Borsa di Studio “Damiano per l’Ematologia” 2018

Proclamazione del Vincitore

L’autrice del progetto vincitore è la Dr.ssa Martina Ferrante, che svolgerà il suo lavoro presso il Laboratorio di Biologia Molecolare, Ematologia Universitaria I, AOU “Città della Salute e della Scienza di Torino”, Presidio Molinette, Dipartimento di Biotecnologie Molecolari e Scienze della Salute, Università degli Studi di Torino, sotto la supervisione del Dr. Simone Ferrero e della Dr.ssa Daniela Drandi.

Il testo del progetto è il seguente:

a) TITLE

Non invasive diagnostic and monitoring of minimal residual disease in patients affected by Waldenström’s macroglobulinemia

b) INTRODUCTION

Waldenström’s macroglobulinemia (WM) is an infrequent, indolent lymphoplasmacytic lymphoma (LPL) characterized by the accumulation in the bone marrow (BM) of monoclonal lymphocytes, lymphoplasmacytic cells and plasma cells, responsible for monoclonal IgM protein secretion.1

In the last years, MYD88L265P mutation has been identified.2,3 Several studies using different techniques such as Sanger sequencing, polymerase chain reaction (PCR) and allele-specific quantitative PCR (AS-qPCR), on CD19-sorted BM samples, observed that about 90% of WM patients carry the MYD88L265P mutation, while it is present only in 6-10% of patients with marginal zone lymphoma (MZL), in 3-5% of chronic lymphocytic leukemia (CLL) and it is absent in multiple myeloma (MM) and in non-IgM monoclonal gammopathies of uncertain significance (MGUS).4–6 MYD88L265P is now considered a hallmark of WM and it might be helpful in the differential diagnosis with other lymphoproliferative neoplasms with overlapping clinical features, as MM or MZL.1,7 Furthermore, it might represent an ideal marker for minimal residual disease (MRD) monitoring in a disease whose therapeutic scenario is rapidly changing, with many new available and highly effective drugs.8–14

Moreover, MYD88L265P has been demonstrated on CD19-sorted BM samples in 50-80% of patients with IgM MGUS, an asymptomatic phase which represents a pre-neoplastic condition, postulating a possible role in disease progression.4,6,15–17 BM biopsy is mandatory for differential diagnosis between WM and IgM MGUS, but patients with asymptomatic M component do not easily accept to undergo such an invasive, painful and time consuming, surgical procedure. The availability of accurate diagnostic tools on more accessible samples, like peripheral blood (PB) or even urine, could overcome this problem and avoid the potential risk of misclassification of patients. Additionally, the current MYD88L265P ASqPCR method lacks sensitivity (up to 1.00E-03) and thus is not suitable for MRD analysis.4,18 Indeed, ASqPCR is suboptimal for testing specimens like unsorted BM or even PB, that harbors low concentrations of circulating tumor cells (especially after immunochemotherapy), neither to assess cell-free tumor DNA (ctDNA), usually present at very low amount in plasma, as well as in other biological fluids, including liquor and pleural effusion.19,20 Recently, digital PCR has been shown to be a powerful technique that provides improved sensitivity, precision and reproducibility overcoming some of qPCR pitfalls.21,22 The project purpose is the application of a highly sensitive droplet digital PCR (ddPCR) assay for the identification of the MYD88L265P mutation in patients affected by WM, IgM-MGUS or other LPL, suitable for screening and MRD monitoring, also on ctDNA. MRD analysis usually requires bone marrow biopsy but preliminary results show that mutational levels of MYD88L265P in plasmatic ctDNA are comparable to BM levels, so ctDNA analysis might be a promising, less invasive and “patient friendly” alternative both for diagnosis and MRD monitoring in WM patients.

c) AIMS OF THE PROJECT

Starting from the following hypotheses:

