT
BACKGROUND: The presence of lymphovascular invasion (LVI) in early esophageal adenocarcinoma (EAC) is associated with more aggressive disease. Molecular markers associated with LVI are still largely unknown. Using a combination of transcriptomic analysis and validation experiments, we sought to describe markers for LVI and survival. METHODS: We performed NanoString expression profiling using RNA from 60 EAC specimens collected from surgery-only cases between 2000 and 2012. Differentially expressed genes (DEGs) were correlated with pathologic characteristics (T and N status and presence of LVI). Kaplan-Meier and Cox regression analyses were used to correlate gene expression with overall survival. Expression of alanyl aminopeptidase, membrane (ANPEP)/CD13 was validated by immunohistochemistry (IHC) in EAC tissue microarray and in EAC cell lines. RESULTS: We identified >20 up-regulated DEGs in tumor samples containing LVI. Multivariable analysis showed depth of invasion and ANPEP/CD13 expression were independently associated with overall survival, whereas nodal status was not. IHC analysis demonstrated overexpression of the ANPEP/CD13 protein in dysplastic Barrett esophagus and EAC tumors. Kaplan-Meier analysis showed that patients with higher RNA expression and strongly positive ANPEP/CD13 membrane IHC-Histoscore staining have shorter survival (P = .002). Down-regulation of ANPEP/CD13 expression by short hairpin RNA vector reduces colony formation, migration, and invasion of FLO-1 EAC cells. Overexpression of CD13 in SKGT4 EAC cells increases colony formation, motility, and invasion in vitro. CONCLUSIONS: Elevated expression of ANPEP/CD13 indicates shorter survival of EAC patients and a more invasive phenotype of cancer cells in vitro. Validation in a larger sample group is required to better understand the clinical significance of ANPEP/CD13 and other candidate genes.
ABSTRACT
Sitravatinib is an immunomodulatory tyrosine kinase inhibitor that can augment responses when combined with programmed death-1 inhibitors such as nivolumab. We report a single-arm, interventional, phase 2 study of neoadjuvant sitravatinib in combination with nivolumab in patients with locally advanced clear cell renal cell carcinoma (ccRCC) prior to curative nephrectomy (NCT03680521). The primary endpoint was objective response rate (ORR) prior to surgery with a null hypothesis ORR = 5% and the alternative hypothesis set at ORR = 30%. Secondary endpoints were safety; pharmacokinetics (PK) of sitravatinib; immune effects, including changes in programmed cell death-ligand 1 expression; time-to-surgery; and disease-free survival (DFS). Twenty patients were evaluable for safety and 17 for efficacy. The ORR was 11.8%, and 24-month DFS probability was 88·0% (95% CI 61.0 to 97.0). There were no grade 4/5 treatment-related adverse events. Sitravatinib PK did not change following the addition of nivolumab. Correlative blood and tissue analyses showed changes in the tumour microenvironment resulting in an immunologically active tumour by the time of surgery (median time-to-surgery: 50 days). The primary endpoint of this study was not met as short-term neoadjuvant sitravatinib and nivolumab did not substantially increase ORR.
Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Humans , Nivolumab/adverse effects , Carcinoma, Renal Cell/drug therapy , Carcinoma, Renal Cell/surgery , Carcinoma, Renal Cell/etiology , Neoadjuvant Therapy , Kidney Neoplasms/drug therapy , Kidney Neoplasms/surgery , Kidney Neoplasms/etiology , Nephrectomy , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Tumor MicroenvironmentABSTRACT
The safety and efficacy of combining the isocitrate dehydrogenase-1 (IDH1) inhibitor ivosidenib (IVO) with the BCL2 inhibitor venetoclax (VEN; IVO + VEN) ± azacitidine (AZA; IVO + VEN + AZA) were evaluated in four cohorts of patients with IDH1-mutated myeloid malignancies (n = 31). Most (91%) adverse events were grade 1 or 2. The maximal tolerated dose was not reached. Composite complete remission with IVO + VEN + AZA versus IVO + VEN was 90% versus 83%. Among measurable residual disease (MRD)-evaluable patients (N = 16), 63% attained MRD--negative remissions; IDH1 mutation clearance occurred in 64% of patients receiving ≥5 treatment cycles (N = 14). Median event-free survival and overall survival were 36 [94% CI, 23-not reached (NR)] and 42 (95% CI, 42-NR) months. Patients with signaling gene mutations appeared to particularly benefit from the triplet regimen. Longitudinal single-cell proteogenomic analyses linked cooccurring mutations, antiapoptotic protein expression, and cell maturation to therapeutic sensitivity of IDH1-mutated clones. No IDH isoform switching or second-site IDH1 mutations were observed, indicating combination therapy may overcome established resistance pathways to single-agent IVO. SIGNIFICANCE: IVO + VEN + AZA is safe and active in patients with IDH1-mutated myeloid malignancies. Combination therapy appears to overcome resistance mechanisms observed with single-agent IDH-inhibitor use, with high MRD-negative remission rates. Single-cell DNA ± protein and time-of-flight mass-cytometry analysis revealed complex resistance mechanisms at relapse, highlighting key pathways for future therapeutic intervention. This article is highlighted in the In This Issue feature, p. 247.
