Stephan Bartels

Routine clinical mutation profiling using next generation sequencing and a customized gene panel improves diagnostic precision in myeloid neoplasms

Microscopic examination of myelodysplastic syndromes (MDS) and myelodysplastic-myeloproliferative neoplasms (MDS/MPN) may be challenging because morphological features can overlap with those of reactive states. Demonstration of clonal hematopoiesis provides a diagnostic clue and has become possible by comprehensive mutation profiling of a number of frequently mutated genes, some of them with large coding regions. To emphasize the potential benefit of NGS in hematopathology we present sequencing results from routinely processed formalin-fixed and paraffin-embedded (FFPE) bone marrow trephines (n = 192). A customized amplicon-based gene panel including 23 genes frequently mutated in myeloid neoplasms was established and implemented. Thereby, 629,691 reads per sample (range 179,847–1,460,412) and a mean coverage of 2,702 (range 707–6,327) could be obtained, which are sufficient for comprehensive mutational profiling. Seven samples failed in sequencing (3.6%). In 185 samples we found in total 269 pathogenic variants (mean 1.4 variants per patient, range 0-5), 125 Patients exhibit at least one pathogenic mutation (67.6%). Variants show allele frequencies ranging from 6.7% up to 95.7%. Most frequently mutated genes were TET2 (28.7%), SRSF2 (19.5%), ASXL1 (8.6%) and U2AF1 (8.1%). The mutation profiling increases the diagnostic precision and adds prognostic information.

SRSF2 mutation is present in the hypercellular and prefibrotic stage of primary myelofibrosis

To the editor: Myeloproliferative neoplasms (MPNs) are characterized by their propensity to progress from overproduction of 1 or more hematopoietic lineages to states of cytopenia associated with myelofibrosis and/or blast excess. Recently, it was demonstrated that in progressed primary

Comprehensive Molecular Profiling of Archival Bone Marrow Trephines Using a Commercially Available Leukemia Panel and Semiconductor-Based Targeted Resequencing

Comprehensive mutation profiling becomes more and more important in hematopathology complementing morphological and immunohistochemical evaluation of fixed, decalcified and embedded bone marrow biopsies for diagnostic, prognostic and also predictive purposes. However, the number and the size of relevant genes leave conventional Sanger sequencing impracticable in terms of costs, required input DNA, and turnaround time. Since most published protocols and commercially available reagents for targeted resequencing of gene panels are established and validated for the analysis of fresh bone marrow aspirate or peripheral blood it remains to be proven whether the available technology can be transferred to the analysis of archival trephines. Therefore, the performance of the recently available Ion AmpliSeq AML Research panel (LifeTechnologies) was evaluated for the analysis of fragmented DNA extracted from archival bone marrow trephines. Taking fresh aspirate as gold standard all clinically relevant mutations (n = 17) as well as 25 well-annotated SNPs could be identified reliably with high quality in the corresponding archival trephines of the training set (n = 10). Pre-treatment of the extracted DNA with Uracil-DNA-Glycosylase reduced the number of low level artificial sequence variants by more than 60%, vastly reducing time required for proper evaluation of the sequencing results. Subsequently, randomly picked FFPE samples (n = 41) were analyzed to evaluate sequencing performance under routine conditions. Thereby all known mutations (n = 43) could be verified and 36 additional mutations in genes not yet covered by the routine work-up (e.g., TET2, ASXL1, DNMT3A), demonstrating the feasibility of this approach and the gain of diagnostically relevant information. The dramatically reduced amount of input DNA, the increase in sensitivity as well as calculated cost-effectiveness, low hands on , and turn-around-time, necessary for the analysis of 237 amplicons strongly argue for replacing Sanger sequencing by this semiconductor-based targeted resequencing approach.

