High Variant Allele Frequency Research Articles

Clonal architecture predicts clinical outcomes and drug sensitivity in acute myeloid leukemia

The impact of clonal heterogeneity on disease behavior or drug response in acute myeloid leukemia remains poorly understood. Using a cohort of 2,829 patients, we identify features of clonality associated with clinical features and drug sensitivities. High variant allele frequency for 7 mutations (including NRAS and TET2) associate with dismal prognosis; elevated GATA2 variant allele frequency correlates with better outcomes. Clinical features such as white blood cell count and blast percentage correlate with the subclonal abundance of mutations such as TP53 and IDH1. Furthermore, patients with cohesin mutations occurring before NPM1, or transcription factor mutations occurring before splicing factor mutations, show shorter survival. Surprisingly, a branched pattern of clonal evolution is associated with superior clinical outcomes. Finally, several mutations (including NRAS and IDH1) predict drug sensitivity based on their subclonal abundance. Together, these results demonstrate the importance of assessing clonal heterogeneity with implications for prognosis and actionable biomarkers for therapy.

Copy-Neutral Loss-of-Heterozygosity Adds Diagnostic and Prognostic Information to the Molecular Profiling of Hematological Malignancies

Background: Copy-neutral loss-of-heterozygosity (CN-LOH) - not detectable by chromosome banding analysis - is gaining importance as a prognostic factor and can either cause the duplication of an activating mutation in an oncogene, the deletion of a tumor suppressor gene or the gain/loss of specific methylated regions. However, examination for possible CN-LOH in hematological diagnostics is at present not routinely performed and, hence, data regarding the occurrence of CN-LOH across different entities as well as the association of relevant genes is limited.Aim: (1) Frequency assessment of CN-LOH by target enrichment sequencing (TES) in a diagnostic setting, (2) evaluation of whole genome sequencing (WGS) data to estimate the prevalence of CN-LOH in a larger cohort, to pinpoint relevant genes for CN-LOHs with so far unknown associations, and to determine cross-entity variability.Patients and Methods: 1196 patients (507 female, 689 male, median age: 66 years), sent between 04/2021-07/2021 for diagnostic work-up, were analyzed by TES with a median coverage of 1765x for the gene panel and 52x for the CNV spike-in panel (IDT, Coralville, IA). Amplification-free WGS libraries of 3851 different patients were sequenced with a median coverage of 102x. Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner). Cnvkit (v 0.9.9) was used to call copy number variations (CNVs) and CN-LOH for TES and HadoopCNV (Yang et al. 2017) was used to call CN-LOH for WGS.Results: 1196 patients were analyzed by TES. For 10% of the patients at least one CN-LOH event was detected without any association to age or gender but a slightly higher incidence in myeloid compared to lymphoid neoplasms (10% vs 6%). In 14 patients, CN-LOH affected more than one chromosome arm. CN-LOH occurred most frequently in 4q (n = 15), 7q (n = 16), 9p (n = 25) and 11q (n = 10). As expected, 4q CN-LOH co-occurred with high variant allele frequencies (VAF) of TET2. Based on WGS data, 4q CN-LOH occurred predominately in AML (35%), CMML (22%), and MDS (20%). In rare cases, 4q CN-LOH was associated with FBXW7 variants in T-ALL. 7q CN-LOH occurred nearly exclusively in myeloid neoplasms (95%) and was associated with high VAFs in EZH2 in 69% of TES and 82% of WGS cases. CUX1 variants with high VAFs were detected in 80% (TES) and 45% (WGS) of the remaining cases, respectively. The well-known 9p CN-LOH led to JAK2V617F homozygosity in all myeloid neoplasms and occurred most often in MPNs. In T-ALL, regions of 9p CN-LOH harbored CDKN2A/B deletions. 11q CN-LOH occurred more often in myeloid than lymphoid neoplasms (79% vs 21%) and was associated with CBL variants in 61% and KMT2A-PTD in 19% of the cases. In contrast, ATM was the relevant gene in all lymphoid cases with 11q CN-LOH. CN-LOH in 11p was detected less frequently and only in 25% of cases an association with WT1 variants could be identified.Our WGS data confirmed the known associations between 1p CN-LOH and high allele burden in MPL, CSF3R and NRAS, 2p CN-LOH and DNMT3A variants, 13q CN-LOH and FLT3-ITD, the near exclusive occurrence of 16p CN-LOH in follicular lymphoma (FL, 98%) with high CREBBP-mutant allele burden , 17p CN-LOH and TP53 homozygosity, and the exclusive occurrence of 21q CN-LOH in AML and its association with RUNX1 mutations. Besides, 12q CN-LOH was associated with KMT2D in FL, with SH2B3 in MDS/MPN overlaps and in rare cases with KDM2B. For 17q CN-LOH the relevant gene was not unequivocally identifiable with high mutant allele variants in SRSF2, STAT5B, and NF1. 18q CN-LOH was a very rare event but consistently associated with a high VAF of MBD2, which presumably influences cell proliferation (Cheng et al. 2018). 19q CN-LOH was mostly (63%) associated with a high VAF of CEBPA variants, except for patients with hairy cell leukemia: in these cases nonsense mutations in CIC (VAF > 90%) were detected. CN-LOH in 22q was more common in myeloid malignancies (65% vs 35%) and associated with PRR14L mutations in the majority of myeloid cases (62%). Of note, this association occurred neither in AML samples nor in lymphoid neoplasms. No recurrent mutations were found for 6p and 14q CN-LOHs. For all other chromosomes, CN-LOH events were very rare.Conclusions: By using a CNV spike-in panel, TES adds additional diagnostic and prognostic information by enabling simultaneous detection of selected gene mutations and genome-wide CNVs, as well as CN-LOH, without increase in sequencing costs and turn-around times. [Display omitted] DisclosuresHaferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Gilteritinib (GILT) Monotherapy with Addition of Decitabine (DEC) in Non-Responders in Older Newly Diagnosed (ND) FLT3 Mutated Acute Myeloid Leukemia (AML) Patients Having High and Low Variant Allele Frequency (VAF): A Phase 2/1b Sub-Study of the Beat AML Master Trial

Gilteritinib (GILT) Monotherapy with Addition of Decitabine (DEC) in Non-Responders in Older Newly Diagnosed (ND) FLT3 Mutated Acute Myeloid Leukemia (AML) Patients Having High and Low Variant Allele Frequency (VAF): A Phase 2/1b Sub-Study of the Beat AML Master Trial

Clonotype-Immunophenotype Relationships in TET2 and IDH-Mutant Myeloid Transformation

