Rapid detection of mutations in CSF-cfTNA with the Genexus Integrated Sequencer.

PURPOSE: Genomic alterations are fundamental for molecular-guided therapy in patients with breast and lung cancer. However, the turn-around time of standard next-generation sequencing assays is a limiting factor in the timely delivery of genomic information for clinical decision-making. METHODS: In this study, we evaluated genomic alterations in 54 cerebrospinal fluid samples from 33 patients with metastatic lung cancer and metastatic breast cancer to the brain using the Oncomine Precision Assay on the Genexus sequencer. There were nine patients with samples collected at multiple time points. RESULTS: Cell-free total nucleic acids (cfTNA) were extracted from CSF (0.1-11.2 ng/µl). Median base coverage was 31,963× with cfDNA input ranging from 2 to 20 ng. Mutations were detected in 30/54 CSF samples. Nineteen (19/24) samples with no mutations detected had suboptimal DNA input (< 20 ng). The EGFR exon-19 deletion and PIK3CA mutations were detected in two patients with increasing mutant allele fraction over time, highlighting the potential of CSF-cfTNA analysis for monitoring patients. Moreover, the EGFR T790M mutation was detected in one patient with prior EGFR inhibitor treatment. Additionally, ESR1 D538G and ESR1::CCDC170 alterations, associated with endocrine therapy resistance, were detected in 2 mBC patients. The average TAT from cfTNA-to-results was < 24 h. CONCLUSION: In summary, our results indicate that CSF-cfTNA analysis with the Genexus-OPA can provide clinically relevant information in patients with brain metastases with short TAT.

Mutation-Agnostic Detection of Colorectal Cancer Using Liquid Biopsy-Based Methylation-Specific Signatures.

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 (n = 20) and non-colorectal cancers (n = 8); and healthy volunteers (n = 4). Median methylation ratio was higher in colorectal cancer compared with non-colorectal cancers (P = .001) and normal donors (P = .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; P = .027).

Clinicopathologic spectrum of myeloid neoplasms with concurrent myeloproliferative neoplasm driver mutations and SRSF2 mutations.

Myeloproliferative neoplasms (MPNs) are frequently associated with classic driver mutations involving JAK2, MPL or CALR. SRSF2 is among the most frequently mutated splicing genes in myeloid neoplasms and SRSF2 mutations are known to confer a poor prognosis in patients with MPNs. In this study, we sought to evaluate the clinicopathologic spectrum of myeloid neoplasms harboring concurrent MPN-driver mutations and SRSF2 mutations. The study cohort included 27 patients, 22 (82%) men and five (19%) women, with a median age of 71 years (range, 51-84). These patients presented commonly with organomegaly (n = 15; 56%), monocytosis (n = 13; 48%), morphologic dysplasia (n = 11; 41%), megakaryocytic hyperplasia and/or clustering (n = 10; 37%) and bone marrow fibrosis >MF-1 (17/22; 77%). About one third of patients either initially presented with acute myeloid leukemia (AML) or eventually progressed to AML. Eighteen (68%) patients had a dominant clone with SRSF2 mutation and nine (33%) patients had a dominant clone with a classic MPN-associated driver mutation. Our data suggest that the presence of an SRSF2 mutation preceding the acquisition of a MPN driver mutations is not a disease-defining alteration nor is it restricted to any specific disease entity within the spectrum of myeloid neoplasms. In summary, patients with myeloid neoplasms associated with concurrent SRSF2 and classic MPN driver mutations have clinical and morphologic features close to that of classic MPNs often with frequent dysplasia and monocytosis.

Long-term results of frontline dasatinib in chronic myeloid leukemia.

