Tracking myeloma tumor DNA in peripheral blood

Journal Pre-proof Tracking myeloma tumor DNA in peripheral blood Johannes M. Waldschmidt, Tushara Vijaykumar, Birgit Knoechel, Jens G. Lohr PII:

S1521-6926(20)30007-4

DOI:

https://doi.org/10.1016/j.beha.2020.101146

Reference:

YBEHA 101146

To appear in:

Best Practice & Research Clinical Haematology

Received Date: 25 November 2019 Accepted Date: 9 January 2020

Please cite this article as: Waldschmidt JM, Vijaykumar T, Knoechel B, Lohr JG, Tracking myeloma tumor DNA in peripheral blood, Best Practice & Research Clinical Haematology, https://doi.org/10.1016/ j.beha.2020.101146. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Tracking myeloma tumor DNA in peripheral blood

Johannes M. Waldschmidt1,2,3, Tushara Vijaykumar1,4, Birgit Knoechel2,3,4, Jens G. Lohr1,2,3

Affiliations: 1

Department of Medical Oncology, Dana-Faber Cancer Institute, Harvard Medical School, Boston, Massachusetts 2 Harvard Medical School, Boston, MA 3 Broad Institute of MIT and Harvard, Cambridge, MA 4 Department of Pediatric Oncology, Dana-Faber Cancer Institute, Harvard Medical School, Boston, Massachusetts

Corresponding author: Dr Jens Lohr E-mail: [email protected]

Abstract Over the past years, the emergence of liquid biopsy technologies has dramatically expanded our ability to assess multiple myeloma without the need for invasive sampling. Interrogation of cell-free DNA from the peripheral blood recapitulates the mutational landscape at excellent concordance with matching bone marrow aspirates. It can quantify disease burden and identify previously undetected resistance mechanisms which may inform clinical management in real-time. The convenience of sample acquisition and storage provides strong procedural benefits over currently available testing. Further investigations will have to define the role of

cell-free DNA as a diagnostic measure by determining clinically relevant tumor thresholds in comparison to existing routine parameters. This review presents an overview of currently available assays and discusses the clinical value, potential and limitations of cell-free DNA technologies for the assessment of this challenging disease.

Key Words cell-free DNA, genomic profiling, liquid biopsy, next-generation sequencing, MRD, early cancer detection

1. Background Multiple myeloma (MM) is a highly heterogeneous disease with multifocal dissemination throughout the body. Over the last two decades, the introduction of novel therapeutic regimens has led to substantial improvement in the management and prognosis of the disease [1,2]. Despite this prolonged survival, MM remains incurable in the large majority of patients, and relapses occur several times over the disease course in a single patient [3]. With a higher number of available treatment options, strategies that allow for more frequent tumor assessment over time are

needed. This may help to identify early signs of therapeutic resistance and enable clinical providers to adjust the therapeutic strategy before relapses become clinically evident.

On a molecular level, MM is characterized by complex cytogenetic and genomic aberrations including primary translocations of the immunoglobin heavy chain (IGH) locus, copy number variations and somatic mutations involving numerous oncogenic signaling pathways [4–6]. As the disease evolves, it typically acquires novel genomic aberrations that drive drug resistance [7–10]. This results in increased genomic complexity which represents a therapeutic challenge but, at the same time, may also provide new therapeutic vulnerabilities [4,7,11,12]. To fully exploit putative therapeutic targets that emerge with the constant evolution of MM, it would be necessary to obtain detailed molecular information about the disease when treatment decisions are to be made. However, beyond conventional monitoring by serum free-light chains (SFLC) and monoclonal paraprotein, no tests are currently performed in clinical routine to repeatedly characterize MM on a molecular level that may inform clinical management in real-time. BM biopsies, in theory, permit adequate monitoring of MM but are associated with discomfort, procedural risks and an inherent spatial sampling bias. Frequent BM biopsies for routine monitoring of MM patients are therefore impractical [13]. First reported in the 1940s, cell-free DNA has recently gained momentum as an innovative source of tumor material in multiple different types of cancer [14–20]. Cell-free DNA is shed into circulation through apoptosis or necrosis. Given its rapid clearance from the bloodstream (half-life ~1.5-2h), it may provide immediate insight into changes in tumor burden and clonal evolution [16].

Most recently, next-generation sequencing (NGS) technologies, including whole genome sequencing (WGS), whole exome sequencing (WES) and targeted sequencing have proven that the identification of tumor-derived cell-free DNA is also feasible and reproducible in MM [21–29]. PCR methodologies may be used to complement NGS for longitudinal quantification of patient-specific mutations in cellfree DNA [21,22,30].

