Central diagnostics, genomics and biobanking
Final Report Abstract
This central project is to provide diagnostics, biobanking, and genomics for the CRU. In the first funding period CLL patients have been phenotypically and molecularly characterized by means of an advanced diagnostic work-up. These investigations included cytological, immunophenotypical, cytogenetic and molecular genetics techniques. Through cytological investigations CLL cases could be clearly separated from B cell prolymphocytic leukemia (B-PLL) and other related B cell malignancies. For flow cytometry a broad panel of different antigens has been analyzed which included standard antigens for the diagnosis of CLL such as CD5, CD19 and CD23, antigens for the diagnosis of related diseases such as CD10, CD11c and FMC7, as well as new antigens, such as CD43, CD200 and ROR1. Furthermore, we performed conventional cytogenetics (karyotyping/banding analyses) at initial diagnosis as well as during relapsing disease in order to search for recurrent or non-recurrent chromosomal aberrations. As we could previously show that complex karyotypes correlate with a significantly adverse prognosis in CLL first line treatment in comorbid patients we extended these investigations also to younger patients and relapses. In given cases multicolor-FISH (mFISH, “chromosomal painting”) has been be performed in order to uncover difficult structural lesions. We performed IgVH sequencing by using IG specific primers for site specific PCR reactions, i.e. according to the Biomed-2 recommendations, followed by Sanger sequencing. Similarly, we have fully established a PCR-based mutational analysis of exon 4 to 10 of the TP53 gene, as well as a NGS technique. Samples from CLL patients who agreed to it have been -after obtaining written consent- characterized by above mentioned approaches and stored at our central biobank and were distributed to the various RPs. In total, another 1,500 samples have been diagnostically characterized, banked and distributed. In the second funding period, we have extended our aims to provide support for genomic analyses and bioinformatics. To this end, we have implemented and established an automated platform to analyze tumor sequencing data. This platform is built on the open-source metadata management system iRODS to securely, consistently, transparently, and reproducibly process large amount of sequencing data. To make this system accessible to non-computational biologists, we also provided a graphical userinterface that allows the management and retrieval of metadata provided and obtained during data processing. As part of our computational pipeline, we have implemented a new method to analyze copy number changes and to infer subclonal populations from whole exome or genome data. Our method combines copy number analysis with mutational clustering (to infer the subclonal populations) and requires significantly less computational resources than alternative approaches. Within the Pan-Cancer Analysis of Whole Genomes conducted by the ICGC, we were able to validate and apply our method on large datasets. In addition, we applied our method to whole exome sequencing data from venetoclax relapsed patients to reconstruct the clonal dynamics towards resistance. Having also collected samples before the venetoclax treatment, we found a highly diverse clonal dynamics leading to venetoclax resistance, including linear, divergent, and convergent evolution. Furthermore, we found recurrent alterations at relapse affecting BTG1 and CDKN2A/B. In addition, functional analyses showed that oncogenic BRAF mutations is substantially lowering the treatment efficacy of venetoclax. Recently, Blombery et al. found a resistance conferring mutation in BCL2 (p.G101V) that remained subclonal after relapse in most cases. Using digital droplet PCR, we found the presence of this mutation in three of our cases that have undergone whole exome sequencing17. However, the allele frequency was far below the detection limit of whole exome sequencing and the pronounced subclonality of the mutations challenging the notion that G101V mutation of BCL2 is solely driving venetoclax resistance in these samples.
Publications
- Therapeutic targeting of a robust non-oncogene addiction to PRKDC in ATM-defective tumors. Sci Transl Med. 2013 Jun 12;5(189):189ra78