Analysis of Mutational Hotspots in Routinely Processed Bone Marrow Trephines by Pyrosequencing

Formalin-fixed, paraffin-embedded (FFPE) bone marrow trephines are widely used in pathology, because they best preserve the morphological details of the bone marrow. However, DNA isolated from FFPE material is fragmented, limiting the size of amplification products, which is a challenge for all sequencing applications.Pyrosequencing(®) is a quantitative and sensitive method for the detection of single-nucleotide variations (SNVs) in DNA samples. Pyrosequencing can easily be performed in a 96-well-plate format with a cost-effective medium-sized throughput.This chapter provides a general outline of SNV detection in FFPE bone marrow trephines, including a detailed protocol of the Pyrosequencing procedure and guidelines for the design of new assays and evaluation of Pyrograms. The strengths of this approach are discussed using myeloproliferative neoplasms as an example.

Archival bone marrow trephines are suitable for high-throughput mutation analysis using next generation sequencing technology

In the May issue of Haematologica, Foa et al.1 provided a comprehensive and authoritative review of the impact of the new sequencing technologies on the clinical management of patients with chronic lymphocytic leukemia. We thoroughly enjoyed studying the review and would like to comment only on a technical point, which is nevertheless of great importance in the field of hematopathology.

De novo CSF3R mutation associated with transformation of myeloproliferative neoplasm to atypical CML

/L), circulating myeloid precursors (>10 %), BCR-ABLnegativity [2, 3]) [4, 5]. We found a newly occurring CSF3R(exons 14, 17) mutation during progression in a case of MPNunclassifiable (MPNu). The 65-year-old male presented withthrombocytosis, splenomegaly, a leukoerythroblastic bloodpicture, anemia, and signs of hemolysis (thrombocytes 602×10

Molecular Analysis of Circulating Cell-Free DNA from Lung Cancer Patients in Routine Laboratory Practice: A Cross-Platform Comparison of Three Different Molecular Methods for Mutation Detection

Circulating cell-free DNA (cfDNA), which is isolated from blood plasma, represents a noninvasive source for the detection of mutations conferring resistance against epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small-cell lung cancer patients. In advanced disease stages, performing regular biopsies is often not possible because of the general health condition of the patients. Furthermore, a biopsy of a single tumor lesion or metastasis may not reflect the heterogeneous genotype of the tumor and its metastases. Plasma cfDNA represents an alternative material for molecular monitoring of patients under therapy. Herein, we present a cross-platform comparison of three different molecular methods [digital PCR, next-generation sequencing (NGS), and quantitative PCR] to detect clinically relevant mutations in cfDNA. We validated our workflow with commercially available cfDNA reference material (5.0%, 1.0%, and 0.1% mutation frequency, respectively). Digital PCR and NGS detect reliably 0.1% allele frequency of the EGFR p.T790M mutation. Furthermore, we analyzed 55 cfDNA preparations from patients with lung cancer to compare reliability and sensitivity of the three methods under routine conditions and achieved 96.0% concordance of p.T790M results. A limit of detection for mutation calling using digital PCR (>0.1%) and NGS (>0.2%) was established. In total, 62.5% of known primary EGFR mutations were successfully detected in cfDNA. In 56.0% of the patients with detectable EGFR primary mutations, we identified a resistance conferring the EGFR p.T790M mutation.