BACKGROUND: Dasatinib is a second-generation tyrosine kinase inhibitor that, when used as frontline therapy, produces more and faster cytogenetic and molecular responses compared with imatinib. The authors report the long-term follow-up from the first study using dasatinib as initial therapy for chronic-phase chronic myeloid leukemia. METHODS: Between November 2005 and August 2014, patients were randomly assigned to receive 100 mg daily or 50 mg twice daily. After June 2009, all patients started with 100 mg daily. RESULTS: With a median follow-up of 6.5 years, 94 of 149 treated patients (63%) were still receiving dasatinib on study. The median patient age was 48 years (interquartile range, 37-55 years), and 9% of patients had a high risk Sokal risk score. The cumulative complete cytogenetic response rate at 11 years was 92.6%, the major molecular response (MR) rate was 88.2%, and the MR4.5 rate (indicating a ≥4.5-log reduction in BCR-ABL1 transcripts) was 79.5%. The median time to a major MR and MR4.5 was 6 and 23 months, respectively. A sustained MR4.5 (≥2 years) was achieved in 82 patients (55%). The 10-year overall survival, transformation-free survival, event-free survival, and failure-free survival rates were 89%, 95%, 86%, and 65%, respectively. Univariate analysis showed that the achievement of a complete MR was associated with improved overall survival. The most common reasons for treatment discontinuation were toxicity and elective discontinuation. The most common treatment-emergent grade 3 and 4 adverse events were fatigue, thrombocytopenia, and infections. CONCLUSIONS: After this long-term follow-up, dasatinib continues to show an excellent safety profile and produces rapid cytogenetic responses and MRs, durable deep MRs, and excellent long-term survival outcomes in patients with chronic-phase chronic myeloid leukemia.

Clinico-pathologic characteristics and outcomes of the World Health Organization (WHO) provisional entity de novo acute myeloid leukemia with mutated RUNX1.

We studied the characteristics of the provisional category de novo acute myeloid leukemia (AML) with mutated RUNX1 (AML-RUNX1mut) proposed by the World Health Organization (WHO). Until now, most published studies have combined de novo and secondary AML-RUNX1mut. We compared the clinicopathologic characteristics and outcomes of WHO-defined de novo AML-RUNX1mut with de novo AML without RUNX1 alterations (AML-RUNX1wt). We performed sequential NGS to assess RUNX1 mutation stability over disease course. We identified 46 de novo AML-RUNX1mut patients [32 (70%) men, 14 (30%) women; median age, 66.5 years] with 54 RUNX1 mutations [median VAF, 32% (2-97%)]. Point mutations clustered within the runt-homology-domain and frame-shift mutations within the transactivation domain. Compared with AML-RUNX1wt, AML-RUNX1mut showed male predominance (p = 0.02), higher frequency of SRSF2 (p = 0.02), and ASXL1 (p = 0.0004) mutations and normal karyotype (p = 0.01), and absent NPM1 mutations (p = 0.0002). De novo AML-RUNX1mut showed no significant difference in overall survival (OS) compared with AML-RUNX1wt (median: 26 vs. 32 months) (p = 0.71). AML-RUNX1mut with clonal RUNX1 mutation (≥20% VAF) had shorter OS than subclonal <20% VAF (23 months vs. undefined; p = 0.04). However, the difference was not significant when compared with AML-RUNX1wt (23 vs. 32 months; p = 0.23). No significant OS difference was noted between de novo AML-RUNX1mut and AML-NOS-RUNX1wt. By sequential multigene mutation profiling, RUNX1 mutation disappeared at relapse in one of ten patients. Overall, the findings support separate categorization of this entity. However, there is no significant outcome difference compared with AML-RUNX1wt.

A phase 2 study of ruxolitinib in combination with azacitidine in patients with myelofibrosis.

Ruxolitinib (RUX)-based combinations may provide benefit for patients with myelofibrosis (MF). In this open-label, nonrandomized, prospective phase 2 study, patients with MF initially received RUX twice per day continuously in 28-day cycles for the first 3 cycles. Azacitidine (AZA) 25 mg/m2 (days 1-5) was added starting with cycle 4 and could be subsequently increased to 75 mg/m2 (days 1-5). Forty-six patients were enrolled with a median follow-up of 28 months (range, 4-50+ months). An International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) response was achieved in 33 patients (72%), with a median time to response of 1.8 months (range, 0.7-19.0 months). One-fourth (7 of 33) of the IWG-MRT responses occurred after the addition of AZA. A reduction of >50% in palpable spleen length at 24 weeks and at any time on the study was achieved in 62% and 71% of the evaluable patients, respectively. Among patients who achieved a >50% reduction in spleen length at 24 weeks, 95% had maintained it at 48 weeks. Notably, improvements in bone marrow reticulin fibrosis grade occurred in 57% of the patients at 24 months. Treatment discontinuations as a result of drug-related toxicities occurred in 4 patients (9%), all as a result of cytopenias. New onset grade 3 to 4 anemia and thrombocytopenia occurred in 35% and 26% of patients, respectively. RUX and AZA were safe, with encouraging spleen response rates and improvement in bone marrow fibrosis in patients with MF. This trial was registered at www.clinicaltrials.gov as #NCT01787487.