- a reliable diagnosis of WM, as well as a discrimination of IgM-MGUS from WM, may be done in clinical setting without the need for invasive procedures, as BM or lymph node biopsy, taking advantage of easy to collect tissues, like PB (for plasma collection) and urine;

- MRD assessment is feasible and predictive of relapse in WM patients receiving therapy, both whether performed on BM and PB samples;

these hypotheses will be tested in 2 Specific Aims:

-Specific Aim 1: to test by MYD88L265P ddPCR paired BM, PB, plasma and urine samples collected at diagnosis of WM and IgM-MGUS, to assess the feasibility of a less invasive diagnostics and of a reliable discrimination between the two entities, possibly identifying the pathogenetic events responsible for disease progression.

- Specific Aim 2: to test MYD88L265P ddPCR as a novel MRD tool on large series of WM patients samples, collected at baseline and after treatment, from multiple, even less invasive, sources (BM, PB, plasma and urine); moreover, as the patients series is fully annotated, to correlate the MRD results with the clinical outcome, to demonstrate the predictive value of this analysis.

d) METHODS

BM, PB and urine samples are being prospectively collected at baseline and during follow-up (FU) from a local series of patients affected by WM, IgM-MGUS and IgG secreting LPL: as of October 2017 paired BM-PB-plasma-urine samples from 35 patients have already been collected. PB samples from 40 healthy subjects and BM samples from 20 MM patients and urine from 20 healthy subjects age-matched will be used as negative controls.

All patients provided written informed consent for the research use of their biological samples, in accordance with Institutional Review Boards requirements, and the Helsinki's declaration.

Genomic (gDNA) from 5x106 cells and cell tumor DNA (ctDNA) from 1 ml of plasma, 1 ml of urine will be extracted by Maxwell automatic system (MaxWell RSC, Promega), following the manufacturer recommendations. Moreover, amount of ctDNA will be evaluated by RnaseP gene.

Mutation detection assay by ddPCR will be performed using a single set of primers combined with two competitive probes, in two assays (CSTM DDPCR HEX/FAM ASSAY BIO-RAD) one for MYD88L265P mutation labeled with FAM and one for MYD88L265P wild type labeled with HEX. ddPCR assay will be performed on the QX100 Droplet Digital PCR system (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, for each replicate, 11 μl of 2X ddPCR Supermix for Probes with no dUTP (Bio-Rad Laboratories), 1.1 μl of each 20X mutation detection assay and 5.5 μl of gDNA (20 ng/μl) or ctDNA were mixed in a total 22 μl reaction volume. Droplets will be generated, by a QX100 droplet generator device, from 20 μl of the reaction mix, and end-point PCR was performed on a T100 Thermal Cycler (Bio-Rad Laboratories) at following conditions: 95°C for 10 minutes, 40 cycles of 94°C for 30 seconds, 55°C for 1 minute followed by 98°C for 10 minutes. Ramp rate was set at 2.5°C/second. PCR products will be loaded into the QX100 droplet reader and analyzed by QuantaSoft v1.6.6.0320 (Bio-Rad Laboratories). Samples will be tested in triplicate and results will be expressed as merge of wells. The cut-off for mutation will be set based on the highest MYD88L265P level detected within the wild type control group. Each experiment will include a known highly mutated positive control sample (MUT 70%), a negative control (healthy donor or MM gDNA) and a no template control (NTC). Gate setting will be performed based on the positive control results.

The associations between categorical variables will be analyzed by the Fisher’ exact test, while the Mann-Whitney and Kruskal-Wallis ones will be used for the inference on continuous variables. All continuous variable results will be expressed as the median (range); ddPCR and ASqPCR results will be expressed as MUT/WT ratio. The interrater agreement on categorical data will be estimated by computing the Fleiss' Kappa index. All reported p-values will be estimated by the 2-sided exact method at the conventional 5% significance level.23

The project will be conducted during a period of 1 year, suitable for the generation of at least solid preliminary results.

e) REFERENCES

1. Swerdlow, S. H. et al. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood 127, 2375–90 (2016).

2. Zhan, C. et al. Conformational dynamics of cancer-associated MyD88-TIR domain mutant L252P (L265P) allosterically tilts the landscape toward homo-dimerization. Protein Eng. Des. Sel. 29, 347–54 (2016).