Subject(s)
Antineoplastic Agents , Neoplasm Recurrence, Local , Humans , Neoplasm Recurrence, Local/chemically induced , Antineoplastic Agents/adverse effects , Azacitidine/adverse effects , Isocitrate Dehydrogenase/geneticsABSTRACT
Detection of methylation patterns in circulating tumor DNA (ctDNA) can offer a novel approach for cancer diagnostics given the unique signature for each tumor type. We developed a next-generation sequencing (NGS)-based assay targeting 32 CpG sites to detect colorectal cancer-specific ctDNA. NGS was performed on bisulfite-converted libraries and status dichotomization was done using median methylation ratios at all targets. We included plasma samples from patients with metastatic colorectal (nâ =â 20) and non-colorectal cancers (nâ =â 8); and healthy volunteers (nâ =â 4). Median methylation ratio was higher in colorectal cancer compared with non-colorectal cancers (Pâ =â .001) and normal donors (Pâ =â .005). The assay detected ctDNA in 85% of patients with colorectal cancer at a specificity of 92%. Notably, we were able to detect methylated ctDNA in 75% of patients in whom ctDNA was not detected by other methods. Detection of methylated ctDNA was associated with shorter median progression-free survival compared to non-detection (8 weeks versus 54 weeks; Pâ =â .027).
Subject(s)
Circulating Tumor DNA , Colorectal Neoplasms , Neoplasms , Humans , Methylation , Circulating Tumor DNA/genetics , Liquid Biopsy , Mutation , Biomarkers, Tumor/genetics , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/geneticsABSTRACT
Epidermal growth factor receptor variant III (EGFRvIII, the deletion of exons 2-7) is a recurrent intragenic EGFR::EGFR.E1E8 fusion that occurs in high-grade gliomas. The presence of EGFRvIII in other solid tumors has not been well characterized. We retrospectively reviewed advanced malignant solid tumor cases tested by a custom hybrid capture 610-gene next-generation sequencing platform from 2021 to 2022. EGFRvIII was identified in 17 of 4331 (0.4%) cases, including 16 of 238 (7%) brain tumors and 1/301 (0.3%) breast tumors. EGFRvIII-positive brain tumors were all glioblastoma IDH-wildtype, most with concurrent TERT promoter mutation (14 of 16), EGFR amplification (13 of 16), and EGFR mutation (8 of 16). The only EGFRvIII-positive breast lesion was a sarcomatoid neoplasm in a young female patient. A separate breast case tested outside our institution with reported EGFRvIII was noted in a young female patient with a malignant phyllodes tumor with stromal overgrowth. Microscopically, both EGFRvIII-positive breast tumors showed high-grade sarcomatoid morphology with brisk mitotic activity. In summary, EGFRvIII is rare, occurring primarily in glioblastoma and rarely in breast sarcomatoid neoplasm, with no instances identified in other tumor types in our series. This select group of patients may benefit from chemotherapy and/or targeted anti-EGFR therapy.