Riabinska A, Daheim M, Herter-Sprie GS, Winkler J, Fritz C, Hallek M, Thomas RK, Kreuzer KA, Frenzel LP, Monfared P, Martins-Boucas J, Chen S, Reinhardt HC
(See online at https://doi.org/10.1126/scitranslmed.3005814) - Obinutuzumab plus chlorambucil in patients with CLL and coexisting conditions. N Engl J Med. 2014 Mar 20;370(12):1101-10
Goede V, Fischer K, Busch R, Engelke A, Eichhorst B, Wendtner CM, Chagorova T, de la Serna J, Dilhuydy MS, Illmer T, Opat S, Owen CJ, Samoylova O, Kreuzer KA, Stilgenbauer S, Döhner H, Langerak AW, Ritgen M, Kneba M, Asikanius E, Humphrey K, Wenger M, Hallek M
(See online at https://doi.org/10.1056/nejmoa1313984) - A complementary role of multiparameter flow-cytometry and high-throughput sequencing for minimal residual disease (MRD) detection in chronic lymphocytic leukemia (CLL): An european research initiative on CLL (ERIC) study. Leukemia, 2015 Dec 7
Rawstron AC, Fazi C, Agathangelidis A, Villamor N, Letestu R, Nomdedeu J, Palacio C, Stehlikova O, Kreuzer KA, Liptrot S, O'Brien D, de Tute RM, Marinov I, Hauwel M, Spacek M, Dobber J, Kater AP, Gambell P, Soosapilla A, Lozanski G, Brachtl G, Lin K, Boysen J, Hanson C, Jorgensen JL, Stetler-Stevenson M, Yuan C, Broome HE, Rassenti L, Craig F, Delgado J, Moreno C, Bosch F, Egle A, Doubek M, Pospisilova S, Mulligan S, Westerman D, Sanders CM, Emerson R, Robins HS, Kirsch I, Shanafelt T, Pettitt A, Kipps TJ, Wierda WG, Cymbalista F, Hallek M, Hillmen P, Montserrat E, Ghia P
(See online at https://doi.org/10.1038/leu.2015.313) - Comprehensive Analysis of Disease-Related Genes in Chronic Lymphocytic Leukemia by Multiplex PCR-Based Next Generation Sequencing. PLoS One. 2015 Jun 8;10(6):e0129544
Vollbrecht C, Mairinger FD, Koitzsch U, Peifer M, Koenig K, Heukamp LC, Crispatzu G, Wilden L, Kreuzer KA, Hallek M, Odenthal M, Herling CD, Buettner R
(See online at https://doi.org/10.1371/journal.pone.0129544) - Organometallic nucleosides induce non-classical leukemic cell death that is mitochondrial-ROS dependent and facilitated by TCL1-oncogene burden. Mol Cancer. 2015 Jun 4;14:114
Prinz C, Vasyutina E, Lohmann G, Schrader A, Romanski S, Hirschhäuser C, Mayer P, Frias C, Herling CD, Hallek M, Schmalz HG, Prokop A, Mougiakakos D, Herling M
(See online at https://doi.org/10.1186/s12943-015-0378-1) - A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells. Mol Cancer Ther. 2016 May;15(5):971-84
Lilienthal N, Lohmann G, Crispatzu G, Vasyutina E, Zittrich S, Mayer P, Herling CD, Tur MK, Hallek M, Pfitzer G, Barth S, Herling M
(See online at https://doi.org/10.1158/1535-7163.mct-15-0685) - Complex karyotypes and KRAS and POT1 mutations impact outcome in CLL after chlorambucil-based chemotherapy or chemoimmunotherapy. Blood. 2016 Jul 21;128(3):395-404
Herling CD, Klaumünzer M, Rocha CK, Altmüller J, Thiele H, Bahlo J, Kluth S, Crispatzu G, Herling M, Schiller J, Engelke A, Tausch E, Döhner H, Fischer K, Goede V, Nürnberg P, Reinhardt HC, Stilgenbauer S, Hallek M, Kreuzer KA
(See online at https://doi.org/10.1182/blood-2016-01-691550) - Long-term remissions after FCR chemoimmunotherapy in previously untreated patients with CLL: updated results of the CLL8 trial. Blood. 2016 Jan 14;127(2):208-15
Fischer K, Bahlo J, Fink AM, Goede V, Herling CD, Cramer P, Langerbeins P, von Tresckow J, Engelke A, Maurer C, Kovacs G, Herling M, Tausch E, Kreuzer KA, Eichhorst B, Böttcher S, Seymour JF, Ghia P, Marlton P, Kneba M, Wendtner CM, Döhner H, Stilgenbauer S, Hallek M
(See online at https://doi.org/10.1182/blood-2015-06-651125) - ATM Deficiency Is Associated with Sensitivity to PARP1- and ATR Inhibitors in Lung Adenocarcinoma. Cancer Res. 2017 Jun 1;77(11):3040-3056
Schmitt A, Knittel G, Welcker D, Yang TP, George J, Nowak M, Leeser U, Büttner R, Perner S, Peifer M, Reinhardt HC
(See online at https://doi.org/10.1158/0008-5472.can-16-3398) - Targeting transcription-coupled nucleotide excision repair overcomes resistance in chronic lymphocytic leukemia. Leukemia. 2017 May;31(5):1177-1186
Lohmann G, Vasyutina E, Bloehdorn J, Reinart N, Schneider JI, Babu V, Knittel G, Crispatzu G, Mayer P, Prinz C, Muenzner JK, Biersack B, Efremov DG, Chessa L, Herling CD, Stilgenbauer S, Hallek M, Schobert R, Reinhardt HC, Schumacher B, Herling M
(See online at https://doi.org/10.1038/leu.2016.294) - Two mouse models reveal an actionable PARP1 dependence in aggressive chronic lymphocytic leukemia. Nat Commun. 2017 Jul 28;8(1):153
