Transfer of next-generation sequencing technology to a Clinical Laboratory Improvement Amendments–certified laboratory requires vigorous validation. Herein, we validated a next-generation sequencing ...screen interrogating 740 mutational hotspots in 46 cancer-related genes using the Ion Torrent AmpliSeq cancer panel and Ion Torrent Personal Genome Machine (IT-PGM). Ten nanograms of FFPE DNA was used as template to amplify mutation hotspot regions of 46 genes in 70 solid tumor samples, including 22 archival specimens with known mutations and 48 specimens sequenced in parallel with alternate sequencing platforms. In the archival specimens, the IT-PGM detected expected nucleotide substitutions ( n = 29) and four of six insertions/deletions; in parallel, 66 variants were detected. These variants, except a single nucleotide substitution, were confirmed by alternate platforms. Repeated sequencing of progressively diluted DNA from two cancer cell lines with known mutations demonstrated reliable sensitivity at 10% variant frequency for single nucleotide variants with high intrarun and inter-run reproducibility. Manual library preparation yielded relatively superior sequencing performance compared with the automated Ion Torrent OneTouch system. Overall, the IT-PGM platform with the ability to multiplex and simultaneously sequence multiple patient samples using low amounts of FFPE DNA was specific and sensitive for single nucleotide variant mutation analysis and can be incorporated easily into the clinical laboratory for routine testing.
Clonal diversity is a consequence of cancer cell evolution driven by Darwinian selection. Precise characterization of clonal architecture is essential to understand the evolutionary history of tumor ...development and its association with treatment resistance. Here, using a single-cell DNA sequencing, we report the clonal architecture and mutational histories of 123 acute myeloid leukemia (AML) patients. The single-cell data reveals cell-level mutation co-occurrence and enables reconstruction of mutational histories characterized by linear and branching patterns of clonal evolution, with the latter including convergent evolution. Through xenotransplantion, we show leukemia initiating capabilities of individual subclones evolving in parallel. Also, by simultaneous single-cell DNA and cell surface protein analysis, we illustrate both genetic and phenotypic evolution in AML. Lastly, single-cell analysis of longitudinal samples reveals underlying evolutionary process of therapeutic resistance. Together, these data unravel clonal diversity and evolution patterns of AML, and highlight their clinical relevance in the era of precision medicine.
SMAD4 is an essential mediator in the transforming growth factor-β pathway. Sporadic mutations of SMAD4 are present in 2.1-20.0% of colorectal cancers (CRCs) but data are limited. In this study, we ...aimed to evaluate clinicopathologic characteristics, prognosis, and clinical outcome associated with this mutation in CRC cases. Data for patients with metastatic or unresectable CRC who underwent genotyping for SMAD4 mutation and received treatment at The University of Texas MD Anderson Cancer Center from 2000 to 2014 were reviewed. Their tumors were sequenced using a hotspot panel predicted to cover 80% of the reported SMAD4 mutations, and further targeted resequencing that included full-length SMAD4 was performed on mutated tumors using a HiSeq sequencing system. Using The Cancer Genome Atlas data on CRC, the characteristics of SMAD4 and transforming growth factor-β pathway mutations were evaluated according to different consensus molecular subtypes of CRC. Among 734 patients with CRC, 90 (12%) had SMAD4 mutations according to hotspot testing. SMAD4 mutation was associated with colon cancer more so than with rectal cancer (odds ratio 2.85; p<0.001), female sex (odds ratio 1.71; p = 0.02), and shorter overall survival than in wild-type SMAD4 cases (median, 29 months versus 56 months; hazard ratio 2.08; p<0.001 log-rank test). SMAD4 mutation was not associated with age, stage at presentation, colonic location, distant metastasis, or tumor grade. A subset of patients with metastatic CRC (n = 44) wild-type for KRAS, NRAS, and BRAF who received anti-epidermal growth factor receptor therapy with mutated SMAD4 (n = 13) had shorter progression-free survival duration than did patients wild-type for SMAD4 (n = 31) (median, 111 days versus 180 days; p = 0.003 log-rank test). Full-length sequencing confirmed that missense mutations at R361 and P356 in the MH2 domain were the most common SMAD4 alterations. In The Cancer Genome Atlas data, SMAD4 mutation frequently occurred with KRAS, NRAS, and BRAF mutations and was more common in patients with the consensus molecular subtype 3 of CRC than in those with the other 3 subtypes. This is one of the largest retrospective studies to date characterizing SMAD4 mutations in CRC patients and demonstrates the prognostic role and lack of response of CRC to anti-epidermal growth factor receptor therapy. Further studies are required to validate these findings and the role of SMAD4 mutation in CRC.