Large scale molecular profiling studies in AML patients have suggested that stepwise acquisition of somatic mutations is crucial in driving leukemic development. High variant allele frequency (VAF) mutations in epigenetic modifier genes, such as TET2 and IDH1/2, are thought to occur early in AML pathogenesis while oncogenic mutations with typically lower VAF mutations, including FLT3 and NRAS, are suggested to occur late in disease evolution. While bulk DNA sequencing has catalogued co-mutations found in individual AMLs, it cannot unveil the heterogeneity and composition of clones that makes up the disease. Elucidating the architecture and clone-specific molecular profiles at the single cell resolution will be key to understanding how sequential and/or parallel mutation acquisition drives myeloid transformation.To assess the clonal architecture of AML, we previously performed single cell DNA sequencing (scDNA seq) in 146 patients with myeloid malignancies. We have further identified specific mutational combinations driving clonal expansion in TET2- or IDH1/2- mutant AML samples. These studies suggest TET2 and IDH1/2 can cooperate to promote clonal expansion with DNMT3A and NPM1 (Figure 1A). However, TET2 or IDH1/2 mutant clones that acquired KRAS mutations underwent minimal clonal expansion, suggesting mutant-pair specific fitness alterations (Figure 1B). To further identify how co-mutational pairing impacted clonal fitness and differentiation, we integrated the scDNA platform with immunophenotypic profiling of 45 cell surface markers and analyzed new TET2- and IDH1/2- mutant AML samples (Figure 1C). We identified clone-specific differences in lineage markers depending on co-mutational partners. NPM1 co-mutant clones were enriched for more primitive markers (CD33), whereas NRAS co-mutant clones possessed high expression of myeloid differentiation markers (CD14/CD11b), suggestive of clone-specific fitness landscapes across hematopoietic differentiation. We also identified divergent clonotype-immunophenotype patterns in TET2- and IDH2-mutant clones harboring NPM1/RAS mutations, suggesting that initiating mutations may prime mutant clones for very different evolutionary trajectories as they acquire similar mutations in leukemogenesis (Figure 1D).To deterministically delineate the relationship between clonal evolution and myeloid transformation, we generated Cre-inducible single (Tet2 -/-), double (Tet2 -/-/Nras G12D and Tet2 -/-/Npm1 cA/wt), and triple (Tet2 -/-/Npm1 cA/wt/Nras G12D) mutant mice and evaluated differences in chimerism, immunophenotype, and survival. We observed a shortened survival for double and triple mutant mice, compared to Tet2 -/- only mice (Figure 1E). As previously reported, Tet2 -/-/Nras G12D mice developed a CMML-like phenotype. Critically, the addition of Npm1 resulted in a more rapid disease onset and transformation to AML (Figure 1F). Moreover, triple mutant WBM transplanted to form a fully penetrant disease into secondary recipients, while double mutant Tet2 -/-/Nras G12D WBM failed to form disease within 3 months of transplant, suggesting a difference in the cell population responsible for disease propagation. Immunophenotypic alterations were evident with Tet2 -/-/ Nras G12D displaying an increase in Mac1 +Gr1 + cells compared to Tet2 -/-/Npm1 cA/wt/Nras G12D mice which possessed increased Mac1 +Gr1 - cells and expansion of lineage negative cells (Figure 1G). These findings align with the clonotype specific expression patterns observed in clinical specimen and suggest that myeloid transformation and maturation biases are influenced by specific mutational combinations. [Display omitted] DisclosuresMiles: Mission Bio: Honoraria, Speakers Bureau. Bowman: Mission Bio: Honoraria, Speakers Bureau. Carroll: Janssen Pharmaceutical: Consultancy; Incyte Pharmaceuticals: Research Funding. Levine: Astellas: Consultancy; Janssen: Consultancy; Auron: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Incyte: Consultancy; Imago: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Prelude: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Morphosys: Consultancy.

The FLT3 F691L Gatekeeper Mutation Promotes Clinical Resistance to Gilteritinib + Venetoclax (GILT + VEN) in AML

Background: FMS-like tyrosine kinase (FLT3) is one the most frequently mutated genes in AML and is associated with poor prognosis. FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations occur in up to 30% and 5-10% of AML, respectively. Several small molecule FLT3 inhibitors (FLT3i) have been developed but their use as single agents is limited due to the development of drug resistance. Our lab developed a two-step model of early and late resistance to FLT3i that recapitulates resistance in AML patients (Traer et al. Cancer Res. 2016; Joshi et al. Cancer Cell 2021). Early resistance, also known as AML persistence, is the stage when residual AML cells are dependent upon the marrow microenvironment for survival and patients are clinically responding. Late resistance to GILT was characterized by expansion of intrinsic mutations, with NRAS mutations being the most frequent mutation, in addition to a few gatekeeper FLT3 mutations. Current therapies are looking at combinations to overcome GILT resistance, including chemotherapy, hypomethylating agents (HMAs), and venetoclax (VEN) +/- HMAs. GILT+VEN, in particular, has shown good initial activity in relapsed/refractory FLT3 AML patients (Daver et al. ASH 2020), however the mechanism of resistance to this combination is unknown.Results: Early resistance cell cultures to GILT+VEN were created by exposing MOLM14 cells to GILT 25nM + VEN 25nM alone or supplemented with microenvironmental ligands FGF2 or FLT3 ligand (FL; N=3/group). Media, drugs, and ligands were replenished twice weekly. After 25 weeks, only the cultures exposed to ligand resumed growth (N=1 for FGF2 and N=3 for FL). Ligands were then removed from these early resistant cultures to induce late resistance. There was an initial drop in cell viability but cells resumed growth after only 3.5 weeks (Fig. 1). In contrast, the time to develop early and late resistance to GILT monotherapy was 8 and 15 weeks, respectively. Immunoblot analysis of GILT + VEN early and late resistant cultures demonstrated restoration of FLT3 signaling, as well as phosphorylation of downstream AKT/MAPK pathways. These results also contrasted to late GILT monotherapy resistant cultures, which had downstream AKT/MAPK activation via outgrowth of NRAS mutations. Since FLT3 appeared to be functionally active, we sequenced FLT3 and found that all early and late GILT + VEN resistance cultures had gatekeeper FLT3 F691L mutations. F691L accounted for only in a minority of resistance cultures to GILT monotherapy. To test if FLT3 signaling was important for resistance, we exposed parental cells to higher concentrations of gilteritinib, which have been shown to partly overcome F691L, as well as the FLT3i FF-10101, which binds FLT3 at a different site and is not affected by the F691L mutation. Both of these approaches restored sensitivity to FLT3i in vitro. As expected, the F691L mutation provided broad resistance to most FLT3i (Fig. 2).To validate this mechanism of resistance in patients, we identified a relapsed FLT3-ITD patient who was treated with GILT monotherapy for 5 months, followed by GILT + HMA for 4 cycles, and then GILT + VEN for resistant proliferative disease. After an initial response to GILT + VEN, the leukemia cells began to increase again in the peripheral blood. A repeat genetic test was ordered and the patient was found to have developed a FLT3 F691L mutation at a high variant allele frequency (Fig. 3).Conclusion: We have developed a robust cell line model of early and late resistance to FLT3i that mimics the timing and expansion of resistance mutations in the clinic. Our model of early and late resistance to GILT combinations can prospectively predict mechanisms of resistance. Although uncommon as a mechanism of resistance to GILT monotherapy, our model and early patient data predicts that F691L mutations are more important for GILT + VEN resistance. [Display omitted] DisclosuresTyner: Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Petra: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Agios: Research Funding; Schrodinger: Research Funding. Traer: ImmunoGen: Membership on an entity's Board of Directors or advisory committees; Schrodinger: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier/Agios: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees.

