Erythroid Predominance in Bone Marrow Biopsies of Acute Myeloid Leukemia Patients after Decitabine Treatment Correlates with Mutation Profile and Complete Remission.

INTRODUCTION: Acute myeloid leukemia (AML) patients may receive hypomethylating agents such as decitabine (DAC) as part of their treatment. Not all patients respond to this therapy, and if they do, the clinical response may occur only after 3-6 courses of treatment. Hence, early biomarkers predicting response would be very useful. METHODS: We retrospectively analyzed a cohort of 22 AML patients who were treated with DAC. Histology of the bone marrow biopsy, pathogenic mutations, and methylation status were related to the treatment response. RESULTS: In 8/22 (36%) patients, an erythroid dominant response (EDR) pattern, defined as a ratio of myeloid cells/erythroid cells <1, was observed. In the remaining 14 cases, a myeloid predominance was preserved during treatment. No difference in the hypomethylating effect of DAC treatment was observed in patients with and without EDR, as global 5-methylcytosine levels dropped similarly in both groups. Mutational analysis by NGS using a panel of commonly mutated genes in AML showed that patients with an early EDR harbored on average less mutations, with U2AF1 mutations occurring more frequently, whereas RUNX1 mutations were underrepresented compared to non-EDR cases. Interestingly, the development of an EDR correlated with complete remission (7/8 cases with an EDR vs. only 2/14 cases without an EDR). CONCLUSION: We conclude that early histological bone marrow examination for the development of an EDR may be helpful to predict response in AML patients during treatment with DAC.

Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos.

Detection of Second Primary Lymphoma in Late Diffuse Large B-cell Lymphoma Recurrences.

Approximately one-third of patients with diffuse large B-cell lymphoma (DLBCL) relapse and often require salvage chemotherapy followed by autologous stem cell transplantation. In most cases, the clonal relationship between the first diagnosis and subsequent relapse is not assessed, thereby potentially missing the identification of second primary lymphoma. In this study, the clonal relationship of 59 paired DLBCL diagnoses and recurrences was established by next-generation sequencing-based detection of immunoglobulin gene rearrangements. Among 50 patients with interpretable results, 43 patients (86%) developed clonally related relapsed disease. This was observed in 100% of early recurrences (<2 years), 80% of the recurrences with an interval between 2 and 5 years, and 73% of late recurrences (≥5 years). On the other hand, 7 (14%) out of 50 patients displayed different dominant clonotypes in primary DLBCL and clinical recurrences, confirming the occurrence of second primary DLBCL; 37% of DLBCL recurrences that occurred ≥4 years after diagnosis were shown to be second primary lymphomas. The clonally unrelated cases were Epstein-Barr virus positive in 43% of the cases, whereas this was only 5% in the relapsed DLBCL cases. In conclusion, next-generation sequencing-based clonality testing in late recurrences should be considered in routine diagnostics to distinguish relapse from second primary lymphoma, as this latter group of patients with DLBCL may benefit from less-intensive treatment strategies.

Impact of molecular tumour board discussion on targeted therapy allocation in advanced prostate cancer.

BACKGROUND: Molecular tumour boards (MTB) optimally match oncological therapies to patients with genetic aberrations. Prostate cancer (PCa) is underrepresented in these MTB discussions. This study describes the impact of routine genetic profiling and MTB referral on the outcome of PCa patients in a tertiary referral centre. METHODS: All PCa patients that received next-generation sequencing results and/or were discussed at an MTB between Jan 1, 2017 and Jan 1, 2020 were included. Genetically matched therapies (GMT) in clinical trials or compassionate use were linked to actionable alterations. Response to these agents was retrospectively evaluated. RESULTS: Out of the 277 genetically profiled PCa patients, 215 (78%) were discussed in at least one MTB meeting. A GMT was recommended to 102 patients (47%), of which 63 patients (62%) initiated the GMT. The most recommended therapies were PARP inhibitors (n = 74), programmed death-(ligand) 1 inhibitors (n = 21) and tyrosine kinase inhibitors (n = 19). Once started, 41.3% had a PFS of ≥6 months, 43.5% a PSA decline ≥50% and 38.5% an objective radiographic response. CONCLUSION: Recommendation for a GMT is achieved in almost half of the patients with advanced prostate cancer, with GMT initiation leading to durable responses in over 40% of patients. These data justify routine referral of selected PCa patients to MTB's.