This review presents an overview of the currently available liquid biopsy approaches and discusses the clinical value, potential and limitations of cell-free DNA technologies for the assessment of MM.

2. Isolation of circulating myeloma DNA One of the great benefits of cell-free DNA is the convenience with which it can be handled. With commercially available phlebotomy tubes that contain formaldehydefree fixatives cell-free DNA remains stable at room temperature for several days, which allows for easy shipping of samples even from remote locations [31]. With ubiquitously available routine EDTA-containing phlebotomy tubes, plasma should be obtained by centrifugation, ideally within several hours after sample acquisition to avoid both DNA degradation and cell death, as DNA from dying white blood cells may diminish the relative concentration of MM-derived cell-free DNA [32]. A small amount of non-tumorous cell-free DNA is detectable in the blood of all healthy individuals [33]. The separation of tumor-specific cell-free DNA from a background of this germline cell-free DNA represents a challenge not exclusively limited to MM. Current approaches in other cancers take advantage of cancerspecific mutations, structural rearrangements, copy number alterations, epigenetic modifications or gene fusions to separate tumor-derived fragments from normal DNA [34]. In MM, targeted sequencing for mutations in key driver genes like KRAS, NRAS, BRAF or TP53 allows for robust identification of MM-derived cell-free DNA [21,22]. This approach has been validated by several groups and can provide sequencing depths up to 20,000X at a comparatively low cost [21]. It is most suitable if specific mutations have previously been identified and can be used to quantify disease burden longitudinally. Similarly, PCR-based technologies, such as digital droplet PCR (ddPCR), are being used to track previously identified mutations at low cost and high reproducibility

[21,22,30].

PCR-based

assessment

of

clonotypic

IgH

gene

rearrangements represents an alternative approach to detect MM-specific cell-free DNA in individual patients [26]. Since copy number variations are detectable in the overwhelming majority of MM patients, we and others have employed low-pass whole genome sequencing (LWPGS) to determine the copy number profile in cell-free DNA and thereby

approximate the MM tumor fraction in the peripheral blood [25,27]. In patients with sufficiently high tumor fraction (typically >5%) discovery-oriented whole exome sequencing (WES) is feasible and has excellent concordance with WES performed in MM cells from the bone marrow (BM) [27]. Using quick and more cost-effective LPWGS as a screening approach to approximate the tumor fraction in a sample allows to predict the success rate of more costly WES or deep WGS approaches (27). This stepwise strategy enables screening of patients in whom informative WES and deep WGS with unbiased genomic discovery and definition of clonal composition can be successfully performed. Lastly, single-gene promoters and CpG panels depict a promising resource to robustly identify MM-specific DNA on an epigenetic level [7]. Such panels have been utilized with great success in other cancer types, but their diagnostic potential in MM still needs to be investigated [35,36]. One of the disadvantages of cell-free DNA is the fact that it cannot be enriched for MM-specific cell-free DNA, unlike MM cells from the BM, which can be enriched by flow sorting or magnetic bead selection. On the other hand, it is far easier to increase the amount of cell-free DNA obtained by increasing the amount of peripheral blood drawn than increasing the amount of BM obtained by biopsy, especially since BM biopsies may be subject to hemodilution.

3. Molecular Rationale From a molecular perspective, interrogation of cell-free DNA holds the potential to overcome the sampling bias that is inherent to a multifocal disease like MM. The mutational landscape in cell-free DNA and corresponding BM is highly concordant, but divergence has been reported to some extent in several studies [22,23,27]. The phenomenon of plasma-only mutations, i.e. mutations that are detectable in cell-free DNA but remain undetectable in the BM compartment, has first been observed in a hybrid-capture assay by Kis and colleagues [21]. In another study by Mithraprabhu and colleagues, 24% of all patients were determined to have plasma-only mutations [22]. If correlated with disease stage, 27% of patients with relapsed/ refractory MM (RRMM), but only 7% of patients with newly diagnosed MM tested positive for such spatially selective mutations, suggesting that liquid biopsy approaches might be particularly useful in the advanced setting when tumor burden is high and molecular

heterogeneity of the disease and genomic complexity are actively evolving [4,13,37,38]. More comprehensive WES has increased our understanding of spatial heterogeneity by confirming divergence in the subclonal composition between the peripheral blood and BM compartment despite high concordance in clonal events [27]. Interestingly, certain aberrations seem to be particularly predominant in cell-free DNA, including mutations of highly prognostic relevance such as PIK3CA and TP53 [21,22]. As a result, interrogation of cell-free DNA may help to better identify patients with an unfavorable risk profile who otherwise would remain undetected by BM sampling alone. Compared to BM biopsies, one shortcoming of cell-free DNA sequencing is that identified mutations cannot be individually assigned to a specific location in the body. However, this limitation may be less relevant since MM is a systemic disease in most cases and effective therapeutic concepts need to address all existing genetic alterations. Consequently, a key advantage of cell-free DNA examination over conventional BM sampling is the potential to capture MM clones that may otherwise be undetectable by conventional BM biopsies. In fact, it must be noted that the molecular heterogeneity associated with resistance may be critically underestimated by a single-lesion BM biopsy [13].