Simplified molecular classification of lung adenocarcinomas based on EGFR, KRAS, and TP53 mutations.

BACKGROUND: Gene expression profiling has consistently identified three molecular subtypes of lung adenocarcinoma that have prognostic implications. To facilitate stratification of patients with this disease into similar molecular subtypes, we developed and validated a simple, mutually exclusive classification. METHODS: Mutational status of EGFR, KRAS, and TP53 was used to define seven mutually exclusive molecular subtypes. A development cohort of 283 cytology specimens of lung adenocarcinoma was used to evaluate the associations between the proposed classification and clinicopathologic variables including demographic characteristics, smoking history, fluorescence in situ hybridization and molecular results. For validation and prognostic assessment, 63 of the 283 cytology specimens with available survival data were combined with a separate cohort of 428 surgical pathology specimens of lung adenocarcinoma. RESULTS: The proposed classification yielded significant associations between these molecular subtypes and clinical and prognostic features. We found better overall survival in patients who underwent surgery and had tumors enriched for EGFR mutations. Worse overall survival was associated with older age, stage IV disease, and tumors with co-mutations in KRAS and TP53. Interestingly, neither chemotherapy nor radiation therapy showed benefit to overall survival. CONCLUSIONS: The mutational status of EGFR, KRAS, and TP53 can be used to easily classify lung adenocarcinoma patients into seven subtypes that show a relationship with prognosis, especially in patients who underwent surgery, and these subtypes are similar to classifications based on more complex genomic methods reported previously.

A Cryptic BCR-PDGFRB Fusion Resulting in a Chronic Myeloid Neoplasm With Monocytosis and Eosinophilia: A Novel Finding With Treatment Implications.

RNA-seq was used to identify the partner gene and confirm the presence of a BCR-PDGFRB fusion. Identification of this fusion product resulted in successful treatment and long-term remission of this myeloid neoplasm. Based on our results, we suggest that despite current WHO recommendations, screening for PDGFRB rearrangement in cases of leukocytosis with eosinophilia and no other etiologic explanation is necessary, even if the karyotype is normal.

Targeted multigene deep sequencing of Bruton tyrosine kinase inhibitor-resistant chronic lymphocytic leukemia with disease progression and Richter transformation.

BACKGROUND: In a proportion of patients with chronic lymphocytic leukemia (CLL), resistance to Bruton tyrosine kinase (BTK) inhibitors (BTKi) is attributed to acquired BTK/phospholipase C gamma 2 (PLCG2) mutations. However, knowledge regarding additional genetic lesions associated with BTK/PLCG2 mutations, and gene mutations in patients lacking BTK/PLCG2 mutations, is limited. METHODS: Using targeted deep sequencing, mutations in 29 genes associated with CLL and/or the BCR signaling pathway were assessed in patients with CLL who developed resistance to BTK inhibition with ibrutinib/acalabrutinib at a single institution. RESULTS: The study group included 29 patients with BTKi-resistant CLL, 23 patients with disease progression, and 6 patients with Richter transformation (RT). The median times to disease progression and RT were 33.3 months and 13.3 months, respectively. In 11 patients, sequencing was possible at both baseline (prior to treatment with BTKi) and at time of disease progression/RT. Of these patients, 4 demonstrated BTK mutations at the time of disease progression/RT; patients without BTK mutations frequently acquired mutations associated with disease progression/RT in TP53, SF3B1, and CARD11, whereas additional mutations were rare in patients with BTK-mutated CLL. Sequencing of all 29 patients at the time of disease progression/RT identified BTK mutations at a frequency of 66%, including a novel V537I mutation. Among patients with disease progression, BTK mutations were observed in 16 patients (70%). The median time to disease progression was shorter in patients without BTK mutations compared with those with BTK-mutated CLL. Among patients with RT, SF3B1 mutations were more frequent than BTK mutations (67% vs 50%). Following BTKi discontinuation, we sequential mutation analysis was performed in 2 patients with RT and 3 patients with disease progression in the setting of persistent disease. Both patients with RT demonstrated disappearance of BTK and expansion of TP53 mutations. All 3 patients with disease progression received venetoclax and demonstrated suppression of BTK mutations. CONCLUSIONS: Longitudinal, targeted, multigene deep sequencing is informative for the clinical monitoring of mutational evolution in patients with CLL who are receiving BTKi.