Impact of DNA damage repair defects and aggressive variant features on response to carboplatin-based chemotherapy in metastatic castration-resistant prostate cancer.

Platinum-based chemotherapy is not standard of care for unselected or genetically selected metastatic castration-resistant prostate cancer (mCRPC) patients. A retrospective assessment of 71 patients was performed on platinum use in the Netherlands. Genetically unselected patients yielded low response rates. For a predefined subanalysis of all patients with comprehensive next-generation sequencing, 30 patients were grouped based on the presence of pathogenic aberrations in genes associated with DNA damage repair (DDR) or aggressive variant prostate cancer (AVPC). Fourteen patients (47%) were DDR deficient (DDRd), of which seven with inactivated BRCA2 (BRCA2mut). Six patients classified as AVPC. DDRd patients showed beneficial biochemical response to carboplatin, largely driven by all BRCA2mut patients having >50% prostate-specific antigen (PSA) decline and objective radiographic response. In the wild-type BRCA2 subgroup, 35% had a >50% PSA decline (P = .006) and 16% radiographic response (P < .001). Median overall survival was 21 months for BRCA2mut patients vs 7 months (P = .041) for those with functional BRCA2. AVPC patients demonstrated comparable responses to non-AVPC, including a similar overall survival, despite the poor prognosis for this subgroup. In the scope of the registration of poly-(ADP)-ribose polymerase inhibitors (PARPi) for mCRPC, we provide initial insights on cross-resistance between PARPi and platinum compounds. By combining the literature and our study, we identified 18 patients who received both agents. In this cohort, only BRCA2mut patients treated with platinum first (n = 4), responded to both agents. We confirm that BRCA2 inactivation is associated with meaningful responses to carboplatin, suggesting a role for both PARPi and platinum-based chemotherapy in preselected mCRPC patients.

Multicenter Comparison of Molecular Tumor Boards in The Netherlands: Definition, Composition, Methods, and Targeted Therapy Recommendations.

BACKGROUND: Molecular tumor boards (MTBs) provide rational, genomics-driven, patient-tailored treatment recommendations. Worldwide, MTBs differ in terms of scope, composition, methods, and recommendations. This study aimed to assess differences in methods and agreement in treatment recommendations among MTBs from tertiary cancer referral centers in The Netherlands. MATERIALS AND METHODS: MTBs from all tertiary cancer referral centers in The Netherlands were invited to participate. A survey assessing scope, value, logistics, composition, decision-making method, reporting, and registration of the MTBs was completed through on-site interviews with members from each MTB. Targeted therapy recommendations were compared using 10 anonymized cases. Participating MTBs were asked to provide a treatment recommendation in accordance with their own methods. Agreement was based on which molecular alteration(s) was considered actionable with the next line of targeted therapy. RESULTS: Interviews with 24 members of eight MTBs revealed that all participating MTBs focused on rare or complex mutational cancer profiles, operated independently of cancer type-specific multidisciplinary teams, and consisted of at least (thoracic and/or medical) oncologists, pathologists, and clinical scientists in molecular pathology. Differences were the types of cancer discussed and the methods used to achieve a recommendation. Nevertheless, agreement among MTB recommendations, based on identified actionable molecular alteration(s), was high for the 10 evaluated cases (86%). CONCLUSION: MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational cancer profiles. We propose a "Dutch MTB model" for an optimal, collaborative, and nationally aligned MTB workflow. IMPLICATIONS FOR PRACTICE: Interpretation of genomic analyses for optimal choice of target therapy for patients with cancer is becoming increasingly complex. A molecular tumor board (MTB) supports oncologists in rationalizing therapy options. However, there is no consensus on the most optimal setup for an MTB, which can affect the quality of recommendations. This study reveals that the eight MTBs associated with tertiary cancer referral centers in The Netherlands are similar in setup and reach a high agreement in recommendations for rare or complex mutational profiles. The Dutch MTB model is based on a collaborative and nationally aligned workflow with interinstitutional collaboration and data sharing.