4. Clinical Value Despite the potential to overcome the spatial sampling bias associated with biopsies from solitary lesions, liquid biopsy approaches today have not replaced BM testing, yet. However, cell-free DNA sequencing may be especially beneficial to several defined patient subpopulations. For patients with extramedullary MM or solitary plasmacytomas, molecular monitoring is challenging either due to the inaccessibility of lesions in the body or the inability to perform invasive sampling repetitively over time. Disease monitoring in these patients is typically ensured by serum markers and imaging technologies. The ability of such monitoring, however, is limited with regard to molecular markers of disease evolution since serological parameters are methodologically uninformative for the detection of genetic resistance mechanisms. A recent case study by Mithraprabhu and colleagues confirmed the utility of cell-free DNA sequencing in extramedullary MM [23]. In this study, WES revealed spatial and temporal heterogeneity between samples from multiple sites of an individual patient. In fact,

only a fraction of single nucleotide variants and indels could be found at all locations in the body. Moreover, driver mutations were constantly detectable in cell-free DNA and increased over the disease course whereas serological parameters pointed toward disease control. These data support that single site biopsies and serological parameters cannot always serve as a reliable genomic representation of the multifocal nature of MM and that cell-free DNA sequencing may provide a more comprehensive approach to monitor patients with extramedullary disease. Patients with non-secretory MM may also benefit from liquid biopsy approaches since SFLCs or monoclonal proteins are not available as follow-up parameters for the monitoring of these patients [39,40]. Technical considerations imply that cell-free DNA sequencing may further be useful for elderly and frail MM patients with significant comorbidities who are less likely to be subjected to frequent BM biopsies. For these patients, cell-free DNA sequencing is particularly attractive, since the technology allows shipping from remote locations at room temperature in preservative-containing tubes, as means to obtain comprehensive molecular information without the need for more invasive biopsies.

5. Monitoring of Response and Tumor Heterogeneity The extent of detectable MM-specific DNA in the peripheral blood correlates with disease activity at the time of sample acquisition [22]. Correspondingly, it has been demonstrated that tumor fractions in cell-free DNA correlate with clinical staging by the R-ISS classification system [25]. In a previous study by our group, a tumor fraction greater than 5% in cell-free DNA was only detectable in MM patients with active disease (initial diagnosis, relapse), but remained <5% in patients with controlled disease (stable disease or better) [27]. On an individual per-patient level, several single case studies or smaller cohort studies sought to determine the clinical relevance of measurable tumor fractions in cell-free DNA [23–27,29]. However, all of these studies were limited by small sample sizes and/or a lack of homogenous treatment and monitoring strategies. LPWGS-based approaches indicate that tumor fractions in cell-free DNA reflect the kinetics of established serological parameters such as SFLCs and serum M protein in the majority of patients [25,27]. In some cases, LPWGS can additionally detect the outgrowth of resistant clones harboring novel amplifications or deletions, thereby demonstrating that cell-free DNA profiling

may provide a powerful tool to follow clonal outgrowth of MM [27]. For more granular assessment of clonal evolution in individual patients, LPWGS needs to be complemented by deeper sequencing methods, e.g. WGS or targeted DNA sequencing. Competitive emergence of drug-resistant clones may also be captured by sequential circulating tumor DNA quantitation using ddPCR [24]. Both methods allow to prioritize and deprioritize competing mutations during the development of relapse and may therefore inform precision medicine approaches. At the same time, ddPCR requires prior identification of resistant mutations by targeted sequencing approaches and is thus not eligible to primarily identify resistance mechanism in an unbiased manner. Cell-free DNA can further be assessed by examining IgH rearrangements [26,29]. In a study by Oberle and colleagues, sequencing of cell-free DNA for such clonotypic events recapitulated the persistence of MM in 91% of nonresponders vs. 41% of responders [26]. Interestingly, responders maintained high paraprotein levels indicating that cell-free DNA provides information about tumor burden in MM patients which is distinct from conventional markers of the disease which are cleared from the blood at a slower rate [41].