ABSTRACT
Purpose: An evolutionary action scoring algorithm (EAp53) based on phylogenetic sequence variations stratifies patients with head and neck squamous cell carcinoma (HNSCC) bearing TP53 missense mutations as high-risk, associated with poor outcomes, or low-risk, with similar outcomes as TP53 wild-type, and has been validated as a reliable prognostic marker. We performed this study to further validate prior findings demonstrating that EAp53 is a prognostic marker for patients with locally advanced HNSCC and explored its predictive value for treatment outcomes to adjuvant bio-chemoradiotherapy. Methods and Materials: Eighty-one resection samples from patients treated surgically for stage III or IV human papillomavirus-negative HNSCC with high-risk pathologic features, who received either radiation therapy + cetuximab + cisplatin (cisplatin) or radiation therapy + cetuximab + docetaxel (docetaxel) as adjuvant treatment in a phase 2 study were subjected to TP53 targeted sequencing and EAp53 scoring to correlate with clinical outcomes. Due to the limited sample size, patients were combined into 2 EAp53 groups: (1) wild-type or low-risk; and (2) high-risk or other. Results: At a median follow-up of 9.8 years, there was a significant interaction between EAp53 group and treatment for overall survival (P = .008), disease-free survival (P = .05), and distant metastasis (DM; P = .004). In wild-type or low-risk group, the docetaxel arm showed significantly better overall survival (hazard ratio [HR] 0.11, [0.03-0.36]), disease-free survival (HR 0.24, [0.09-0.61]), and less DM (HR 0.04, [0.01-0.31]) than the cisplatin arm. In high-risk or other group, differences between treatments were not statistically significant. Conclusions: The docetaxel arm was associated with better survival than the cisplatin arm for patients with wild-type or low-risk EAp53. These benefits appear to be largely driven by a reduction in DM.
ABSTRACT
Little is known of the geospatial architecture of individual cell populations in lung adenocarcinoma (LUAD) evolution. Here, we perform single-cell RNA sequencing of 186,916 cells from five early-stage LUADs and 14 multiregion normal lung tissues of defined spatial proximities from the tumors. We show that cellular lineages, states, and transcriptomic features geospatially evolve across normal regions to LUADs. LUADs also exhibit pronounced intratumor cell heterogeneity within single sites and transcriptional lineage-plasticity programs. T regulatory cell phenotypes are increased in normal tissues with proximity to LUAD, in contrast to diminished signatures and fractions of cytotoxic CD8+ T cells, antigen-presenting macrophages, and inflammatory dendritic cells. We further find that the LUAD ligand-receptor interactome harbors increased expression of epithelial CD24, which mediates protumor phenotypes. These data provide a spatial atlas of LUAD evolution, and a resource for identification of targets for its treatment. SIGNIFICANCE: The geospatial ecosystem of the peripheral lung and early-stage LUAD is not known. Our multiregion single-cell sequencing analyses unravel cell populations, states, and phenotypes in the spatial and ecologic evolution of LUAD from the lung that comprise high-potential targets for early interception.This article is highlighted in the In This Issue feature, p. 2355.
Subject(s)
Adenocarcinoma of Lung/pathology , CD8-Positive T-Lymphocytes , Lung Neoplasms/pathology , Tumor Microenvironment , Humans , Single-Cell AnalysisABSTRACT
PURPOSE: Consensus molecular subtyping (CMS) of colorectal cancer has potential to reshape the colorectal cancer landscape. We developed and validated an assay that is applicable on formalin-fixed, paraffin-embedded (FFPE) samples of colorectal cancer and implemented the assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. EXPERIMENTAL DESIGN: We performed an in silico experiment to build an optimal CMS classifier using a training set of 1,329 samples from 12 studies and validation set of 1,329 samples from 14 studies. We constructed an assay on the basis of NanoString CodeSets for the top 472 genes, and performed analyses on paired flash-frozen (FF)/FFPE samples from 175 colorectal cancers to adapt the classifier to FFPE samples using a subset of genes found to be concordant between FF and FFPE, tested the classifier's reproducibility and repeatability, and validated in a CLIA-certified laboratory. We assessed prognostic significance of CMS in 345 patients pooled across three clinical trials. RESULTS: The best classifier was weighted support vector machine with high accuracy across platforms and gene lists (>0.95), and the 472-gene model outperforming existing classifiers. We constructed subsets of 99 and 200 genes with high FF/FFPE concordance, and adapted FFPE-based classifier that had strong classification accuracy (>80%) relative to "gold standard" CMS. The classifier was reproducible to sample type and RNA quality, and demonstrated poor prognosis for CMS1-3 and good prognosis for CMS2 in metastatic colorectal cancer (P < 0.001). CONCLUSIONS: We developed and validated a colorectal cancer CMS assay that is ready for use in clinical trials, to assess prognosis in standard-of-care settings and explore as predictor of therapy response.
Subject(s)
Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/genetics , Colorectal Neoplasms/diagnosis , Gene Expression Regulation, Neoplastic , Support Vector Machine , Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Profiling , Humans , Male , Middle Aged , Prognosis , Reproducibility of Results , Risk Assessment/methods , TranscriptomeABSTRACT
Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIß (PI4KIIIß). Molecular, biochemical, and cell biological studies show that PI4KIIIß-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIß-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIß antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIß-dependent secretion for cancer cell survival and tumor progression.