Absence of MGMT promoter methylation in diffuse midline glioma, H3 K27M-mutant

Letter According to the revised 4th edition of the WHO classification of tumors of the CNS, diffuse midline gliomas, H3 K27M-mutant (DMG) are molecularly defined as tumors with a predominantly astrocytic differentiation carrying mutations in the histone H3 encoding genes H3F3A (histone H3.3), HIST1H3B (H3.1) or HIST1H3C (H3.2) [9]. The vast majority of DMG demonstrate typical features of glioblastomas WHO grade IV (GBM): Malignant astrocytic morphology, necrosis and/ or microvascular proliferation. However, due to the poor clinical course of patients with DMG these tumors are assigned WHO grade IV irrespectively of GBM features. DMG are usually observed in children and young adults and occur in midline structures like thalamus, brainstem and spinal cord [9]. Most DMG carry H3F3A mutations; a smaller fraction shows HIST1H3B alterations, whereas HIST1H3C and, as recently shown, HIST2H3C mutations were identified only in single cases [10]. Around 80% of all diffuse intrinsic pontine gliomas (DIPG) exhibit the molecular profile of DMG [8]. Surgical intervention in DMG is often challenging and may lead to incomplete resection or even unsuccessful attempt failing to do a biopsy in many cases. Thus, radiotherapy and chemotherapy have a significant therapeutic importance in these patients compared with those with supratentorial GBM. Nowadays most patients with GBM receive radiotherapy and concomitant/adjuvant chemotherapy with temozolomide (TMZ) [1]. Around 40% of these cases feature hypermethylation of the promoter region of O-6-methylguanine DNA methyltransferase (MGMT) gene showing a positive response to TMZ treatment in comparison to those with absent MGMT methylation [1]. The MGMT gene on the chromosomal arm 10q26 consists of five exons and a CpG-rich island with 98 CpG sites covering exon 1 and most parts of the promoter (Fig. 1). Since CpG methylation pattern is not always homogenous, distinct assays may lead to conflicting results depending on the CpG sites analyzed [13]. For further understanding the contribution of each of the 98 CpG sites to MGMT expression, different studies have focused on sequencing large areas of the CpG island. By analyzing glioma cells without MGMT expression upstream and downstream highly methylated regions (UHMR, DHMR) were identified in the CpG island as well as a region in between containing a varying methylation rate (Fig. 1) [11]. Furthermore, through analyzing 52 CpG sites, the methylation status of six CpG sites was found to highly correlate with MGMT mRNA expression (Fig. 1) [5]. Because of the GBM-like histological appearance of most DMG, patients receive in many institutions the same treatment as those with supratentorial GBM. However, the MGMT promoter methylation status has not systematically been studied in patients with DMG and only few data have been reported so far [2, 3, 7, 12]. To clarify the frequency of MGMT promoter methylation in DMG we analyzed a retrospective series of 143 astrocytic midline tumors for H3F3A and HIST1H3B codon 27 mutations by pyrosequencing (MHH ethic board vote #1707–2013 & #6960). We identified H3F3A K27M mutations in 46/143 tumors including 25 males (54%) and 21 females (46%) with a median age of 23 at diagnosis and a range of 1–68 years. No HIST1H3B mutation was found. Next, we tested these 46 DMG for MGMT promoter methylation. For this purpose, DNA underwent bisulfite treatment and 14 CpG sites in the distal promoter region were analyzed by pyrosequencing (Fig. 1). A mean methylation level of 10% was defined as * Correspondence: hartmann.christian@mh-hannover.de; https://www.mhhannover.de/pathologie-neuropat.html Institute of Pathology, Department of Neuropathology, Hannover Medical School (MHH), Carl-Neuberg-Str. 1, D-30625 Hannover, Germany Full list of author information is available at the end of the article