Routine sequencing in CLL has prognostic implications and provides new insight into pathogenesis and targeted treatments.

Chronic lymphocytic leukaemia (CLL) is a genetically heterogeneous disease characterised by genomic alterations and gene mutations that may portend worse survival or resistance to treatments. A total of 680 blood or bone marrow samples underwent targeted sequencing of 29 genes previously identified as being mutated in CLL, which were correlated to known prognostic clinical characteristics. Overall, 400 (59%) patients were treatment-naïve (TN) and 280 (41%) were relapsed/refractory (R/R). Most patients (70%) had ≥1 mutation, with TP53 (22%), SF3B1 (18%), NOTCH1 (13%) and ATM (13%) being the most commonly mutated genes. A higher proportion of R/R patients had mutations in SF3B1 (P = 0·01) and TP53 (P < 0·001). Patients with mutated IGHV CLL more often had mutations in KLHL6 (P = 0·001) and MYD88 (P < 0·001). Pairwise associations showed mutational co-occurrences in the TN group including SF3B1/ATM [false discovery rate (FDR) < 0·05] and NOTCH1/POT1 (FDR < 0·01). Recurrent mutations resulting in premature truncation prior to the ubiquitination domains of NOTCH1 in its PEST domain and BIRC3 in its RING domain can produce proteins that constitutively activate CLL. Frequent missense mutations, such as K700E in SF3B1 and E571K in XPO1, have unknown function but are most likely to be activating mutations. Future directions include using these mutations to identify pathways for therapeutic targeting and rational drug design.

Association of gene mutations with time-to-first treatment in 384 treatment-naive chronic lymphocytic leukaemia patients.

This study correlated somatic mutation results and known prognostic factors with time-to-first treatment (TTFT) in 384 treatment-naïve (TN) chronic lymphocytic leukaemia (CLL) patients to help determine disease-specific drivers of early untreated CLL. CLL DNA from either peripheral blood or bone marrow underwent next generation targeted sequencing with a 29-gene panel. Gene mutation data and concurrent clinical characteristics, such as Rai/Binet stage, fluorescence in situ hybridisation (FISH), ZAP70/CD38, karyotype and IGHV mutation, status were analysed in univariable and multivariable analyses to identify associations with TTFT. TTFT was defined as time from diagnosis to initial treatment. In univariable analyses, mutated ATM (P < 0·001), NOTCH1 (P < 0·001) and SF3B1 (P = 0·002) as well as unmutated IGHV (P < 0·001), del(11q) (P < 0·001) and trisomy 12 (P < 0·001) by hierarchal FISH and advanced Rai (P = 0·05) and Binet (P < 0·001) stages were associated with shorter TTFT. Importantly, del(17p), mutated TP53 and complex karyotype were not associated with shorter TTFT. In a reduced multivariable analysis, mutated ATM (P < 0·001) and unmutated IGHV status (P < 0·001) remained significant, showing their importance in early leukaemogenesis. High-risk prognostic markers such as del(17p), mutated TP53 and complex karyotype, were not correlated with TTFT, suggesting that these abnormalities have limited roles in early disease progression but are more important in relapsed CLL.

Targeted next-generation sequencing of endometrial cancer and matched circulating tumor DNA: identification of plasma-based, tumor-associated mutations in early stage patients.

There is currently no blood-based marker in routine use for endometrial cancer patients. Such a marker could potentially be used for early detection, but it could also help to track tumor recurrence following hysterectomy. This is important, as extra-vaginal recurrence of endometrial endometrioid adenocarcinoma is usually incurable. This proof-of-principle study was designed to determine if tumor-associated mutations could be detected in cell-free DNA from the peripheral blood of early and late stage endometrial endometrioid carcinoma patients. Approximately 90% of endometrioid carcinomas have at least one mutation in the genes CTNNB1, KRAS, PTEN, or PIK3CA. Using a custom panel targeting 30 hotspot amplicons in these four genes, next-generation sequencing was performed on cell-free DNA extracted from plasma obtained from a peripheral blood draw at the time of hysterectomy and the matching tumor DNA from 48 patients with endometrioid endometrial carcinomas. At least one mutation in the tumor was detected in 45/48 (94%) of patients. Fifteen of 45 patients (33%) had a mutation in the plasma that matched a mutation in the tumor. These same mutations were not detected in the matched negative control buffy coat. Presence of a plasma mutation was significantly associated with advanced stage at hysterectomy, deep myometrial invasion, lymphatic/vascular invasion, and primary tumor size. Detecting a plasma-based mutation was independent of the amount of cell-free DNA isolated from the plasma. Overall, 18% of early stage patients had a mutation detected in the plasma. These results demonstrate that mutations in genes relevant to endometrial cancer can be identified in the peripheral blood of patients at the time of surgery. Future studies can help to determine the post-operative time course of mutation clearance from the peripheral blood and if mutation re-emergence is predictive of recurrence.