Preclinical models have shown that blocking PD-1/PD-L1 pathways enhances antileukemic responses. Azacitidine upregulates PD-1 and IFNγ signaling. We therefore conducted this single-arm trial, in ...which patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) were treated with azacitidine 75 mg/m
days 1 to 7 intravenously or subcutaneously with nivolumab 3 mg/kg intravenously on days 1 and 14, every 4 to 6 weeks. For the seventy patients who were treated, the median age was 70 years (range, 22-90) and the median number of prior therapies received was 2 (range, 1-7). The overall response rate (ORR) was 33%, including 15 (22%) complete remission/complete remission with insufficient recovery of counts, 1 partial response, and 7 patients with hematologic improvement maintained >6 months. Six patients (9%) had stable disease >6 months. The ORR was 58% and 22%, in hypomethylating agent (HMA)-naïve (
= 25) and HMA-pretreated (
= 45) patients, respectively. Grade 3 to 4 immune-related adverse events occurred in 8 (11%) patients. Pretherapy bone marrow and peripheral blood CD3 and CD8 were significantly predictive for response on flow cytometry. CTLA4 was significantly upregulated on CD4
Teff in nonresponders after 2 and 4 doses of nivolumab. Azacitidine and nivolumab therapy produced an encouraging response rate and overall survival in patients with R/R AML, particularly in HMA-naïve and salvage 1 patients. Pretherapy bone marrow aspirate and peripheral blood CD3 percentage may be biomarkers for patient selection. SIGNIFICANCE: Azacitidine in combination with nivolumab appeared to be a safe and effective therapy in patients with AML who were salvage 1, prior hypomethylator-naïve, or had increased pretherapy CD3
bone marrow infiltrate by flow cytometry or IHC. Bone marrow CD3 and CD8 are relatively simple assays that should be incorporated to select patients in future trials.
.
Atypical chronic myeloid leukemia (aCML) is a rare subtype of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) largely defined morphologically. It is, unclear, however, whether aCML-associated ...features are distinctive enough to allow its separation from unclassifiable MDS/MPN (MDS/MPN-U). To study these 2 rare entities, 134 patient archives were collected from 7 large medical centers, of which 65 (49%) cases were further classified as aCML and the remaining 69 (51%) as MDS/MPN-U. Distinctively, aCML was associated with many adverse features and an inferior overall survival (12.4 vs 21.8 months, P = .004) and AML-free survival (11.2 vs 18.9 months, P = .003). The aCML defining features of leukocytosis and circulating myeloid precursors, but not dysgranulopoiesis, were independent negative predictors. Other factors, such as lactate dehydrogenase, circulating myeloblasts, platelets, and cytogenetics could further stratify MDS/MPN-U but not aCML patient risks. aCML appeared to have more mutated RAS (7/20 35% vs 4/29 14%) and less JAK2p.V617F (3/42 7% vs 10/52 19%), but was not statistically significant. Somatic CSF3R T618I (0/54) and CALR (0/30) mutations were not detected either in aCML or MDS/MPN-U. In conclusion, within MDS/MPN, the World Health Organization 2008 criteria for aCML identify a subgroup of patients with features clearly distinct from MDS/MPN-U. The MDS/MPN-U category is heterogeneous, and patient risk can be further stratified by a number of clinicopathological parameters.
•Within MDS/MPN, the WHO 2008 criteria for aCML identify a subgroup of patients with aggressive clinical features distinct from MDS/MPN-U.•The MDS/MPN-U category is heterogeneous, and patient risk can be further stratified by a number of clinicopathological parameters.
Increasing use of fine needle aspiration for oncological diagnosis, while minimally invasive, poses a challenge for molecular testing by traditional sequencing platforms due to high sample ...requirements. The advent of affordable benchtop next-generation sequencing platforms such as the semiconductor-based Ion Personal Genome Machine (PGM) Sequencer has facilitated multi-gene mutational profiling using only nanograms of DNA. We describe successful next-generation sequencing-based testing of fine needle aspiration cytological specimens in a clinical laboratory setting. We selected 61 tumor specimens, obtained by fine needle aspiration, with known mutational status for clinically relevant genes; of these, 31 specimens yielded sufficient DNA for next-generation sequencing testing. Ten nanograms of DNA from each sample was tested for mutations in the hotspot regions of 46 cancer-related genes using a 318-chip on Ion PGM Sequencer. All tested samples underwent successful targeted sequencing of 46 genes. We showed 100% concordance of results between next-generation sequencing and conventional test platforms for all previously known point mutations that included BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, RET and TP53, deletions of EGFR and wild-type calls. Furthermore, next-generation sequencing detected variants in 19 of the 31 (61%) patient samples that were not detected by traditional platforms, thus increasing the utility of mutation analysis; these variants involved the APC, ATM, CDKN2A, CTNNB1, FGFR2, FLT3, KDR, KIT, KRAS, MLH1, NRAS, PIK3CA, SMAD4, STK11 and TP53 genes. The results of this study show that next-generation sequencing-based mutational profiling can be performed on fine needle aspiration cytological smears and cell blocks. Next-generation sequencing can be performed with only nanograms of DNA and has better sensitivity than traditional sequencing platforms. Use of next-generation sequencing also enhances the power of fine needle aspiration by providing gene mutation results that can direct personalized cancer therapy.