Subject(s)
Adenocarcinoma of Lung/metabolism , Chromosomes, Human, Pair 1/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Adenocarcinoma of Lung/genetics , Animals , Chromosomes, Human, Pair 1/genetics , Enzyme-Linked Immunosorbent Assay , Golgi Apparatus/metabolism , Humans , In Vitro Techniques , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , X-Ray MicrotomographyABSTRACT
Endometrial mesonephric-like carcinomas (MLCa) are uncommon with <50 reported cases thus far. Previous studies have characterized the histologic, immunohistochemical, and molecular features of MLCa; however, there is limited information with respect to outcome. This single-institution study of 23 uterine MLCas characterizes the behavior of such a neoplasm. Uterine MLCas (2004-present) had review of histologic features, immunohistochemical results, molecular profile, and clinical information (stage, treatment, follow-up). The behavior of MLCa was compared with low-grade endometrioid carcinomas (ECas) and uterine serous carcinomas (USCs) treated at our institution from 2004 to present. All MLCas had a mixture of previously described architectural and cytologic features most notably ductal and/or tubular architecture (21/23), nuclei resembling those of papillary thyroid carcinoma (18/23), and at least focal intraluminal eosinophilic secretions (20/23). Immunoperoxidase studies facilitated diagnosis in 22 cases: CD10, 10/10; calretinin, 5/15; estrogen receptor (≥10% nuclei), 6/21; progesterone receptor, 1/15; GATA-3, 15/16; TTF-1, 11/16. Fourteen of 17 tested cases had a KRAS mutation (7 as the only alteration; 7 with additional mutations including PIK [n=5]; PTEN [n=2], CTNNB1 [n=1]).One case had mutations in PTEN, PIK, and CTNNB1 without KRAS; 2 cases had no detectable somatic mutation. Overall, 48% of patients presented with International Federation of Gynecology and Obstetrics (FIGO) stage 3 or 4 disease with the following uterine risk factors: >50% myometrial invasion, 20/23; lymphovascular space invasion, 16/23; cervical stromal invasion, 7/23. Twenty patients had adjuvant therapy (7 radiation only; 13 chemotherapy±radiation), whereas 3 patients had either unknown or declined therapy. Follow-up was known for 21 patients: 17 patients had recurrences or never achieved remission with the lung being the most common recurrence site (n=9); 7 patients died of disease. The median progression-free survival was 18.2 months for MLCa compared with 183 months for ECa and 67.1 months for USC. The median overall survival for MLCa was 70.6 months compared with 139.1 months for USC (median survival for ECa not reached). Uterine MLCa is uncommon with most tumors recognized by architectural heterogeneity, vesicular, overlapping nuclei with grooves, and eosinophilic luminal secretions. The typical immunoprofile includes low to absent expression of hormone receptors but at least focal expression of GATA-3 and/or TTF-1. Most tested cases had a KRAS mutation although genetic mutations typically associated with ECa are not uncommon. Compared with more commonly encountered types of ECa, MLCa is more aggressive with a tendency towards earlier and distant recurrence.
Subject(s)
Carcinoma/secondary , Endometrial Neoplasms/pathology , Wolffian Ducts/pathology , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Carcinoma/chemistry , Carcinoma/genetics , Carcinoma/therapy , Disease Progression , Endometrial Neoplasms/chemistry , Endometrial Neoplasms/genetics , Endometrial Neoplasms/therapy , Female , Humans , Lung Neoplasms/secondary , Middle Aged , Mutation , Neoplasm Grading , Neoplasm Invasiveness , Neoplasm Recurrence, Local , Progression-Free Survival , Risk Factors , Time Factors , Wolffian Ducts/chemistryABSTRACT
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.