TP53 Allelic Status in Myeloid Malignancies

IntroductionTP53 is a tumor suppressor gene involved in regulating cell division and apoptosis in response to DNA damage. In hematologic malignancies, TP53 alterations are present in both myeloid and lymphoid malignancies. TP53 alterations including both sequence level mutations and deletions occur in 8-10% of de novo acute myeloid leukemia (AML), and are significantly enriched in patients with therapy-related myeloid neoplasms with a frequency of 25-40%. TP53 alterations are associated with complex karyotype, resistance to traditional cytotoxic chemotherapy and dismal outcome, and are well established poor prognostic markers for both AML and myelodysplastic syndrome (MDS). While both biallelic and monoallelic TP53 alterations are seen, biallelic TP53 alteration is more frequent in both AML and MDS. In the instances of monoallelic TP53 alterations, the remaining wild-type allele can be inhibited by the dominant negative effect of the mutant p53.The clinical implication of TP53 allelic status remains controversial. Biallelic TP53 alterations and/or high TP53 mutation variant allele frequency (VAF) are associated with older patients, complex karyotype, few co-occurring mutations and poor outcome in both AML and MDS. Other studies demonstrate that biallelic vs. monoallelic TP53 alterations or high vs. low TP53 VAF have similar prognosis in myeloid malignancies. Current European LeukemiaNet (ELN) recommends testing TP53 deletion using karyotype analysis and TP53 mutations by molecular testing.Methods:We performed a retrospective review of patients with myeloid malignancies, myeloid next generation sequencing (NGS) panel and cytogenetic tests performed at ARUP Laboratories. We identified 18 patients with myeloid malignancies and TP53 mutations. We used a combination of karyotype analysis, FISH, chromosomal microarray (CMA) and NGS to determine the TP53 allelic status.Results:18 patients diagnosed with myeloid malignancies and TP53 mutations were identified. Among them, 6 were diagnosed with AML, 10 had MDS, one with CMML and one had post essential thrombocythemia myelofibrosis. Age range is from 31 to 78 with a median age 65.5 years.23 TP53 mutations were identified among 18 patients. The majority (78%) of TP53 mutations are located in the DNA binding domain. Co-occurring mutations are uncommon in patients with TP53 mutations. 11 out of 18 patients did not have co-occurring mutations in other myeloid malignancy related genes. Three cases had 1, three cases had 2, and one case had 3 co-occurring mutations at the time of diagnosis.Karyotype analysis and FISH were performed on all 18 patients. CMA was performed on 9 patients. 17p abnormalities, defined by the deletion or copy-neutral loss of heterozygosity (CN-LOH) of 17p, were seen in 9 out of 18 cases. The 17p abnormalities in 8 out of these 9 cases were visible by karyotype. Case 11 had a CN-LOH identified by CMA which was cryptic by karyotype. Typical complex karyotype was seen in 16 out of 18 cases.Biallelic TP53 alterations are defined by either the presence of a TP53 mutation with a 17p abnormality (deletion or CN-LOH), or two TP53 mutations with similar VAF, or one TP53 mutation with VAF >50%. Biallelic TP53 alterations were seen more frequently compared with monoallelic TP53 alterations (14 vs. 4 patients) and enriched in older patients (Figure 1). All 6 AML patients had biallelic TP53 alterations.Outcome data is available in 10 patients including 8 patients with biallelic and 2 patients with monoallelic TP53 alterations. Patients with biallelic TP53 alterations have dismal outcome (Figure 2). Patient 7 represents an atypical patient with TP53 alteration in our study cohort. She was diagnosed with MDS at the age of 31. She had monoallelic TP53 mutation with a relatively low VAF and normal karyotype through her disease course. Her MDS never progressed and she remained in remission after transplant for more than four years and still doing well.Conclusion:In this study, we evaluated the TP53 allelic status in 18 patients with myeloid malignancies. Biallelic TP53 alterations are more frequent than monoallelic TP53 alterations, and are associated with older patients, and fewer co-occurring mutations. Biallelic TP53 alterations are associated with typical complex karyotype and dismal outcome. Our study supports the importance to differentiate between biallelic and monoallelic TP53 alterations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Circulating Tumor DNA Profiling From Breast Cancer Screening Through to Metastatic Disease.

PURPOSEWe investigated the utility of the Oncomine Breast cfDNA Assay for detecting circulating tumor DNA (ctDNA) in women from a breast screening population, including healthy women with no abnormality detected by mammogram, and women on follow-up through to advanced breast cancer.MATERIALS AND METHODSBlood samples were taken from 373 women (127 healthy controls recruited through breast screening, 28 ductal carcinoma in situ, 60 primary breast cancers, 47 primary breast cancer on follow-up, and 111 metastatic breast cancers [MBC]) to recover plasma and germline DNA for analysis with the Oncomine Breast cfDNA Assay on the Ion S5 platform.RESULTSOne hundred sixteen of 373 plasma samples had one or more somatic variants detected across eight of the 10 genes and were called ctDNA-positive; MBC had the highest proportion of ctDNA-positive samples (61; 55%) and healthy controls the lowest (20; 15.7%). ESR1, TP53, and PIK3CA mutations account for 93% of all variants detected and predict poor overall survival in MBC (hazard ratio = 3.461; 95% CI, 1.866 to 6.42; P = .001). Patients with MBC had higher plasma cell-free DNA levels, higher variant allele frequencies, and more polyclonal variants, notably in ESR1 than in all other groups. Only 15 individuals had evidence of potential clonal hematopoiesis of indeterminate potential mutations.CONCLUSIONWe were able detect ctDNA across the breast cancer spectrum, notably in MBC where variants in ESR1, TP53, and PIK3CA predicted poor overall survival. The assay could be used to monitor emergence of resistance mutations such as in ESR1 that herald resistance to aromatase inhibitors to tailor adjuvant therapies. However, we suggest caution is needed when interpreting results from a single plasma sample as variants were also detected in a small proportion of HCs.

