Liquid biopsy: A promising tool for driving strategies and predicting failures in patients with classic Hodgkin lymphoma

Classic Hodgkin lymphoma (cHL) consists of a heterogeneous group of haematological disorders that covers undifferentiated B cell neoplasms originating from germinal centre B cells. The HL molecular characterization still represents an ongoing challenge due to the low fraction of tumour Hodgkin and Reed–Sternberg cells mixed with a plethora of non‐tumour haematological cells. In this scenario, next generation sequencing of liquid biopsy samples is emerging as a useful tool in HL patients' management. In this review, we aimed to overview the clinical and methodological topics regarding the implementation of molecular analysis in cHL, focusing on the role of liquid biopsy in diagnosis, follow‐up, and response prediction.


| INTRODUC TI ON
2][3] In this scenario, molecular profiling is considered a new weapon aimed at integrating morphological diagnosis.
[6] Although formalin-fixed paraffin-embedded (FFPE) samples still remain the most suitable diagnostic material for the implementation of molecular tests, liquid biopsy is considered an emerging tool able to integrate conventional tissue specimens into the clinical setting.
Here, we overview clinical and methodological topics related to the implementation of molecular analysis in cHL patients.

| HL MOLECUL AR ANALYS IS: E XPERIEN CE S WITH FFPE MATERIAL
To date, several assays have been developed to routinely perform molecular analysis in cHL patients.First, Küppers et al 7 focused on the evaluation of the mRNA expression profile in preclinical models in order to establish a common signature in cHL patients.This effort was based on the implementation of U95A microarrays (Affymetrix Inc.) on the corresponding platform.Data processing was carried out by using Affymetrix Microarray Suite 4.0 software selecting an arbitrary cutoff of 20 to determine expression level variations. 7Briefly, cell lines from different haematological malignancies highlighted a distinct expression profile able to distinguish cHL from the other groups.Of note, five of the upregulated genes (PRAME, IPL, FER, Rab13, and EAR3) from a previously detected positive group were confirmed in tissue samples from nine cHL patients by adopting a customised real time polymerase chain reaction (RT-PCR) assay. 7milarly, Malec et al 8 investigated the role of inflammation in cHL patients by inspecting pro-inflammatory cytokine levels in 15 patients with heterogeneous morphological features.In this case, an optimised RT-PCR system was enabled to perform molecular analysis.Overall, a matched cytokines expression pattern between preclinical models and patients' samples was identified.Briefly, IL-10, IL-1b, IL-15 and IL-12p35 were more highly expressed than matched control samples. 8These data demonstrated that cHLs drastically impact the modulation of both the mRNA expression profile of tumour cells and modifications of cytokines levels.These strategies highlighted some significant technical and clinical challenges.First, these technical approaches were able to work successfully only when starting from frozen samples.In addition, the identification of non-recurrent expression signatures indicated that the clinical role of other promising biomarkers should be investigated.9][10] Recently, a whole exome sequencing approach has been validated to comprehensively analyse molecular profiles (DNA and RNA) in a set of 10 cHL patients.In brief, Reichel et al optimised KAPA Biosystems technical workflow integrated with a sorting system in order to selectively improve technical sensitivity in target cells. 10markably, a median number of 244 somatic mutations were detected in each case.Among them, B2M harboured different molecular alterations that inactivated protein function. 10It was observed that B2M loss of function plays a pivotal role as a diagnostic (NS subtype 86/115 cases; 75%) and prognostic biomarker (B2M expression is significantly associated with stage III-IV; p = 0.001) in cHL patients. 10Similarly, Mata et al implemented a deep NGS platform based on a semiconductor system for the molecular analysis of cancer-related genes by adopting a customised panel integrated with a dedicated bioinformatic pipeline. 9Focusing on 57 FFPE samples from cHL patients, CSF2RB, EP300, STAT6, and BTK showed the highest mutation rate according to the most frequent deregulated pathway in cHL patients. 9Moreover, a not negligible percentage of cases (10.3%) was affected by somatic alterations in members of the B cell receptor pathway, considered an emerging target for therapeutic approaches. 9In another experiment, Mata et al 11