Clonal evolution and outcomes in myelofibrosis after ruxolitinib discontinuation.

Despite significant improvements in the signs and symptoms of myelofibrosis (MF), and possible prolongation of patients' survival, some have disease that is refractory to ruxolitinib and many lose their response over time. Furthermore, patients with ≥3 mutations are less likely to respond to ruxolitinib. Here we describe outcomes after ruxolitinib discontinuation in MF patients enrolled in a phase 1/2 study at our center. After a median follow-up of 79 months, 86 patients had discontinued ruxolitinib (30 of whom died while on therapy). The median follow-up after ruxolitinib discontinuation for the remaining 56 patients was 32 months, with median survival after discontinuation of 14 months. Platelets <260 × 109/L at the start of therapy or <100 × 109/L at the time of discontinuation were associated with shorter survival after discontinuation. Of 62 patients with molecular data at baseline and follow-up, 22 (35%) acquired a new mutation while receiving ruxolitinib (14 [61%] in ASXL1). Patients showing clonal evolution had significantly shorter survival after discontinuation (6 vs 16 months). Transfusion dependency was the only clinical variable associated with clonal evolution. These findings underscore the need for novel therapies and suggest that clonal evolution or decreasing platelet counts while on ruxolitinib therapy may be markers of poor prognosis.

Long-term outcome of acute promyelocytic leukemia treated with all-trans-retinoic acid, arsenic trioxide, and gemtuzumab.

The combination of all-trans-retinoic acid (ATRA) plus arsenic trioxide (ATO) has been shown to be superior to ATRA plus chemotherapy in the treatment of standard-risk patients with newly diagnosed acute promyelocytic leukemia (APL). A recent study demonstrated the efficacy of this regimen with added gemtuzumab ozogamicin (GO) in high-risk patients. We examined the long-term outcome of patients with newly diagnosed APL treated at our institution on 3 consecutive prospective clinical trials, using the combination of ATRA and ATO, with or without GO. For induction, all patients received ATRA (45 mg/m2 daily) and ATO (0.15 mg/kg daily) with a dose of GO (9 mg/m2 on day 1) added to high-risk patients (white blood cell count, >10 × 109/L), as well as low-risk patients who experienced leukocytosis during induction. Once in complete remission, patients received 4 cycles of ATRA plus ATO consolidation. One hundred eighty-seven patients, including 54 with high-risk and 133 with low-risk disease, have been treated. The complete remission rate was 96% (52 of 54 in high-risk and 127 of 133 in low-risk patients). Induction mortality was 4%, with only 7 relapses. Among low-risk patients, 60 patients (45%) required either GO or idarubicin for leukocytosis. Median duration of follow-up was 47.6 months. The 5-year event-free, disease-free, and overall survival rates are 85%, 96%, and 88%, respectively. Late hematological relapses beyond 1 year occurred in 3 patients. Fourteen deaths occurred beyond 1 year; 12 were related to other causes. This study confirms the durability of responses with this regimen.

Persistent IDH1/2 mutations in remission can predict relapse in patients with acute myeloid leukemia.

Persistence of IDH1 or IDH2 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 IDH1 R132 or IDH2 R140/R172 mutations and assessed their bone marrow at the time of remission to determine the potential impact of persistent IDH1/2 mutations. Approximately 40% of acute myeloid leukemia patients given standard treatment in this cohort had persistent mutations in IDH1/2 Patients with an IDH1/2 mutation had an increased risk of relapse after 1 year of follow-up compared to patients without a detectable IDH1/2 mutation (59% versus 24%; P<0.01). However, a persistent mutation was not associated with a shorter time to relapse. High IDH1/2 mutation burden (mutant allelic frequency ≥10%) did not correlate with relapse rate (77% versus 86% for patients with a low burden, i.e., mutant allelic frequency <10%; P=0.66). Persistent mutations were also observed in NPM1, DNMT3A and FLT3 during remission, but IDH1/2 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 IDH1/2 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.