Knittel G, Rehkämper T, Korovkina D, Liedgens P, Fritz C, Torgovnick A, Al-Baldawi Y, Al-Maarri M, Cun Y, Fedorchenko O, Riabinska A, Beleggia F, Nguyen PH, Wunderlich FT, Ortmann M, Montesinos-Rongen M, Tausch E, Stilgenbauer S, Frenzel LP, Herling M, Herling C, Bahlo J, Hallek M, Peifer M, Buettner R, Persigehl T, Reinhardt HC
(See online at https://doi.org/10.1038/s41467-017-00210-6) - A mechanistic classification of clinical phenotypes in neuroblastoma. Science. 2018 Dec 7;362(6419):1165-1170
Ackermann S, Cartolano M, Hero B, Welte A, Kahlert Y, Roderwieser A, Bartenhagen C, Walter E, Gecht J, Kerschke L, Volland R, Menon R, Heuckmann JM, Gartlgruber M, Hartlieb S, Henrich KO, Okonechnikov K, Altmüller J, Nürnberg P, Lefever S, de Wilde B, Sand F, Ikram F, Rosswog C, Fischer J, Theissen J, Hertwig F, Singhi AD, Simon T, Vogel W, Perner S, Krug B, Schmidt M, Rahmann S, Achter V, Lang U, Vokuhl C, Ortmann M, Büttner R, Eggert A, Speleman F, O'Sullivan RJ, Thomas RK, Berthold F, Vandesompele J, Schramm A, Westermann F, Schulte JH, Peifer M, Fischer M
(See online at https://doi.org/10.1126/science.aat6768) - Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia. Nat Commun. 2018 Feb 20;9(1):727
Herling CD, Abedpour N, Weiss J, Schmitt A, Jachimowicz RD, Merkel O, Cartolano M, Oberbeck S, Mayer P, Berg V, Thomalla D, Kutsch N, Stiefelhagen M, Cramer P, Wendtner CM, Persigehl T, Saleh A, Altmüller J, Nürnberg P, Pallasch C, Achter V, Lang U, Eichhorst B, Castiglione R, Schäfer SC, Büttner R, Kreuzer KA, Reinhardt HC, Hallek M, Frenzel LP, Peifer M
(See online at https://doi.org/10.1038/s41467-018-03170-7) - Copy-number analysis and inference of subclonal populations in cancer genomes using Sclust. Nat Protoc. 2018 Jun;13(6):1488-1501
Cun Y, Yang TP, Achter V, Lang U, Peifer M
(See online at https://doi.org/10.1038/nprot.2018.033) - Acquisition of the recurrent Gly101Val mutation in BCL2 confers resistance to venetoclax in patients with progressive chronic lymphocytic leukemia (Comment to Tausch et al.). Haematologica. 2019 Nov;104(11):e540
Weiss J, Peifer M, Herling CD, Frenzel LP, Hallek M
(See online at https://doi.org/10.3324/haematol.2019.232835) - iRODS metadata management for a cancer genome analysis workflow. BMC Bioinformatics. 2019 Jan 15;20(1):29
Nieroda L, Maas L, Thiebes S, Lang U, Sunyaev A, Achter V, Peifer M
(See online at https://doi.org/10.1186/s12859-018-2576-5) - UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors. Cell. 2019 Jan 24;176(3):505-519.e22
Jachimowicz RD, Beleggia F, Isensee J, Velpula BB, Goergens J, Bustos MA, Doll MA, Shenoy A, Checa-Rodriguez C, Wiederstein JL, Baranes-Bachar K, Bartenhagen C, Hertwig F, Teper N, Nishi T, Schmitt A, Distelmaier F, Lüdecke HJ, Albrecht B, Krüger M, Schumacher B, Geiger T, Hoon DSB, Huertas P, Fischer M, Hucho T, Peifer M, Ziv Y, Reinhardt HC, Wieczorek D, Shiloh Y
(See online at https://doi.org/10.1016/j.cell.2018.11.024) - ATM activity in T cells is critical for immune surveillance of lymphoma in vivo. Leukemia. 2020 Mar;34(3):771-786
Riabinska A, Lehrmann D, Jachimowicz RD, Knittel G, Fritz C, Schmitt A, Geyer A, Heneweer C, Wittersheim M, Frenzel LP, Torgovnick A, Wiederstein JL, Wunderlich CM, Ortmann M, Paillard A, Wößmann W, Borkhardt A, Burdach S, Hansmann ML, Rosenwald A, Perner S, Mall G, Klapper W, Merseburg A, Krüger M, Grüll H, Persigehl T, Wunderlich FT, Peifer M, Utermöhlen O, Büttner R, Beleggia F, Reinhardt HC
(See online at https://doi.org/10.1038/s41375-019-0618-2) - CaMuS: simultaneous fitting and de novo imputation of cancer mutational signature. Sci Rep. 2020 Nov 9;10(1):19316
Cartolano M, Abedpour N, Achter V, Yang TP, Ackermann S, Fischer M, Peifer M
(See online at https://doi.org/10.1038/s41598-020-75753-8) - Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1. Blood. 2021 Feb 4;137(5):646-660
Kohlhaas V, Blakemore SJ, Al-Maarri M, Nickel N, Pal M, Roth A, Hövelmeyer N, Schäfer SC, Knittel G, Lohneis P, Nikolic M, Wiederstein JL, Franitza M, Georgomonolis T, Reinart N, Herling M, Herling C, Hartmann EM, Rosenwald A, Klapper W, Büttner R, Moia R, Rossi D, Boldorini R, Gaidano G, Frenzel LP, Reinhardt HC, Brüning JC, Hallek M, Krüger M, Peifer M, Pallasch CP, Wunderlich FT
(See online at https://doi.org/10.1182/blood.2020005734)