| CHL MOLECUL AR ANALYS IS: E XPERIEN CE S ON LI QU ID B I OPSY
The liquid biopsy consists of drawn peripheral blood, providing a less invasive, ready-to-use and dynamic sampling modality to investigate a plethora of different biomarkers. 12,13It was observed that circulating tumour DNA levels (ctDNA) are age-and gender-dependent in lymphoma patients 14,15 with a statistically significant rate higher levels in advanced stage and low responder patients. 14,16In this regard, NGS platforms include an heterogenous group of sequencing systems that allow simultaneous detection of a broad spectrum of cancer-related genes starting from a low number of nucleic acids fragments. 13,14,17Of note, one of the most frequently adopted methods is an ultra-sensitive hybrid selection-based panel (SeqCap EZ Choice Library) on the MiSeq platform (Illumina).8][19] Interestingly, Spina et al 20 20 As previously demonstrated, this approach enabled the selective identification of most recurrent cancer-related molecular alterations at very low frequencies. 18,19Data analysis was optimised according to molecular alteration recurrence.In particular, a mutant allele fraction (MAF) ≥5.0% and ranged between 1.0% and 5.0% for unknown and well-established cancer-related genes were taken in account, respectively. 20Molecular analysis was approached on liquid biopsy samples at different clinically relevant timing points: at baseline, during the course (interim) and at the conclusion of a conventional ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy regimen, or at progression in resistant cHL patients.
In addition, genomic DNA extracted from white cells on matched plasma specimens and from neoplastic cells on tissue specimens without any HRS cell selection was collected for molecular testing.
Remarkably, TNFAIP3, ITPKB, GNA13, and B2M driver alterations not yet reported in the literature were found in cell free DNA (cfDNA) samples of 15 cHL patients. 20A similar molecular spectrum between newly diagnosed and relapsing cHL patients was observed; this point demonstrated the technical feasibility of this approach for the selective identification of molecular alterations settled in tumour cells.In addition, biopsy specimens also highlighted an agreement of matched results according to cfDNA-based detected alterations (87.5%).On the basis of these results, NGS showed a median of five somatic mutations in 81.2% of newly diagnosed cHL cases; then a median MAF was observed as 5.5% while the vast majority of molecular alterations had a frequency higher than 1.0% (87.3%). 20Moreover, different histological groups matched with specific molecular profiles.

TA B L E 1 List of available technical approaches for the molecular analysis of cHL patients in clinical routine practice
Briefly, a retrospective series of 60 newly diagnosed cHL patients was evaluated by using a semiconductor-based NGS platform that covers hotspot molecular alterations in pivotal genes for haematological malignancies patients. 21Data analysis was carried out by filtering MAF below 0.5%.Overall, a concordance rate of 83.3% (61 variants) was found between tissue and matched cfDNA samples; then, a higher amount of cfDNA was observed in patients with a worse clinical outcome, and in fact, a statistically significant correlation was observed with tumour volume. 21

