Comparison of sequencing performance of the corresponding fresh and archival patient samples.

<p>Mapped reads, reads on target, mean coverage, and mean read length of the sequenced sample sets (each n = 10) of the aspirate DNA and three isolation methods of FFPE samples are shown. Additionally total reported variants are shown and the number of C>T/G>A variants below 10%.</p

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

<div><p>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., <i>TET2</i>, <i>ASXL1</i>, <i>DNMT3A</i>), 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.</p></div

Overview of verified known pathogenic mutations and additional detected variants in the validation cohort (n = 41).

<p>All variants are reported as mutations by Cartagenia Bench Lab NGS software (Version 3.1.2).</p

Technical run data from Torrent Server.

<p>(A) Loading intensity of 318 v2 chip with 6 aspirate samples. (B) Loading intensity of 318 v2 chip with 6 FFPE samples isolated with the standard protocol. Read length histogram represent selected samples from aspirate (C), standard protocol (D), GeneRead Kit (E), and standard protocol + UNG pre-treatment (F) sample sets.</p

Flow diagram of sample processing from the training cohort.

<p>Flow diagram of sample processing from the training cohort.</p

Estimation of the turn-around time and the costs for 12 samples processed with the AML panel and IonTorrent PGM semiconductor sequencer.

<p>Reagent costs are calculated from net list prices for Germany.</p

Verification of mutations from training cohort by Sanger sequencing.

<p>(A) Mapped reads of Patient 10 loaded in the IGV browser (Version 2.3.34). <i>CBL</i> p.T377A (c.1129G>A) variant with 50.6% variant frequency is shown. (B) Results from Sanger sequencing of <i>CBL</i> Exon 8. (C) Mapped reads of Patient 2 loaded in the IGV browser. <i>TP53</i> p.S241T (c.721T>A) with 24.7% variant frequency is shown (D) Results from Sanger sequencing of <i>TP53</i> Exon 7.</p

Comparison of sequencing performance of the corresponding fresh and archival patient samples.

<p>Mapped reads, reads on target, mean coverage, and mean read length of the sequenced sample sets (each n = 10) of the aspirate DNA and three isolation methods of FFPE samples are shown. Additionally total reported variants are shown and the number of C>T/G>A variants below 10%.</p

Verification of mutations from validation cohort by Sanger sequencing.

<p>(A) Mapped reads of a Patient with RCMD loaded in the IGV browser (Version 2.3.34). <i>TET2</i> p.L541X (c.1620delT) variant with 29.1% variant frequency in 592 reads is shown (B) Results from Sanger sequencing of <i>TET2</i> Exon 3. (C) Mapped reads of a Patient with AML loaded in the IGV browser. <i>NPM1</i> p.W288CfsX12 (c.860_863dupTCTG) mutation with 43.7% variant frequency is shown (D) Results from Sanger sequencing of <i>NPM1</i> Exon 12. Please note that Sanger sequencing shows the reverse sequence.</p

Is Upregulation of BCL2 a Determinant of Tumor Development Driven by Inactivation of CDH1/E-Cadherin?

<div><p>Inactivation of <i>CDH1</i>, encoding E-cadherin, promotes cancer initiation and progression. According to a newly proposed molecular mechanism, loss of E-cadherin triggers an upregulation of the anti-apoptotic oncoprotein BCL2. Conversely, reconstitution of E-cadherin counteracts overexpression of BCL2. This reciprocal regulation is thought to be critical for early tumor development. We determined the relevance of this new concept in human infiltrating lobular breast cancer (ILBC), the prime tumor entity associated with <i>CDH1</i> inactivation. BCL2 expression was examined in human ILBC cell lines (IPH-926, MDA-MB-134, SUM-44) harboring deleterious <i>CDH1</i> mutations. To test for an intact regulatory axis between E-cadherin and BCL2, wild-type E-cadherin was reconstituted in ILBC cells by ectopic expression. Moreover, BCL2 and E-cadherin were evaluated in primary invasive breast cancers and in synchronous lobular carcinomas <i>in situ</i> (LCIS). MDA-MB-134 and IPH-926 showed little or no BCL2 expression, while SUM-44 ILBC cells were BCL2-positive. Reconstitution of E-cadherin failed to impact on BCL2 expression in all cell lines tested. Primary ILBCs were almost uniformly E-cadherin-negative (97%) and were frequently BCL2-negative (46%). When compared with an appropriate control group, ILBCs showed a trend towards an increased frequency of BCL2-negative cases (<i>P = </i>0.064). In terminal duct-lobular units affected by LCIS, the E-cadherin-negative neoplastic component showed a similar or a reduced BCL2-immunoreactivity, when compared with the adjacent epithelium. In conclusion, upregulation of BCL2 is not involved in lobular breast carcinogenesis and is unlikely to represent an important determinant of tumor development driven by <i>CDH1</i> inactivation.</p></div