Somatic mosaics in hereditary tumor predisposition syndromes

Historically, it is estimated that 5–10% of cancer patients carry a causative genetic variant for a tumor predisposition syndrome. These conditions have high clinical relevance as they are actionable regarding risk-specific surveillance, predictive genetic testing, reproductive options, and – in some cases – risk reducing surgery or targeted therapy. Every individual is born with on average 0.5–1 exonic mosaic variants prevalent in single or multiple tissues. Depending on the tissues affected, mosaic conditions can abrogate the clinical phenotype of a tumor predisposition syndrome and can even go unrecognized, because it can be impossible or difficult to detect them with routine genetic testing in blood/leucocytes. On the other hand, it is estimated that at least 4% of presumed de novo variants are the result of low-level mosaicism (variant allele frequency <10%) in a parent, while around 7% are true mosaic variants with a higher variant allele frequency, which can sometimes be confused for heterozygous variants. Clonal hematopoiesis however can simulate a mosaic tumor predisposition in genetic diagnostics and has to be taken into account, especially for TP53 variants.Depending on the technique, variant allele frequencies of 2–3% can be detected for single nucleotide variants by next generation sequencing, copy number variants with variant allele frequencies of 5–30% can be detected by array-based technologies or MLPA.Mosaic tumor predisposition syndromes are more common than previously thought and may often remain undiagnosed. The clinical suspicion and diagnostic procedure for several cases with mosaic tumor predisposition syndromes are presented.

Bio-O2-08 - Mutational landscape of nodal peripheral T-cell lymphoma subtypes

<h3>Introduction:</h3> Nodal peripheral T-cell lymphoma (PTCL) subclassification (Angioimmunoblastic TCL, PTCL-with T Follicular Helper phenotype and PTCL-NOS) and therapeutic targeting is still controversial. Overall survival (OS) is only around 30% after 5 years. <h3>Methods:</h3> We performed targeted next-generation sequencing of 61 selected genes in 76 PTCL patients. Among the 76cases, 44 were classified as AITL, 18 as PTCL-TFH and 14 as PTCL-NOS. <h3>Results:</h3> Our analysis revealed a wide variety of genetic variants that possibly drive the development of AITL, PTCL-TFH and/or PTCL-NOS. We identified an average of 4 variants per patient, mainly clustered in genes regulating chromatin conformation (TET2, DNMT3A) and T-cell differentiation (RHOA, MTOR, IDH2, VAV1 and NOTCH1). More frequently mutated genes were TET2 (76,31%) and DNMT3A (27,63%). Multiple mutations were found in TET2, VAV1, RHOA, NOTCH1, MTOR, JAK1, ZEB1, ATM, DNMT3A and ARID1B. Main difference among the three Nodal PTCL classes was the higher frequency of RHOAG17V mutations (p<0.0001), present approximately 2 times more frequently in AITL cases (34,09%) than in PTCL-TFH (16,66%), and they were not detected in PTCL-NOS. All three Nodal PTCL subtypes share finding suggesting Clonal Hematopoiesis (CH) in TET2 and DNMT3A genes. Cases with higher variant allele frequencies (VAF)s in TET2 and DNMT3A genes vs. other mutated genes were attributed to CH. In our study we found this phenomenon to take place in all Nodal PTCL subtypes (AITL: 31,8%; PTCL-TFH: 50%; PTCL-NOS: 35,71%). A correlation was not found between specific mutations, mutational index, mutations attributed to CH and clinical outcome. <h3>Conclusions:</h3> There is a common molecular basis for the three Nodal PTCL with very frequent mutations in genes regulating chromatin conformation associated with mutations in genes governing T-cell differentiation. RHOA G17V mutations were highly significant enriched in AITL. Findings suggesting Clonal Hematopoiesis were found in the three types of nodal PTCL.

PIK3CA mutations in plasma circulating tumor DNA predict survival and treatment outcomes in patients with advanced cancers

BackgroundOncogenic mutations in PIK3CA are prevalent in diverse cancers and can be targeted with inhibitors of the phosphoinositide-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Analysis of circulating tumor DNA (ctDNA) provides a minimally invasive approach to detect clinically actionable PIK3CA mutations.Patients and methodsWe analyzed PIK3CA hotspot mutation frequency by droplet digital PCR (QX 200; BioRad) using 16 ng of unamplified plasma-derived cell-free DNA from 68 patients with advanced solid tumors (breast cancer, n = 41; colorectal cancer, n = 13; other tumor types, n = 14). Results quantified as variant allele frequencies (VAFs) were compared with previous testing of archival tumor tissue and with patient outcomes.ResultsOf 68 patients, 58 (85%) had PIK3CA mutations in tumor tissue and 43 (74%) PIK3CA mutations in ctDNA with an overall concordance of 72% (49/68, κ = 0.38). In a subset analysis, which excluded samples from 26 patients known not to have disease progression at the time of sample collection, we found an overall concordance of 91% (38/42; κ = 0.74). PIK3CA-mutated ctDNA VAF of ≤8.5% (5% trimmed mean) showed a longer median survival compared with patients with a higher VAF (15.9 versus 9.4 months; 95% confidence interval 6.7-17.1 months; P = 0.014). Longitudinal analysis of ctDNA in 18 patients with serial plasma collections (range 2-22 time points, median 5) showed that those with a decrease in PIK3CA VAF had a longer time to treatment failure (TTF) compared with patients with an increase or no change (10.7 versus 2.6 months; P = 0.048).ConclusionsDetection of PIK3CA mutations in ctDNA is concordant with testing of archival tumor tissue. Low quantity of PIK3CA-mutant ctDNA is associated with longer survival and a decrease in PIK3CA-mutant ctDNA on therapy is associated with longer TTF.

Clinical Outcomes in Non-Small-Cell Lung Cancer Patients Treated With EGFR-Tyrosine Kinase Inhibitors and Other Targeted Therapies Based on Tumor Versus Plasma Genomic Profiling.

PURPOSETo compare clinical outcomes in a cohort of patients with advanced non–small-cell lung cancer (NSCLC) with targetable genomic alterations detected using plasma-based circulating tumor DNA (ctDNA) or tumor-based next-generation sequencing (NGS) assays treated with US Food and Drug Administration–approved therapies at a large academic research cancer center.METHODSA retrospective review from our MD Anderson GEMINI database identified 2,224 blood samples sent for ctDNA NGS testing from 1971 consecutive patients with a diagnosis of advanced NSCLC. Clinical, treatment, and outcome information were collected, reviewed, and analyzed.RESULTSOverall, 27% of the ctDNA tests identified at least one targetable mutation and 73% of targetable mutations were EGFR-sensitizing mutations. Among patients treated with first-line epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapies, there were no significant differences in progression-free survival of 379 days and 352 days (P value = .41) with treatment based on tissue (n = 40) or ctDNA (n = 40), respectively. Additionally, there were no differences in progression-free survival or objective response rate among those with low (n = 8, 0.01%-0.99%) versus high (n = 16, ≥ 1%) levels of ctDNA of the targetable mutation as measured by variant allele frequency (VAF). Overall, there was excellent testing concordance (n = 217 tests) of > 97%, sensitivity of 91.7%, and specificity of 99.7% between blood-based ctDNA NGS and tissue-based NGS assays.CONCLUSIONThere were no significant differences in clinical outcomes among patients treated with approved EGFR-TKIs whose mutations were identified using either tumor- or plasma-based comprehensive profiling and those with very low VAF as compared with high VAF, supporting the use of plasma-based profiling to guide initial TKI use in patients with metastatic EGFR-mutant NSCLC.