3. Treon, S. P. et al. MYD88 L265P somatic mutation in Waldenström’s macroglobulinemia. N. Engl. J. Med. 367, 826–33 (2012).

4. Xu, L. et al. MYD88 L265P in Waldenstrom macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction. Blood 121, 2051–2058 (2013).

5. Ngo, V. N. et al. Oncogenically active MYD88 mutations in human lymphoma. Nature 470, 115–9 (2011).

6. Varettoni, M. et al. Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstrom’s macroglobulinemia and related lymphoid neoplasms. Blood 121, 2522–2528 (2013).

7. Treon, S. P. et al. Somatic mutations in MYD88 and CXCR4 are determinants of clinical presentation and overall survival in Waldenstrom macroglobulinemia. Blood 123, 2791–2796 (2014).

8. Treon, S. P. How I treat Waldenström macroglobulinemia. Blood 114, 2375–85 (2009).

9. Treon, S. P., Xu, L. & Hunter, Z. MYD88 Mutations and Response to Ibrutinib in Waldenström’s Macroglobulinemia. N. Engl. J. Med. 373, 584–586 (2015).

10. Leblond, V. et al. Treatment recommendations from the Eighth International Workshop on Waldenström’s Macroglobulinemia. Blood 128, 1321–8 (2016).

11. Castillo, J. J. et al. Future therapeutic options for patients with Waldenström macroglobulinemia. Best Pract. Res. Clin. Haematol. 29, 206–215 (2016).

12. Abeykoon, J. P., Yanamandra, U. & Kapoor, P. New developments in the management of Waldenström macroglobulinemia. Cancer Manag. Res. 9, 73–83 (2017).

13. Hunter, Z. R. et al. Genomics, Signaling, and Treatment of Waldenström Macroglobulinemia. J. Clin. Oncol. 35, 994–1001 (2017).

14. Tedeschi, A. et al. Bendamustine and rituximab combination is safe and effective as salvage regimen in Waldenström macroglobulinemia. Leuk. Lymphoma 56, 2637–42 (2015).

15. Kyle, R. A. Long-term follow-up of IgM monoclonal gammopathy of undetermined significance. Blood 102, 3759–3764 (2003).

16. Landgren, O. & Staudt, L. MYD88 L265P somatic mutation in IgM MGUS. N. Engl. J. Med. 367, 2255-6-7 (2012).

17. Varettoni, M. et al. MYD88 (L265P) mutation is an independent risk factor for progression in patients with IgM monoclonal gammopathy of undetermined significance. Blood 122, 2284–2285 (2013).

18. Jiménez, C. et al. Detection of MYD88 L265P Mutation by Real-Time Allele-Specific Oligonucleotide Polymerase Chain Reaction. Appl. Immunohistochem. Mol. Morphol. 22, 768–773 (2014).

19. Gustine, J. N. et al. MYD88 mutations can be used to identify malignant pleural effusions in Waldenström macroglobulinaemia. Br. J. Haematol. (2016). doi:10.1111/bjh.14386

20. Komatsubara, K. M. & Sacher, A. G. Circulating Tumor DNA as a Liquid Biopsy: Current Clinical Applications and Future Directions. Oncology (Williston Park). 31, 618–27 (2017).

21. Hindson, B. J. et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal. Chem. 83, 8604–10 (2011).

22. Huggett, J. F. & Whale, A. Digital PCR as a novel technology and its potential implications for molecular diagnostics. Clin. Chem. 59, 1691–3 (2013).

23. Fleiss, J. L., Levin, B. & Paik, M. C. Statistical Methods for Rates and Proportions. (John Wiley & Sons, Inc., 2003). doi:10.1002/0471445428