| LIQU ID B I OPSY IN CHL : A PROG NOS TI C TOOL
Currently, there are no biomarkers that are predictive of cHL. 22andard lymphadenectomy or tissue biopsy is the recommended method for lymphoma diagnosis and is a routine method of oncogene profiling, but the rarity of HRS cells increases the complexity of pathological diagnosis in small biopsy samples.In fact, to date, the genetic landscape of cHL has been only incompletely described because HRS tumour cells are very few. 23Despite the scarcity of HRS cells and a typical lower tumour volume, many studies suggest that cHL seems to display a higher trend of releasing cfDNA than diffuse large B cell lymphoma (DLBCL).This finding may be related to an increased fraction of tumour cells in apoptosis but also related to the nature of the alterations in the nuclear DNA of HRS cells. 24r patients with cHL it is very crucial to discriminate the responders from non-responders at the end of frontline chemotherapy with ABVD, in order to intensify treatment with salvage regimens, and to detect possible relapse in a timely manner. 25rrently, the 2-deoxy-2[F-18]fluoro-D-glucose positron emission tomography (FDG-PET) is considered the standard of care for remission assessment in HL 26 and is driving strategies of treatment in patients with cHL.However, the cfDNA baseline assessment might foresee the patient's outcome.Indeed, there is a link between the tumour burden (expressed either as metabolic tumour volume [MTV] or as total metabolic tumour surface or as volume of the bounding box including tumour [TumBB]) and the amount of cfDNA released.Thus, cfDNA could be complementary to FDG-PET staging. 27 20%-30% of patients interim FDG-PET results do not correspond with the final outcome.In these cases in particular, ct/cfDNA may come to the aid, being potentially complementary or even a convenient substitute for imaging. 14,27 a matter of fact, in the recent years, studies on liquid biopsy have proven that the peripheral blood level of ctDNA at interim (after two cycles) or at the end of treatment (EOT) strictly correlates with disease response and that it could be a potential tool for evaluating the MRD in the real-world setting.Spina et al 20  According to many trials (GHSG HD18 trial, LYSA AHL2011, RATHL trial) interim PET/CT results guide the subsequent treatment, either in terms of intensification to improve the response (GHSG HD18 trial), or in terms of de-escalation when a response is already successful (LYSA AHL2011, RATHL trial). 29e real prognostic significance of interim PET/CT in HL is yet to be completely validated; thus, patients would probably benefit from the inclusion of additional strategies to complement the therapeutic strategy of PET indication.Decazes et al 27 prospectively analysed the correlation between cfDNA and PET/TC parameters in 50 patients with cHL.In particular, two burden parameters as total lesion glycolysis (TLG) and total MTV presented the highest correlation with ctDNA.Among the dispersion parameters, TumBB (which reflects the dispersion volume of the tumours) was highly correlated with ctDNA in cHL.PET/ctDNA correlation is nowadays a growing trend. 27

| LI QU ID B I OPSY IN CHL : PRED I C TI ON FOR CLONAL E VOLUTION
Assessment of cfDNA may help to identify clonal evolution in cHL.
The panel used by Camus et al was likely too narrow and insufficiently sensitive to detect certain subclonal mutations of low allelic frequency, close to the noise threshold of the NGS sequencer (variant allele frequency [VAF] 0.1%).In addition, it was insufficient for detecting somatic alterations in all patients due to the limited number of included genes. 21However, Spina et al also genotyped longitudinal ctDNA samples from 13 patients collected at diagnosis, at time of relapse, and during salvage therapies.In all cases they pointed out clonal shifts between pretreatment and relapse samples.In particular they inferred that an ancestral clone persisted analysed 35 mostly mutated genes in 20 cHL patients by adopting a semiconductor-based NGS platform for a series of matched original pretreatment and relapse FFPE biopsy samples.Overall, an increasing rate of high impacting p53 molecular alterations and inactivating molecular alterations in epigenetic regulators EP300 and CREBBP were detected in the refractory samples.This study demonstrated the dynamic evolution of the molecular landscape in cHL patients after a conventional treatment strategy.11 focused on the comprehensive role of liquid biopsy in the diagnosis of cHL without any previous HRS selection, for the identification of the molecular evolution of tumour cells and the detection of minimal residual disease (MRD) after conventional therapeutic strategies.The authors designed and developed a customised NGS panel able to detect hotspot exons and intronic sites in 77 mostly mutated genes in mature B cell tumours on a series of 80 naïve and 32 relapsed or refractory (R/R) first line cHL patients.
28monstrated that a 2-log drop in cfDNA between diagnosis and at the end of the second ABVD course can predict metabolic complete response, in particular when the cfDNA trend did not match with interim PET/TC results.The 2-log drop threshold was borrowed from expertise on DLBCL and is named early molecular response-EMR (after 1 cycle of treatment).Another threshold used for DLBCL is a 2.5-log drop after 2 cycles of treatment, named major molecular response (MMR).28In the same way, in the report by Camus et al a consistent de-