Abstract 638: Enhancing the molecular diagnosis, prognosis, and treatment monitoring of retinoblastoma using cell free DNA in aqueous humor

Abstract Purpose: All previous studies of retinoblastoma (RB) aqueous humor (AH) analysis for tumor-derived cell-free DNA (cfDNA) have focused on sampling AH from eyes undergoing therapy; results from AH liquid biopsy at the time of diagnosis have not yet been published. Herein, we detail the diagnostic and prognostic genomic biomarkers found in the AH cfDNA from 7 RB eyes at diagnosis, each with longitudinal evaluation of over 12 months in follow-up. Methods: Subjects included 7 eyes of 6 RB patients who underwent AH sampling at diagnosis. CfDNA from each AH sample was isolated and sequenced to assess genome-wide somatic copy number alterations (SCNAs), followed by targeted resequencing and mutation detection using a custom hybridization panel for RB1 and MYCN. Results were compared to peripheral blood RB1 testing and matched tumor samples, when available. Tumor fraction (TFx) was calculated using ichorCNA. Results: Five of 7 diagnostic AH samples contained highly recurrent RB SCNAs, 4 with large scale alterations and 1 with a focal RB1 gene deletion. Mutational analysis of AH cfDNA identified pathogenic somatic variants in 5 diagnostic AH samples with high variant allele frequency, while the remaining 2 diagnostic AH samples had either a high TFx from SCNAs or a focal MYCN amplification. Taken together, somatic tumoral genomic information was detected in all 7 diagnostic AH samples. The 2 eyes that required enucleation had poor prognostic biomarkers (chromosome 6p gain and MYCN amplification) present at the time of diagnosis. TFx from longitudinal AH sampling corresponded to treatment response over time in all cases for which sequential AH samples were available. Conclusions: This study demonstrates that AH sampling at diagnosis is both feasible and safe. Molecular profiling of AH provides a plethora of diagnostic and prognostic information from only a single 0.1 mL AH sample. 1 Citation Format: Liya Xu, Mary E. Kim, Ashley Polski, Rishvanth K. Prabakar, Chen-Ching Peng, Patricia Chévez-Barrios, Jonathan W. Kim, Rachana Shah, Rima Jubran, Peter Kuhn, David Cobrinik, James Hicks, Jesse L. Berry. Enhancing the molecular diagnosis, prognosis, and treatment monitoring of retinoblastoma using cell free DNA in aqueous humor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 638.

Clonal Analysis of Acute Myeloid Leukemia Secondary to Myeloproliferative Neoplasms

[Background] Prognosis of patients with secondary acute myeloid leukemia (sAML) transformed from myeloproliferative neoplasms (MPNs) is extreamly poor. It has been demonstrated that several additional genetic alterations, which are engaged in epigenetic regulation, cell-growth signaling and RNA splicing machinery, cooperate with JAK2, CALR and MPL mutations for the development of sAML from MPN. The order of mutation acquisition also influences the clonal evolution in MPN. However, little is known about the mechanisms of its leukemic transformation. In previous studies, both JAK2 -mutated and -unmutated AML clones were identified in sAML cases transformed from JAK2 -mutated MPNs, suggesting the diversity of the clonal evolution during the leukemic transformation from MPNs. In this study, we investigated genetic alterations and clonal evolutions in sAML from MPN, especially in JAK2 -unmutated sAML, to elucidate the mechanisms of the leukemic transformation.[Methods] We performed targeted deep sequencing of 54 genes in six paired MPN and sAML samples. Whole-exome sequencing (WES) was also performed in a paired MPN, sAML and normal tissue cells. For a clonal analysis, bone marrow (BM) cells at complete remission (CR) stage after chemotherapy were sorted into hematopoietic stem cell (HSC) and hematopoietic progenitor fractions such as common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP) and megakaryocytic/erythroid progenitor (MEP) fractions, and analyzed the mutations detected in targeted deep sequencing or WES. Furthermore, we sorted single cells from the HSC fraction and performed single-cell mutational analysis to clarify the clonal evolutions precisely during the development of sAML. In addition, we intravenously inoculated BM cells at the sAML stage into NOD/SCID/IL-2Rγnull (NOG) mice, and compared mutation status between primary AML cells and engrafted leukemia cells to evaluate the clonal hierarchy of leukemia cells.[Results] Four JAK2 -mutated and two CALR -mutated MPNs were analyzed in this study. Targeted deep sequencing identified TP53 mutations in five of six sAML samples with high variant allele frequencies (VAFs). In two of the four JAK2 -mutated MPNs, VAFs of JAK2 -V617F were significantly decreased in sAML cells compared with those in MPN cells, whereas VAFs of TP53 mutations were emerged or increased in sAML cells, suggesting that sAML clones with TP53 mutations were raised from JAK2-V617F negative hematopoietic cells in both cases. Moreover, we identified the mutations which was common to MPN and AML cells in both cases. These mutations formed common initiating clones prior to MPN and sAML, and additional mutation in JAK2 or TP53 was associated with their MPN or sAML development. The single-cell analysis in CD34+/CD38- HSC fractions revealed that a small part of the cells harbored TP53 -R248W and JAK2 -wild type (WT), half of the cells harbored homozygous JAK2 -V617F and TP53 -WT, and the other half cells harbored JAK2 - and TP53 -WT. Notably, three common mutations detected both in MPN and in sAML with the same VAFs were mainly found in JAK2 -WT cells. Thus, we validated that JAK2 and TP53 mutations were mutually exclusive HSC fractions, and confirmed that sAML clones were raised from JAK2 -unmutated clone. In patient derived xenotransplant models using JAK2 -WT sAML cells following JAK2 -mutated MPN or CALR -mutated sAML, engrafted leukemia cells showed the same mutational pattern as primary sAML cells. These results suggested that sAML clones but not MPN clones were able to engraft and proliferate in mice.[Conclusions] The genetic analysis in paired MPN and sAML samples revealed the diverse clonal evolution patterns during the development of sAMLs from MPNs. TP53 mutation was strongly associated with leukemic transformations both in JAK2 -V617F mutated and CALR -mutated MPNs. However, we demonstrated two cases in whom driver mutations in MPNs were not preserved in sAML clones. In those cases, there might be independent common initiating clones corresponding to MPN or sAML, and each initiating clone might be acquired JAK2 or TP53 mutation before the development of MPN or sAML, respectively. Our data also suggested the importance of single-cell analysis which enable us to clarify the clonal evolutions in leukemic transformation from MPN, that were not identified in targeted deep sequencing as well as WES. DisclosuresKiyoi:Phizer Japan Inc.: Honoraria, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Meiji Seika Pharma Co.,Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Eisai Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding.