Evolution of chronic myelomonocytic leukemia to myeloproliferative neoplasm

Dear Editor, Chronic myelomonocytic leukemia (CMML) is characterized by sustained non-reactive monocytosis (≥1 × 10μl, ≥10 % monocytes of peripheral blood leucocytes), frequently accompanied by thrombocytopenia and/or anemia. Progression to acute leukemia (AML) takes place in 30– 40 % of cases [1]. Here, we present for the first time a series of four CMML cases which did not progress to AML but evolved to myeloproliferative neoplasm (MPN). The four patients were followed by sequential bone marrow biopsies and mutational studies for 2–11 years (median 5 years, 4–6 sequential biopsies per patient). CMML had been known for 2–6 years (median 3.5 years). During follow-up, all four patients showed an increase of platelet counts (from median 99.5 × 10μ l -1 , range 49–279 × 10μ l -1 to median 497.5×10μl, range 210–560×10μl Table 1). In two patients, previous thrombocytopenia was converted into thrombocytosis (cases 1 and 4, Table 1). By contrast, an improvement of anemia could not be noticed (Table 1). Leucocyte counts increased in three patients while absolute and relative monocytosis persisted in all four patients. The monocyte counts before increase of platelet counts (median 3.2 × 10μl, 20 % of peripheral leucocytes; range 1.0– 7.8×10μl, 18.5–28.9 % of peripheral leucocytes) did not differ from the values afterwards (median 3.3 × 10μl, 16.6 % of peripheral leucocytes; range 1.1–7.4 × 10μl, 10.9–18.7 % of peripheral leucocytes, Table 1). Evolution to MPN became evident by a profound change of bone marrow morphology as shown in Fig. 1. Before, as typical for CMML, there had been a predominant proliferation of granuloand monopoiesis accompanied by a small number of dysplastic small megakaryocytes (Fig. 1a, b). The most impressive change in bone marrow histology, indicating evolution to MPN, was provided by an increase in megakaryocyte number and size with cluster formation and fibrosis (Fig. 1c, d). The transformation of bonemarrowmorphology was paralleled by the occurrence of MPN driver mutations such as JAK2 or MPL (Table 1). Mutation profiling of 23 genes was performed as previously described [2]. All four patients exhibited TET2 mutations with a high allelic burden, probably affecting both alleles (Table 1). Three cases showed SRSF2 mutations, which are typically found in CMML [3]. Furthermore, a mutation of CBL was noticed (Table 1). Case 1, which unexpectedly revealed a homozygous SRSF2 mutation (96.2 % p.P95_P96del) was studied with Sanger sequencing and PyrosequencingTM with a different primer set and yielded identical results. The four cases demonstrate that CMML can undergo transformation to a clinical presentation and histological picture that fulfill the criteria of MPN. The phenotypic transformation appears not to be caused by a replacement of the CMML clone by a MPN clone because the characteristic SRSF2 and TET2 mutations persisted. The MPNtypical driver mutations, such as JAK2 and MPL appeared to be acquired by the pre-existing neoplastic clone which had given rise to CMML. In one case, the JAK2 mutation was detectable already at presentation but revealed an increase of allelic burden from 4.7 to 64.8 % which probably caused the transformation of the histological picture. In all the other cases, evolution to MPN could be explained by de novo mutation of either JAK2 or MPL. The detection threshold of NGS is approximately 1 %; consequently, it cannot be excluded that very small sub-clones had * H. Kreipe Kreipe.Hans@MH-Hannover.de

Estrogen receptor ( ESR1 ) mutation in bone metastases from breast cancer

Activating mutations of estrogen receptor α gene (ESR1) in breast cancer can cause endocrine resistance of metastatic tumor cells. The skeleton belongs to the metastatic sides frequently affected by breast cancer. The prevalence of ESR1 mutation in bone metastasis and the corresponding phenotype are not known. In this study bone metastases from breast cancer (n=231) were analyzed for ESR1 mutation. In 27 patients (12%) (median age 73 years, range: 55–82 years) activating mutations of ESR1 were detected. The most frequent mutation was p.D538G (53%), no mutations in exon 4 (K303) or 7 (S463) were found. Lobular breast cancer was present in 52% of mutated cases (n=14) and in 49% of all samples (n=231), respectively. Mutated cancers constantly displayed strong estrogen receptor expression. Progesterone receptor was positive in 78% of the mutated cases (n=21). From 194 estrogen receptor-positive samples, 14% had ESR1 mutated. Except for one mutated case, no concurrent HER2 overexpression was noted. Metastatic breast cancer with activating mutations of ESR1 had a higher Ki67 labeling index than primary luminal cancers (median 30%, ranging from 5 to 60% with 85% of cases revealing ≥20% Ki67-positive cells). From those patients from whom information on endocrine therapy was available (n=7), two had received tamoxifen only, 4 tamoxifen followed by aromatase inhibitors and one patient had been treated with aromatase inhibitors only. We conclude that ESR1 mutation is associated with estrogen receptor expression and high proliferative activity and affects about 14% of estrogen receptor-positive bone metastases from breast cancer.