DDX41 mutations in myeloid neoplasms are associated with male gender, TP53 mutations and high-risk disease.

Myeloid neoplasms with germline DDX41 mutations have been incorporated into the 2017 WHO classification. Limited studies describing the clinicopathologic features and mutation profile are available. We searched for myeloid neoplasms with a DDX41 gene mutation tested by an 81-gene next-generation sequencing panel over a 7-month period. We identified 34 patients with myeloid neoplasms with DDX41 abnormalities; 26 (76%) men and 8 women (24%) [median age, 70 years], 20 acute myeloid leukemia (AML), 10 myelodysplastic syndrome (MDS), 1 chronic myelomonocytic leukemia (CMML) and 3 myeloproliferative neoplasms (MPN). Fifty-nine DDX41 variants were detected: 27 (46%) appeared somatic and 32 (54%) were presumably germline mutations. The majority of presumed germline mutations were upstream of the Helicase 2 domain (93%) and involved loss of the start codon (30%). The majority of somatic mutations were within the Helicase 2 domain (78%), with the missense mutation p.R525H being most common (67%). There was a significant difference in the location of germline or somatic mutations (P < .0001). Concomitant mutations were detected involving 19 genes, but only TP53 (n = 11, 32%), ASXL1 (n = 8, 24%), and JAK2 (n = 4, 12%) were recurrent. Twenty (59%) patients showed diploid cytogenetics. Twenty-three (68%) patients presented with AML or MDS-EB-2, suggesting an association with high-grade myeloid neoplasm. Patients with myeloid neoplasms carrying DDX41 mutations show male predominance (3:1), higher age at presentation, association with TP53 mutations, and association with high-grade myeloid neoplasms in our cohort at a referral cancer center setting. These findings support the recognition of myeloid neoplasms with DDX41 mutation as unique, need for germline confirmation, and further assessment of family members.

Novel enriched pathways in superficial malignant peripheral nerve sheath tumours and spindle/desmoplastic melanomas.

Superficial malignant peripheral nerve sheath tumour (MPNST) is a rare, soft tissue neoplasm that shares morphological features and some molecular events with spindle/desmoplastic melanoma (SDM). Herein, we sought to identify molecular targets for therapy by using targeted RNA/DNA sequencing and gene expression of key immunological players. DNA and RNA from formalin-fixed paraffin-embedded tissue were extracted and processed. Massive high-throughput deep parallel sequencing was performed with the Oncomine comprehensive panel, enabling detection of relevant single-nucleotide variants, copy number variations, gene fusions and indels for 143 unique genes on the Ion torrent sequencer for clinical trial research programmes. Gene expression analysis was carried out with a customized 770-gene expression panel combining markers for 24 different immune cell types and 30 common cancer antigens, including key checkpoint blockade genes analysed with the Ncounter system. Fifty-one patients (SDM, 16/11; MPNST, 24; male, n = 37; female, n = 16) had sufficient DNA and RNA for testing. NF1 deleterious mutations and/or deep/homozygous deletions were identified in 73% of MPNSTs and 67% of SDMs, with 50% of the mutations involving the RAS-binding domain. Inactivating/deleterious mutations of TSC1/TSC2 were identified in 40% and 41% of MPNSTs and SDMs, respectively. Activating mutations affecting the EGFR-like and the negative regulatory domains of NOTCH1 and KDR (VEGFR2) were identified in 45% and 40% of SDMs and in 30% and 8% of MPNSTs, respectively. Differential gene expression and gene clustering analysis showed significantly perturbed immune pathway components, including nuclear factor-κB (NF-κB), JAK-STAT, and CXCL12-CXCR4, and differentially expressed CD274 and CTLA4, in both SDM and MPNST. Angiogenesis (KDR and NOTCH1) and mammalian target of rapamycin complex (mTORC) pathways offer a rationale for anti-angiogenic and selective mTORC inhibition as treatment strategies for MPNST and SDM. Cytokines and the JAK-STAT, TNF and NF-κB axes were perturbed in both SDM and MPNST. These pathways have been targeted in haematological malignancies and present promising targets for these tumours. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

ATRX protein loss and deregulation of PI3K/AKT pathway is frequent in pilocytic astrocytoma with anaplastic features.