6. Detection of Minimal Residual Disease The detectability of minimal residual disease (MRD) in patients with MM has emerged as a useful prognostic marker which is associated with improved progression-free survival in many studies [42–44]. MRD can be detected in the BM by multi-color flow cytometry and NGS-based clonotype detection with a sensitivity as low as 10-5 to 10-6 [43,45]. The frequency at which such MRD detection may be performed, however, is limited by the pain and complications caused by BM biopsies. Interrogation of cell-free DNA may allow for more frequent testing of patients, as it decreases the risk and discomfort associated with invasive sampling. Similarly, liquid biopsy approaches also hold the potential to reflect residual tumor burden in previously unidentified locations of the body. Current cell-free DNA based technologies are limited for their use in MRD detection by simple quantitative considerations [46]. The amount of cell-free DNA from the plasma of MM patients is highly variable [25]. The highest quantity of ideally extractable cell-free DNA has been reported at a maximum of 92,000 genome equivalents per sample [21]. More commonly, the average yield is rather measured

at ~1,000 to ~10,000 genome equivalents [21,28]. MRD detection by targeted sequencing approaches will thus remain intrinsically underpowered to identify single mutations at a sensitivity comparable to MRD testing from the BM. To overcome this technical challenge, future paths for MRD detection in cell-free DNA may improve detection power by combining multiple parameters, e.g. by integrating patient-specific mutation

panels,

methylation

patterns,

copy

number

alterations

and

Ig

rearrangements in parallel. The sensitivity of PCR-based approaches may be increased by allele-specific oligonucleotide (ASO) real-time quantitative (RQ)-PCR that utilizes patient-specific primers designed from the complementarity determining region 3 (CDR3) sequence of the Ig genes [47]. With the implementation of these methods, the incorporation of unique barcode identifiers holds the potential to critically enable error suppression at a frequency <10-5, thereby considerably improving the technically achievable sensitivity for MRD detection [48]. Notably, even at sensitivity levels that are attainable to date, the presence of MM-specific tumor fractions in cell-free DNA may help to identify patients who are misclassified as having achieved MRD negativity by BM assessment.

7. Early Cancer Detection In theory, interrogation of cell-free DNA holds the potential for early cancer detection through a simple blood draw. The sensitivity and specificity of liquid biopsy approaches, however, remain a major limitation to date [49]. MM primarily affects patients of more advanced age who are at an increased risk of co-existing malignancies or precursor diseases. Driver genes of MM such as NRAS, KRAS, BRAF and TP53 are shared with other cancer types and if identified in a liquid biopsy, cannot be unambiguously assigned to MM or its precursor state, monoclonal gammopathy of undetermined significance (MGUS) [4,7,8,50]. Recent reports suggest that early precursor states of melanoma can produce cell-free DNA with tumor-specific BRAF mutations [51]. Clonal hematopoiesis of indeterminate potential (CHIP) represents another example of a benign, but highly prevalent condition that coincides with MM [52]. CHIP increases exponentially with age, with a frequency >10% among healthy individuals aged 70 years or older. The overlap between the mutational spectrum of CHIP and MM or other plasma cell precursor diseases makes it an integral confounder in the age group typically affected by MM. In summary, the

inability to assign mutational alterations to a specific, previously unidentified tumor disease poses a major challenge to the utility of targeted sequencing approaches for the early detection of MM. Genome-wide copy number alterations may provide a more unbiased metric for the early detection of MM-specific aberrations. A recent study by Manier and colleagues identified a fraction of MGUS and SMM patients with detectable MM-derived cell-free DNA, and the median tumor fraction correlated with disease progression from MGUS to SMM and overt MM [25]. Further validation in larger prospective studies will be needed to specify the positive and negative predictive value of this approach for the detection of plasma cell precursor conditions to determine the risk of developing MM in otherwise healthy individuals.

8. Alternatives to cell-free DNA Liquid biopsy approaches in MM are not limited to cell-free DNA. Several other methodologies have been described, including the querying of molecular information from i.) circulating MM cells (CMMCs), ii) cell-free RNA and iii) microRNAs [24,45,53–57]. CMMCs provide a rich resource for minimally invasive molecular profiling of MM. Our group

has

recently

developed

a

CD138+/CD45−cell

enrichment

single-cell

micromanipulation platform using serial dilution and fluorescence microscopy [45]. This technology allows to detect CMMCs down to a frequency of <1:106. Similar to cell-free DNA, mutational profiles of CMMCs are highly concordant with the BM compartment but show relevant disparity at a subclonal level [25,45,53]. Compared to cell-free DNA, CMMCs have certain merits, as they are by definition intact cells that can simultaneously be assessed for many parameters such as morphology and surface protein expression by multiparameter flow and mass cytometry [58,59]. By assessing CMMCs for the co-expression of typical MM surface markers, they can be reliably assigned to the actual disease which is a clear advantage over cell-free DNA sequencing with its potentially confounding bias caused by co-existing malignancies. With the advent of NGS, CMMCs may also be subjected to single-cell RNA sequencing. This technology allows to identify cancer drivers and mechanisms of molecular drug resistance at unprecedented granularity (3,500-4,000 genes on average) which seems particularly attractive for the monitoring of emerging immunotherapies [60].