CSF3R Mutation in Acute Leukemia and MDS Patients

Introduction: CSF3R somatic mutations has been included in the 2016 revision of the WHO classification of myeloid neoplasms and acute leukemia as a specific molecular diagnostic marker of CNL. Besides CNL, CSF3R somatic mutation have been reported in about 30% of Severe Congenital Neutropenia (SCN) and in a low frequency of other myeloid malignancies (about 4% of CMML, 1.7% of adult AML, 1.9% of children AML), but not in MDS, JMML and ET (Lavallée Et al., Blood 2016; Sano H et al., Br J Haematol 2015). Lavallée et al. reported for the first time that CSF3R mutation are associated with AML with biallelic mutated CEBPA (4/14, 29%) (Lavallée et al., Blood 2016).We aimed to assess the frequency of CSF3R mutation in MDS, AML and ALL patients and the association with other genetic abnormalities.Methods: A retrospective analysis of 974 patients with hematological malignancies, including 297 cases of de novo AML, 144 cases of relapsed AML, 268 cases of de novo ALL, 191 cases of relapsed ALL and 74 cases of MDS patients. 58 genes which common mutated in hematological malignancies were tested by amplicon-based NGS with IonTorrent platform (PGM, Life Technology) and the average sequencing depth was 1000x. 36 leukemia related fusion genes were screened by RT-PCR.Results: We identified CSF3R mutation in 19 patients, which was found in 3.0% (9/297) of de novo AML, 5.6% (8/144) of relapsed AML, 0.5% (1/191) of relapsed ALL and 1.4% (1/74) of MDS. No CSF3R mutation was detected in de novo ALL. All 19 cases had no history of MPN, MDS/MPN or SCN. A total of 8 mutations of CSF3R were detected in 19 patients, 11 cases with T618I mutation, 3 cases with T615A mutation, 1 case with Y779H, Q781*, P809Hfs*8, S810Qfs*6, S813Lfs*2 and D837* mutation respectively, among which 1 patient accompanied with CSF3R T618I and Y779H mutation.In AML cohort, a total of 17 cases with mutant CSF3R, the median age was 20 years (rang, 6-49) and the frequency of CSF3R mutation have no difference between children and adult. Cytomorphology revealed AML M2 (n=11), AML M4 (n=3), AML M5 (n=1), and 1 case have no data available. The median of CSF3R variant allele frequency (VAF) was 25% (3%-49%), 3 cases with VAF <10%, 10 cases with VAF comparable to the concomitant gene mutation with highest VAF. CSF3R mutation was more common as the predominant clone in relapsed AML (87.5% VS 33.3%, P=0.05). The median of concomitant gene mutations was 2 (range 0-3), double-mutated CEBPA (CEBPAdm) was the most frequently concomitant partner of CSF3R mutation (7/17, 41.2% VS 51/382, 13.4%, P <0.05). We also identified 9 cases with AML1-ETO fusion gene which were mutually exclusive with CEBPAdm. In totally, 16 cases of 17 (94.1%) CSF3R mutated AML patient concomitant with core-binding factor gene abnormalities (Fig.1).CSF3R mutation have not been reported in ALL, we identified CSF3R mutation in one patient with B-ALL who was 10 years old and relapsed at 43 months after diagnosed. Bone marrow morphology and immunophenotyping at relapse shows no abnormal neutrophils and myeloid lineage expression were found. No fusion genes were detected. CSF3R T618I and ASXL1 Q803* mutations were detected with VAF of 50% and 51%, respectively, which were confirmed of somatic mutation rather than germline origin. As lack of genetic data at diagnosis, we could not analyze the order of mutation acquisition and clone evolution comprehensively. At the time of reporting, the patients have received allo-HSCT for 4 months and acquired sustained remission. We also detected 1 case of MDS patients (MDS-U) carried U2AF1 S34F, CSF3R Q781* and CBL G413S mutations with VAF of 45%, 32% and 31%, respectively.Due to the limited number of CSF3R mutated cases, we did not perform survival analysis in this study.Conclusion: (1) CSF3R mutation was found in 3.9% of total AML and 3% of de novo AML cohort, rarely seen in MDS and ALL. (2) In AML, CSF3R mutation are prevalent in M2 and M4, and all concomitant with core-binding factor gene abnormalities (CEBPAdm and AML1-ETO fusion gene) and NPM1 mutation which were associated with favorable-risk prognosis. (3) The CSF3R mutation could appeared as predominant clone or subclone in AML and seemly more common as the predominant clone in relapsed AML. As CSF3R mutation also have implications for therapeutic that JAK inhibitors may be efficacious in treating CSF3R-mutant CNL. The implications of prognostic and target therapy (eg. JAK inhibitors) of CSF3R mutation in AML warranting further investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic Impact of Clonal Hematopoiesis in Acute Myeloid Leukemia Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation in Complete Remission

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) is defined as presence of hematologic malignancy-associated mutations, e.g. in the genes DNMT3A, TET2, ASXL1, but absence of hematologic neoplasms & is a frequent phenomenon in healthy individuals with increasing age. Some individuals with CHIP eventually develop acute myeloid leukemia (AML) & persistent clonal hematopoiesis-associated mutations (CH-mutations) in complete remission (CR) may give rise to relapse. To date in AML little is known about the biological & clinical implications of the presence of CH-mutations in CR, especially in the context of allogeneic hematopoietic stem cell transplantation (HSCT).Patients & Methods: We analyzed peripheral blood samples of 113 AML patients (median age 63.6 years, range 31.9-75.8 years) in CR (CR1 61.9%, CR2 14.2%) or CR with incomplete peripheral recovery (CRi; 23.9%) for the presence of CH-mutations by targeted amplicon sequencing (mean amplicon coverage per sample 7205x) prior to HSCT. Genes evaluated for somatic CH-mutations based on database entries (dbSNP & COSMIC) & with a variant allele frequency (VAF) of ≥3% were DNMT3A, TET2, ASXL1, SRSF2, SF3B1, IDH1, IDH2, JAK2, PPM1D, &IKZF1 . Codon 646 ASXL1 mutations were validated using proof-reading polymerase Sanger sequencing. For 76 patients (67.3%) diagnostic bone marrow samples were available for applying a targeted amplicon panel comprising 54 recurrently mutated genes in myeloid malignancies (mean amplicon coverage per sample 8624x). At diagnosis, mutations in the genes NPM1, CEBPA, presence of FLT3 -ITD, & cytogenetics were determined using standard techniques. All patients received HSCT after non-myeloablative conditioning at our institution between 2001 & 2015. Samples were collected within 30 days before HSCT. Median follow-up for patients alive was 4.4 years after HSCT.Results: We identified 70 CH-mutations present in CR/CRi in 48 AML patients (42.5%) with a mean VAF of 19.1% (58.6% of mutations with VAF>10%). The genes DNMT3A (31.4%), TET2 (28.6%) &ASXL1 (14.3%) were found mutated most frequently. We observed no associations between present CH-mutations in CR/CRi & clinical characteristics, except that patients with ≥1 CH-mutation were more often transplanted in CR2 compared to patients without CH-mutation by trend (20.8% vs 9.2%, P=0.10). The presence of ≥1 CH-mutation in CR/CRi did not impact leukemia-free survival (LFS; P=0.95, Figure 1A) or OS (P=0.37, Figure 1B), whereas patients with ≥2 CH-mutations had longer LFS (P=0.02, Figure 1C) & OS (P=0.007, Figure 1D) compared to patients with no or 1 CH-mutation. In CR/CRi DNMT3A mutations did not impact LFS (P=0.73) or OS (P=0.71), the presence of TET2 mutations did not influence LFS (P=0.25) but associated with longer OS (P=0.06) &ASXL1 mutations associated with longer LFS (P=0.11) & OS (P=0.13) by trend. At diagnosis, we identified 83 CH-mutations in 56/76 AML patients (73.7%) of which 29 (34.9%) persisted in 19/76 patients (25.0%) in CR/CRi. Mutations in the genes JAK2 (80.0%), SRSF2 (57.1%) &DNMT3A (50.0%) were most likely to persist. We identified 9 CH-mutations (3 DNMT3A, 3 TET2, 2 ASXL1, 1 SF3B1 mutation) in CR/CRi in 8 patients (10.5%), which were not detectable at diagnosis, possibly following the expansion of subclones under chemotherapy selection pressure. Whether patients had persistent, newly detected or lost CH-mutation in CR/CRi did not impact LFS (P=0.22) or OS (P=0.83).Conclusion: In AML patients in CR/CRi CH-mutations are frequently present (42.5%) & show high VAFs (mean 19.1%) indicating presence of clonal hematopoiesis. Compared to diagnosis 34.9% of CH-mutations persisted in CR/CRi. 10.5% of patients had CH-mutations only detectable in CR/CRi prior to HSCT. While we did not observe a negative prognostic impact of presence of ≥1 CH-mutation in CR/CRi, the presence of distinct mutations (TET2, ASXL1) may positively influence outcome. Patients with ≥2 CH-mutations had longer LFS & OS. This finding may be explained by an increased immunogenic potential with higher number of CH-mutations leading to potent graft vs leukemia effects in the HSCT context. [Display omitted] DisclosuresLange:Novartis: Honoraria, Research Funding. Franke:Takeda: Consultancy; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Schwind:Novartis: Consultancy.