ABSTRACT
Potential applications of cell-free DNA (cfDNA)-based molecular profiling have used in patients with diverse malignant tumors. However, capturing all cfDNA that originates from tumor cells and identifying true variants present in this minute fraction remain challenges to the widespread application of cfDNA-based liquid biopsies in the clinical setting. In this study, we evaluate a systematic approach and identify key components of wet bench and bioinformatics strategies to address these challenges. We found that concentration of enrichment oligonucleotides, elements of the library preparation, and the structure of adaptors are critical for achieving high enrichment of target regions, retaining variant allele frequencies accurately throughout all involved steps of library preparation, and obtaining high variant coverage. We developed a dual molecular barcode-integrated error elimination strategy to remove sequencing artifacts and a background error correction strategy to distinguish true variants from abundant false-positive variants. We further describe a clinical application of this cfDNA-based duplex sequencing approach that can be used to monitor disease progression in patients with stage IV colorectal cancer. The findings also suggest that cfDNA-based molecular testing observations are highly concordant with observations obtained by traditional imaging methods. Overall, the findings presented in this study have potential implications for early detection of cancer, identification of minimal residual disease, and evaluation of therapeutic responses in patients with cancer.
Subject(s)
Adenocarcinoma/genetics , Cell-Free Nucleic Acids/genetics , Colorectal Neoplasms/genetics , Computational Biology/methods , High-Throughput Nucleotide Sequencing/methods , Adenocarcinoma/pathology , Artifacts , Cell Line, Tumor , Colorectal Neoplasms/pathology , Gene Frequency , Gene Library , Humans , In Situ Hybridization , Liquid Biopsy , Mutation , Reproducibility of ResultsABSTRACT
Circulating cell-free DNA (ccfDNA) in plasma provides an easily accessible source of circulating tumor DNA (ctDNA) for detecting actionable genomic alterations that can be used to guide colorectal cancer (CRC) treatment and surveillance. The goal of this study was to test the feasibility of using a traditional amplicon-based next-generation sequencing (NGS) on Ion Torrent platform to detect low-frequency alleles in ctDNA and compare it with a digital NGS assay specifically designed to detect low-frequency variants (as low as 0.1%) to provide evidence for the standard care of CRC. The study cohort consisted of 48 CRC patients for whom matched samples of formalin-fixed, paraffin-embedded tumor tissue, plasma, and peripheral blood mononuclear cells were available. DNA samples from different sources were sequenced on different platforms using commercial protocols. Our results demonstrate that the ccfDNA sequencing with the traditional NGS can be reliably used in an integrated workflow to detect low-frequency somatic variants in CRC. We found a high degree of concordance between traditional NGS and digital NGS in profiling mutant alleles in ccfDNA. These findings suggest that the traditional NGS is a viable alternative to digital sequencing of ccfDNA at allele frequency above 1%. ccfDNA sequencing can not only provide real-time monitoring of CRC, but also lay the basis for its application as a clinical diagnostic test to guide personalized therapy.
Subject(s)
Cell-Free Nucleic Acids/blood , Colorectal Neoplasms/genetics , Genotype , Neoplasm Metastasis/genetics , Cohort Studies , Colorectal Neoplasms/pathology , Colorectal Neoplasms/therapy , Humans , Precision MedicineABSTRACT
The emergence of highly sensitive molecular diagnostic approaches, such as droplet digital PCR, has allowed the accurate identification of low-frequency variant alleles in clinical specimens; however, the multiplex capabilities of droplet digital PCR for variant detection are inadequate. The incorporation of molecular barcodes or unique IDs into next-generation sequencing libraries through PCR has enabled the detection of low-frequency variant alleles across multiple genomic regions. However, rational library preparation and sequencing data analytic strategies that integrate molecular barcodes have rarely been applied to clinical settings. In this study, we evaluated the parameters that are crucial in the use of molecular barcodes in next-generation sequencing for genotyping clinical specimens from patients with hematologic malignancies. The uniform incorporation of molecular barcodes into DNA templates through PCR was found to be crucial, and the extent of uniformity was governed by multiple interdependent variables. An error elimination strategy was developed for removing sequencing background errors by using molecular barcode sequence information as an alternative to the conventional error correction approach. This approach was successfully used to identify mutations with frequencies as low as 0.15%, and the clonal heterogeneity of hematologic malignancies was revealed. These findings have implications for elucidating heterogeneity and temporal and spatial clonal evolution, evaluating response to therapy, and monitoring relapse in patients with hematologic malignancies.
Subject(s)
DNA Barcoding, Taxonomic , Hematologic Neoplasms/genetics , High-Throughput Nucleotide Sequencing , Mutation Rate , Mutation/genetics , Cell Line, Tumor , Humans , Limit of Detection , Multiplex Polymerase Chain ReactionABSTRACT
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.