Furthermore, identification of cell-free RNA may, in theory, represent a therapyspecific proxy for early response evaluation. In a recent proof-of-concept study on 24 RRMM

patients

receiving

oral

azacytidine/

lenalidomide/

dexamethasone,

Mitraprabhu and colleagues observed a correlation between survival and a decrease of cell-free RNA transcripts for genes known to be modulated by lenalidomide after five days of treatment [24]. Technical shortcomings of such RNA-based approaches however remain i) the inferior stability of cell-free RNA as compared to cell-free DNA and ii) the inability to differentiate MM-specific cell-free RNA from non-cancerous background RNA. Lastly, the role of circulating miRNAs in MM has been another area of active investigation over the past years. Arrays for sets of miRNAs have the potential to complement current liquid biopsy technologies. Past studies have identified miRNA signatures for all stages of MM precursor diseases, ranging from MGUS to SMM, MM and plasma cell leukemia [54–57]. Despite a multitude of currently available micro-array platforms, all of these methodologies, however, have not yet succeeded in establishing uniform signatures that remain consistent across independent study cohorts. Consequently, current microRNA arrays require better standardization and further investigation is needed to demonstrate their clinical and biological value.

9. Summary and Future Challenges Tracking of MM by examination of cell-free DNA in the peripheral blood enables (i) identification and robust quantification of MM-derived genetic material from background germline DNA, (ii) characterization of the mutational landscape of MM at excellent concordance with BM without the need for invasive BM biopsy (iii) identification of previously unidentified resistance mechanisms, and (iv) detection of genomic aberrations in a quantitative manner. Current approaches utilize genomic (targeted DNA sequencing, low-pass WGS, WES) and molecular techniques (multiplex PCR, ddPCR) that are highly scalable, e.g. by the integration of broader sequencing panels, patient-specific PCR primers and cell-free RNA sequencing. In its current state, cell-free DNA sequencing represents a promising methodology to complement BM testing and to obtain robust molecular information of MM when BM testing is not performed. Large-scale technologies are becoming more inexpensive and will likely enable the implementation of cell-free DNA assessment into the routine

diagnostic work-up for MM. The integration of cell-free DNA sequencing into multicenter clinical trials will determine its value as an independent prognostic parameter for diagnosis and clinical monitoring of MM beyond currently available markers. The convenience and ease of a simple blood draw over a BM biopsy is of immeasurable benefit for patients and strongly warrants further investigation on the value of such approaches in the context of MM.

Practice Points: -

Cell-free DNA allows for frequent molecular monitoring without the need for invasive BM biopsies.

-

Cell-free DNA reflects tumor burden and may be useful to monitor disease activity in patients for which current clinical parameters are not available (extramedullary/ non-secretory MM).

-

Cell-free DNA characterizes the genomic landscape at excellent concordance with matching BM.

-

The sensitivity of cell-free DNA for longitudinal molecular monitoring and detection of the genomic landscape of MM depends on the amount of cell-free DNA in the circulation and the sequencing technology used. Detectability of MM-derived cell-free DNA is therefore challenging in patients with very low disease burden.

-

Samples can be stored at room temperature until further assessment by NGS and PCR.

Research Agenda: -

Cell-free DNA has the potential to complement laboratory tests that are currently used in the routine monitoring of MM.

-

Future trials are required to determine the value of cell-free DNA as an independent prognostic and predictive parameter for MM.

-

Sensitivity levels for detecting low disease burden, including MRD, remain to be defined and standardized.

Acknowledgements: J.M.W. is supported by a postdoctoral fellowship of Deutsche Forschungsgemeinschaft (German Research Foundation, 391926441). J.G.L. is supported by the NCI (K08CA191026) and the V foundation for Cancer Research. B.K. is supported by the NCI (K08CA191091), the St. Baldrick’s Foundation, Hyundai Hope on Wheels Foundation, and the AACR Mark Foundation. Authorship Contributions: All authors participated in discussing and writing the manuscript. Conflict of Interest Disclosures: J.G.L.: Consultant for T2 Biosystems. All other authors declare no conflicts of interest.

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