Effect of DNMT3A variant allele frequency and double mutation on clinicopathologic features of patients with de novo AML

The clinicopathologic features of DNA methyltransferase 3A (DNMT3A)-mutated de novo acute myeloid leukemia (AML), and the significance of variant type, variant allele frequency (VAF), and multiple concomitant DNMT3A mutations, remain poorly defined. We examined 104 DNMT3A-mutated de novo AML patients from 2 major centers. Most (82%) had normal karyotype (NK); R882H variants were frequent(38%). The most commonly comutated genes included nucleophosmin (NPM1; 53%), Fms-related tyrosine kinase 3 (FLT3)–internal tandem duplication (25%), IDH1 (23%), IDH2 (23%), and TET2 (21%). Patients with high DNMT3A VAF at diagnosis (≥44%; DNMT3AHIGH) had more significant leukocytosis and higher blast counts in peripheral blood and bone marrow. DNMT3AHIGH cases were associated with much shorter event-free survival (EFS; 14.1 vs 56.8 months) and overall survival (OS; 18.3 months vs not reached) compared with cases of patients with low DNMT3A (DNMT3ALOW). Thirteen patients had 2 DNMT3A variants and similar VAFs at diagnosis that tracked together at multiple time points after chemotherapy and/or stem cell transplantation (SCT). In multivariable analyses performed in NK patients who received standard induction chemotherapy, presence of 2 DNMT3A mutations (hazard ratio [HR] = 3.192; P = .038) and SCT in first complete remission (HR = 0.295; P = .001) independently affected EFS; increasing marrow blast percentage (HR = 1.026; P = .025), high DNMT3A VAF (HR = 3.003; P = .010), and 2 DNMT3A mutations (HR = 4.816; P = .020) had independent effects on OS. These data support the adverse prognostic significance of DNMT3AHIGH reveal a novel association between 2 concomitant DNMT3A mutations and inferior outcome in DNMT3A-mutated de novo AML with a NK.

Association of cell-free DNA dominant clone allele frequency with poor outcomes in advanced biliary cancers treated with platinum-based chemotherapy.

4079 Background: Cell-free circulating tumor DNA (ctDNA) holds significant promise and is being used for clinical decision making in multiple tumors. This study aimed to evaluate the prognostic value of pre-treatment ctDNA in metastatic biliary tract cancers (BTC) treated with platinum based first-line chemotherapy treatment. Methods: We performed a retrospective analysis of 67 patients who underwent ctDNA testing before platinum-based chemotherapy for first-line treatment for metastatic BTC. For analysis we considered the detected gene with highest variant allele frequency (VAF). Results of ctDNA analysis were correlated with patients’ demographics, progression-free survival (PFS) and overall survival (OS). Results: The median age of patients was 67 y/o (27-90). 54 (80.6%) of 67 patients evaluated had intrahepatic cholangiocarcinoma; seven had extrahepatic cholangiocarcinoma and six gallbladder cancer. 46 (68.6%) of the patients were treated with cisplatin plus gemcitabine, 14 (21%) patients received gemcitabine and other platinum (carboplatin or oxaliplatin) combinations while 7 (10.4%) patients were treated on a clinical trial with gemcitabine and cisplatin plus additional targeted agents (CX4945 or PEGPH20). TP53, KRAS, APC, FGFR2 and IDH1 were the genes with highest frequency as dominant clone. The median dominant clone allele frequency (DCAF) was 3% (0-97%). DCAF &gt;3% was associated with statistically significant worse PFS (median PFS: 4.05 vs. 7.70 months, p=0.046) and OS (median OS: 10.8 vs. 18.8 months, p=0.056). Each 1% increase in DCAF is associated with a hazard ratio of 26.21 in OS when adjusting for subtypes, age, treatment type, and CA19-9 [Table]. Conclusions: Patients with metastatic BTC with DCAF &gt; 3% at diagnosis have worse PFS and OS compared to patients with low ctDNA when treated with upfront platinum-based chemotherapy.[Table: see text]

Safety and efficacy of neoadjuvant pembrolizumab in mismatch repair deficient localized/locally advanced solid tumors.