INTRODUCTION: Pilocytic astrocytoma (PA) with anaplastic features (PAAF) is a rare entity associated with decreased survival. It is characterized by hypercellularity, atypia, brisk mitotic activity, variable necrosis, and association with a classic PA component or anaplastic transformation in a recurrent tumor with a previously-documented classic PA. MATERIALS AND METHODS: We present 5 PAAF cases with clinical, radiological, pathological, and molecular correlation. We interrogated ATRX, IDH, TP53, PTEN, EGFR, BRAF, 6q23, p16(Ink4a) by sequencing, FISH, and immunohistochemistry. RESULTS: Four tumors were located in the cerebellum, and 1 was supratentorial. All showed ATRX protein loss by immunohistochemistry, loss of heterozygosity for PTEN, and had no IDH/TP53/BRAF mutations, nor EGFR amplification. Two of 5 tumors showed BRAF duplication by pyrosequencing. All showed loss of PTEN nuclear expression in subsets of tumor cells, which was associated with variable cytoplasmic positivity for pS6. There was a relative correlation between loss of PTEN expression and pS6 cytoplasmic expression. p53 was expressed in ~ 50% of tumor cells in all tumors. P16 was variably lost in all cases. One tumor showed MYB/6q23 deletion. CONCLUSION: We confirm ATRX protein loss suggestive of ATRX alteration as well as dysregulation of the PI3K/AKT pathway and, less often, of the MAPK/ERK pathway in PAAF.
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TP53 mutations are common in mantle cell lymphoma, including the indolent leukemic non-nodal variant.

INTRODUCTION: Mantle cell lymphoma (MCL) is an aggressive B-cell neoplasm, but clinically indolent subtypes are also recognized. Data on the utility of mutation profiling in the context of routine workup and its role in risk-stratification of MCL patients are limited. In this study, we describe the mutational landscape and clinicopathologic correlates of a series of MCL cases at a single-institution setting. METHODS: Samples from 26 patients with MCL were evaluated by NGS using DNA extracted from peripheral blood (PB) or bone marrow (BM). Evaluation of extent of PB or BM involvement was performed using flow cytometry immunophenotyping. RESULTS: The study group included 17 (65%) men and 9 (35%) women with a median age of 65 years (range, 50-94). Twenty-one (81%) patients had nodal MCL (N-MCL) and 5 (19%) had the "leukemic variant" (L-MCL). Mutated genesincluded TP53 (35%), ATM (27%), CARD11 (10%); and FBXW7, NOTCH1, SPEN, BIRC3 (~5% each). Most mutations were clonal in nature. Ten unique TP53 mutations were identified in 9 samples, including 3 L-MCL cases. There was no difference in the frequency of TP53 mutations between L-MCL and N-MCL groups (p = 0.3), but TP53 mutations were subclonal in 2/3 L-MCL cases. Identification of clonal TP53 alterations in L-MCL patients prompted initiation of therapy despite low tumor burden. CONCLUSIONS: TP53 is commonly mutated in MCL. TP53 mutations may be clonal or subclonal. Seemingly indolent L-MCL may harbor subclonal TP53 mutations which may serve as a useful biomarker for prognostication, therapeutic planning, follow-up monitoring, and early detection of clonal expansion.

Improving the detection of patients with inherited predispositions to hematologic malignancies using next-generation sequencing-based leukemia prognostication panels.

Recognizing and referring patients with possible inherited cancer predisposition syndromes for appropriate genetic evaluation and testing provides insights into optimal patient treatment approaches and also can provide education and testing opportunities for family members. Next-generation sequencing (NGS)-based, targeted genotyping for somatic mutations is increasingly used in the diagnosis, prognostication, and treatment selection for patients with hematologic malignancies. However, certain mutations that may be somatically acquired can also be present as germline mutations in some individuals and families. Whether the results of NGS-based leukemia panels can be used to inform decisions and subsequent evaluation of patients with possible inherited cancer predispositions has not been described previously. Because a normal control often is not available when using NGS panels in patients with hematologic malignancies, NGS panel results offer both an opportunity and a challenge to determine the origin and pathogenicity of identified mutations. In the absence of a matched germline control, variant allele frequency (VAF) estimation and data from publically available data sets provide important clues to the possible germline origin of a variant. Careful annotation and review of NGS panels in patients with hematologic malignancies can provide a useful screening tool to systematically improve upon the detection of potentially pathogenic germline variants. Cancer 2018;124:2704-2713. © 2018 American Cancer Society.