Routine molecular testing in acute myeloid leukemia involves screening several genes of therapeutic and prognostic significance for mutations. A comprehensive analysis using single-gene assays ...requires large amounts of DNA, is cumbersome and timely consolidation of results for clinical reporting is challenging. High throughput, next-generation sequencing platforms widely used in research have not been tested vigorously for clinical application. Here we describe the clinical application of MiSeq, a next-generation sequencing platform to screen mutational hotspots in 54 cancer-related genes including genes relevant in acute myeloid leukemia (NRAS, KRAS, FLT3, NPM1, DNMT3A, IDH1/2, JAK2, KIT and EZH2). We sequenced 63 samples from patients with acute myeloid leukemia/myelodysplastic syndrome using MiSeq and compared the results with those obtained using another next-generation sequencing platform, Ion-Torrent Personal Genome Machine and other conventional testing platforms. MiSeq detected a total of 100 single nucleotide variants and 23 NPM1 insertions that were confirmed by Ion Torrent or conventional platforms, indicating complete concordance. FLT3-internal tandem duplications (n=10) were not detected; however, re-analysis of the MiSeq output by Pindel, an indel detection algorithm, did detect them. Dilution studies of cancer cell-line DNA showed that the quantitative accuracy of mutation detection was up to an allelic frequency of 1.5% with a high level of inter- and intra-run assay reproducibility, suggesting potential utility for monitoring response to therapy, clonal heterogeneity and evolution. Examples demonstrating the advantages of MiSeq over conventional platforms for disease monitoring are provided. Easy work-flow, high throughput multiplexing capability, 4-day turnaround time and simultaneous assessment of routinely tested and emerging markers make MiSeq highly applicable for clinical molecular testing in acute myeloid leukemia.
Persistence of
or
mutations in remission bone marrow specimens of patients with acute myeloid leukemia has been observed, but the clinical impact of these mutations is not well known. In this study, ...we evaluated 80 acute myeloid leukemia patients with known
R132 or
R140/R172 mutations and assessed their bone marrow at the time of remission to determine the potential impact of persistent
mutations. Approximately 40% of acute myeloid leukemia patients given standard treatment in this cohort had persistent mutations in
Patients with an
mutation had an increased risk of relapse after 1 year of follow-up compared to patients without a detectable
mutation (59%
24%;
<0.01). However, a persistent mutation was not associated with a shorter time to relapse. High
mutation burden (mutant allelic frequency ≥10%) did not correlate with relapse rate (77%
86% for patients with a low burden, i.e., mutant allelic frequency <10%;
=0.66). Persistent mutations were also observed in
,
and
during remission, but
mutations remained significant in predicting relapse by multivariate analysis. Flow cytometry was comparable and complementary to next-generation sequencing-based assay for predicting relapse. Monitoring for persistent
mutations in patients with acute myeloid leukemia in remission can provide information that could be used to justify early interventions, with the hope of facilitating longer remissions and better outcomes in these patients.
TP53 mutations are associated with poor outcomes in acute myeloid leukemia (AML). The prognostic impact of mutant TP53 (TP53mut) variant allelic frequency (VAF) is not well established, nor is how ...this information might guide optimal frontline therapy. We retrospectively analyzed 202 patients with newly diagnosed TP53-mutated AML who underwent first-line therapy with either a cytarabine- or hypomethylating agent (HMA)–based regimen. By multivariate analysis, TP53mut VAF >40% was independently associated with a significantly higher cumulative incidence of relapse (P = .003) and worse relapse-free survival (P = .001) and overall survival (OS; P = .003). The impact of TP53mut VAF on clinical outcomes was driven by patients treated with a cytarabine-based regimen (median OS, 4.7 vs 7.3 months for VAF >40% vs ≤40%; P = .006), whereas VAF did not significantly affect OS in patients treated with HMA. The addition of venetoclax to HMA did not significantly affect OS compared with HMA without venetoclax, both in the entire TP53-mutated population and in patients stratified by TP53mut VAF. Among patients with TP53mut VAF ≤40%, OS was superior in those treated with higher-dose cytarabine, whereas OS was similarly poor for patients with TP53mut VAF >40% regardless of therapy. The best long-term outcomes were observed in those with 1 TP53 mutation with VAF ≤40% who received a frontline cytarabine-based regimen (2-year OS, 38% vs 6% for all others; P < .001). In summary, TP53mut VAF provides important prognostic information that may be considered when selecting frontline therapy for patients with newly diagnosed TP53-mutated AML.
•TP53mut VAF >40% was independently associated with higher rates of relapse and worse relapse-free survival and OS.•Treatment with a cytarabine-based regimen preferentially benefited patients with 1 TP53 mutation with VAF ≤40%.
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