2520 Background: Pembrolizumab (Pembro), anti-PD1 therapy, is FDA approved for refractory microsatellite instability high (MSI-H)/deficient mismatch repair (dMMR) advanced/metastatic solid tumors. The robust activity of anti-PD1 therapy in these tumors argues for a neoadjuvant organ-sparing approach. However, the role of anti-PD1 monotherapy in the neoadjuvant setting is unknown. Methods: This is a phase 2 open-label, single center trial (NCT04082572) of MSI-H/dMMR non-metastatic solid tumors with localized unresectable or high risk resectable (defined as ≥ 20% recurrence) with measurable disease per RECISTv1.1 and ECOG PS 0/1. Treatment is Pembro 200mg every 3 wks for 8 cycles (6 months) followed by surgical resection with option to continue therapy for 18 cycles (12 months) followed by observation. First restaging is at 6 wks and includes baseline and 3-week 70-gene ctDNA assessment. To continue on study, patients are required to have PR/CR, SD with tumor shrinkage or SD with decline in ctDNA [highest variant allele frequency (VAF) baseline mutation]. The co-primary endpoints are safety and pathological complete response (pCR). Key secondary endpoints are response rate and organ-sparing at one year for patients who declined surgery. Results: Between 12/2019 and 2/2021, 32 pts were enrolled and treated. Enrolment goal of 35 anticipated to be met by 4/2021. Baseline characteristics included 13 females, median age of 63 yrs (range 26 - 91), Lynch syndrome in 12 pts, BRAF V600E mutation in 11 pts. Tumor type included 24 CRC and 8 non-CRC (1 endometrial, 1 gastric, 1 meningeal, 2 duodenal, 1 ampullary, 2 pancreatic). At baseline disease was resectable in 23 (72%). Among 30 evaluable pts, best overall response rate was 77%: 30% CR (n = 9), 47% PR (n = 14), 20% SD (n = 6), 3% PD (n = 1). Only one pt progressed after initial SD of -18%. Median follow-up is 6.1 months (range 0.1 - 14). Among the 6 (20%) pts who underwent surgery, pCR was seen in 3 (50%). A non-operative approach (pembro for 12 months) has been chosen in 15 pts and 1-year organ-sparing was seen in 2/2 evaluable pts. Treatment-related grade 3/4 immune adverse events (TRAE) were seen in 3 (9%) pts: grade 3 immune hepatitis (2) and grade 3 type 1 diabetes (1). Baseline ctDNA was positive in 17 (53%) pts with a median of 4 mutations per pt (1 - 35) and median highest VAF of 0.9% (range 0.3% to 38.2%). Among 26 pts with successful tumor tissue testing, median tumor mutations were 10.5, range 1 to 21 (Oncomine 134 gene panel). ctDNA decline at 3 weeks was seen in 14/17 (82%) patients. Luminal disease was present in 24 pts with endoscopic response of: CR in 13 (54%), major response 1, pending follow-up evaluation 6, not evaluated 3, and no response in 1. Conclusions: Neoadjuvant pembrolizumab is safe with encouraging clinical activity and this data suggests that a non-operative management for dMMR/MSI-H localized solid tumors should receive further investigation. Clinical trial information: NCT04082572.

The AIM-HN Study: A pivotal study evaluating the efficacy of tipifarnib in patients with recurrent or metastatic head and neck squamous cell carcinoma with HRAS mutations.

TPS6087 Background: Head and neck squamous cell carcinoma (HNSCC) accounts for more than 830,000 new cancer cases each year worldwide. The prognosis for recurrent and/or metastatic (R/M) HNSCC patients remains poor with an estimated median overall survival (mOS) of 7-15 months in the first line setting and 5-8 months in the second line setting and beyond. Approximately 4-8% of HNSCC tumors are driven by gain-of-function mutations in the HRAS (m HRAS) proto-oncogene. Tipifarnib is a potent and selective farnesyltransferase inhibitor that disrupts HRAS function by blocking required protein membrane localization, and subsequent cellular growth and survival. Data from a prior phase 2 study (RUN-HN; NCT02383927) of tipifarnib in R/M m HRAS HNSCC patients in the second line plus setting demonstrated encouraging efficacy, with an objective response rate (ORR) of 55% and mOS of 15.4 months for patients with mHRAS variant allele frequency (VAF) ≥ 20%, providing support for pursuing a pivotal trial in this patient population. Methods: AIM-HN (NCT03719690) is a global, open-label single-arm pivotal study evaluating the efficacy and tolerability of tipifarnib in second line plus R/M m HRAS HNSCC patients. The primary objective is to determine the ORR in patients with a m HRAS VAF ≥ 20% (High VAF population), as assessed using RECIST v1.1 by Independent Review Facility. Key secondary objectives include the ORR for patients of all VAF levels, and the duration of responses for both VAF≥ 20% and all VAF levels. Key inclusion criteria include: histologically confirmed head and neck cancer of squamous histology not amenable to local therapy with curative intent; known tumor missense HRAS mutation (with VAF determined and available) detected by Next Generation Sequencing; ECOG performance status of 0-1; measurable disease by RECIST v1.1; and adequate organ function. Key exclusion criteria include: salivary gland, thyroid, (primary) cutaneous squamous or non-squamous histologies; intolerable Grade 2 or ≥ Grade 3 neuropathy or unstable neurological symptoms within 4 weeks of Cycle 1 Day 1; or active, uncontrolled infections requiring systemic therapy. Tipifarnib is administered at a dose of 600 mg, orally with a meal twice a day for 7 days in alternating weeks (Days 1-7 and 15-21) of 28-day cycles until discontinuation criteria are met. All patients are being followed for safety through the End of Treatment visit, roughly 30 days after treatment discontinuation or immediately before the administration of another anticancer treatment, whichever occurs first. Upon therapy discontinuation, all patients are being followed approximately every 12 weeks for survival status, and the use of subsequent therapy. The IDMB last reviewed data in October 2020 and recommended the trial continue as planned. AIM-HN is continuing to enroll patients globally. Ho et al, JCO, accepted. Clinical trial information: NCT03719690.

High BRAF variant allele frequencies are associated with distinct pathological features and responsiveness to target therapy in melanoma patients

BackgroundBRAF mutant melanoma patients are commonly treated with anti-BRAF therapeutic strategies. However, many factors, including the percentage of BRAF-mutated cells, may contribute to the great variability in patient outcomes.Patients and methodsThe BRAF variant allele frequency (VAF; defined as the percentage of mutated alleles) of primary and secondary melanoma lesions, obtained from 327 patients with different disease stages, was assessed by pyrosequencing. The BRAF mutation rate and VAF were then correlated with melanoma pathological features and patients’ clinical characteristics. Kaplan–Meier curves were used to study the correlations between BRAF VAF, overall survival (OS), and progression-free survival (PFS) in a subset of 62 patients treated by anti-BRAF/anti-MEK therapy after metastatic progression.ResultsA highly heterogeneous BRAF VAF was identified (3%-90%). Besides being correlated with age, a higher BRAF VAF level was related to moderate lymphocytic infiltration (P = 0.017), to melanoma thickness according to Clark levels, (level V versus III, P = 0.004; level V versus IV, P = 0.04), to lymph node metastases rather than cutaneous (P = 0.04) or visceral (P = 0.03) secondary lesions. In particular, a BRAF VAF >25% was significantly associated with a favorable outcome in patients treated with the combination of anti-BRAF/anti-MEK drug (OS P = 0.04; PFS P = 0.019), retaining a significant value as an independent factor for the OS and the PFS in the multivariate analysis (P = 0.014 and P = 0.003, respectively).ConclusionThese results definitively support the role of the BRAF VAF as a potential prognostic and predictive biomarker in melanoma patients in the context of BRAF inhibition.