ABSTRACT
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 PIK3CAE545K mutation as conferring drug resistance. We demonstrate that PIK3CAE545K preexisted in a rare subpopulation that was missed by both clinical and research testing, but was revealed upon multiregion sampling due to PIK3CAE545K 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 PIK3CAE545K-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 PIK3CAE545K 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 PIK3CAE545K-expressing NRAS-mutant melanoma cells to MEKi + CDK4i. Cancer Discov; 8(5); 556-67. ©2018 AACR.See related commentary by Sullivan, p. 532See related article by Teh et al., p. 568This article is highlighted in the In This Issue feature, p. 517.
Subject(s)
Class I Phosphatidylinositol 3-Kinases/genetics , Drug Resistance, Neoplasm/genetics , GTP Phosphohydrolases/genetics , Melanoma/genetics , Melanoma/metabolism , Membrane Proteins/genetics , Mutation , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction , Animals , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers , Cell Line, Tumor , Class I Phosphatidylinositol 3-Kinases/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Female , GTP Phosphohydrolases/metabolism , Humans , Melanoma/diagnosis , Melanoma/drug therapy , Membrane Proteins/metabolism , Mice , Middle Aged , Models, Biological , Phosphorylation , Positron-Emission Tomography , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Signal Transduction/drug effectsABSTRACT
Myeloid/lymphoid neoplasms with FGFR1 rearrangement are a rare entity. We present a multicenter experience of 17 patients with FISH-confirmed FGFR1 rearrangement. The clinical presentation at diagnosis included myeloproliferative neoplasm (MPN) in 4 (24%) patients, acute leukemia (AL) in 7 (41%), and concomitant MPN with AL in 6 (35%). The two most frequently observed cytogenetic abnormalities were t(8;13)(p11.2;q12)(partner gene ZMYM2) and t(8;22)(p11.2; q11.2)(BCR). Seventy-eight percent of tested patients had a RUNX1 mutation, of whom all had AL. Overall response rate to frontline therapy was 69%, and 76% of patients subsequently received allogeneic stem cell transplant (ASCT). After a median follow-up of 11 months, median progression-free survival was 15 months and median overall survival was not reached. In conclusion, FGFR1-rearranged hematologic malignancies present with features of MPN and/or AL. FGFR1 and RUNX1 are therapeutic targets for ongoing and future clinical trials.
Subject(s)
Biomarkers, Tumor , Gene Rearrangement , Leukemia, Lymphoid/genetics , Lymphoma/genetics , Myeloproliferative Disorders/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Translocation, Genetic , Adolescent , Adult , Aged , Biomarkers , Child , Child, Preschool , DNA Mutational Analysis , Female , Genetic Association Studies , Genetic Predisposition to Disease , Humans , In Situ Hybridization, Fluorescence , Infant , Karyotype , Leukemia, Lymphoid/diagnosis , Leukemia, Lymphoid/mortality , Leukemia, Lymphoid/therapy , Lymphoma/diagnosis , Lymphoma/mortality , Lymphoma/therapy , Male , Middle Aged , Myeloproliferative Disorders/diagnosis , Myeloproliferative Disorders/mortality , Myeloproliferative Disorders/therapy , Prognosis , Young AdultABSTRACT
Context: Interpretation of calcitonin measurement to predict the prognosis of medullary thyroid carcinoma (MTC) requires multiple measurements over an extended time period, making it an imperfect biomarker for evaluating prognosis or disease behavior. Single circulating cell-free DNA (cfDNA) values have been shown to be a valuable prognostic marker for several solid tumors. Objective: We tested the hypothesis that cfDNA containing the RET M918T mutation could be detected in the blood of patients with advanced MTC whose tumor harbored an M918T mutation and would be able to predict overall survival more reliably than calcitonin. Design: The level of cfDNA containing RET M918T mutation was measured in the plasma of patients with MTC via droplet digital polymerase chain reaction. Patients: Patients had a confirmed sporadic MTC diagnosis, a serum calcitonin measurement >100 pg/mL, and tumor tissue biopsy results providing RET M918T mutation status. There were 75 patients included in this study, 50 of whom harbored an RET M918T mutation by tissue biopsy. Results: RET M918T cfDNA was detected in 16 of 50 patients (32%) with a positive tissue biopsy. The detection of RET M918T cfDNA strongly correlated with worse overall survival and more accurately predicted a worse outcome than calcitonin doubling time. Conclusions: Liquid biopsy is able to detect RET M918T mutations in patient plasma with high specificity but low sensitivity. In patients with established somatic RET M918T mutations, the allelic fraction of circulating tumor DNA is prognostic for overall survival and may play a role in monitoring response to treatment.