Mutational signature analysis in non-small cell lung cancer patients with a high tumor mutational burden.

BACKGROUND: Lung cancer is the leading cause of cancer death worldwide. With the growing number of targeted therapies and the introduction of immuno-oncology (IO), personalized medicine has become standard of care in patients with metastatic disease. The development of predictive and prognostic biomarkers is of great importance. Mutational signatures harbor potential clinical value as predictors of therapy response in cancer. Here we set out to investigate particular mutational processes by assessing mutational signatures and associations with clinical features, tumor mutational burden (TMB) and targetable mutations. METHODS: In this retrospective study, we studied tumor DNA from patients with non-small cell lung cancer (NSCLC) irrespective of stage. The samples were sequenced using a 2 megabase (Mb) gene panel. On each sample TMB was determined and defined as the total number of single nucleotide mutations per Mb (mut/Mb) including non-synonymous mutations. Mutational signature profiling was performed on tumor samples in which at least 30 somatic single base substitutions (SBS) were detected. RESULTS: In total 195 samples were sequenced. Median total TMB was 10.3 mut/Mb (range 0-109.3). Mutational signatures were evaluated in 76 tumor samples (39%; median TMB 15.2 mut/Mb). SBS signature 4 (SBS4), associated with tobacco smoking, was prominently present in 25 of 76 samples (33%). SBS2 and/or SBS13, both associated with activity of the AID/APOBEC family of cytidine deaminases, were observed in 11 of 76 samples (14%). SBS4 was significantly more present in early stages (I and II) versus advanced stages (III and IV; P = .005). CONCLUSION: In a large proportion of NSCLC patients tissue panel sequencing with a 2 Mb panel can be used to determine the mutational signatures. In general, mutational signature SBS4 was more often found in early versus advanced stages of NSCLC. Further studies are needed to determine the clinical utility of mutational signature analyses.

Prediction of clinical benefit from androgen deprivation therapy in salivary duct carcinoma patients.

Androgen deprivation therapy (ADT) is first-line palliative treatment in androgen receptor-positive (AR+) salivary duct carcinoma (SDC), and response rates are 17.6-50.0%. We investigated potential primary ADT resistance mechanisms for their predictive value of clinical benefit from ADT in a cohort of recurrent/metastatic SDC patients receiving palliative ADT (n = 30). We examined mRNA expression of androgen receptor (AR), AR splice variant-7, intratumoral androgen synthesis enzyme-encoding genes AKR1C3, CYP17A1, SRD5A1 and SRD5A2, AR protein expression, ERBB2 (HER2) gene amplification and DNA mutations in driver genes. Furthermore, functional AR pathway activity was determined using a previously reported Bayesian model which infers pathway activity from AR target gene expression levels. SRD5A1 expression levels and AR pathway activity scores were significantly higher in patients with clinical benefit from ADT compared to those without benefit. Survival analysis showed a trend toward a longer median progression-free survival for patients with high SRD5A1 expression levels and high AR pathway activity scores. The AR pathway activity analysis, and not SRD5A1 expression, also showed a trend toward better disease-free survival in an independent cohort of locally advanced SDC patients receiving adjuvant ADT (n = 14) after surgical tumor resection, and in most cases a neck dissection (13/14 patients) and postoperative radiotherapy (13/14 patients). In conclusion, we are the first to describe that AR pathway activity may predict clinical benefit from ADT in SDC patients, but validation in a prospective study is needed.

A novel next generation sequencing approach to improve sarcoma diagnosis.

Sarcoma is a rare disease affecting both bone and connective tissue and with over 100 pathologic entities, differential diagnosis can be difficult. Complementing immune-histological diagnosis with current ancillary diagnostic techniques, including FISH and RT-PCR, can lead to inconclusive results in a significant number of cases. We describe here the design and validation of a novel sequencing tool to improve sarcoma diagnosis. A NGS DNA capture panel containing probes for 87 fusion genes and 7 genes with frequent copy number changes was designed and optimized. A cohort of 113 DNA samples extracted from soft-tissue and bone sarcoma FFPE material with clinical FISH and/or RT-PCR results positive for either a translocation or gene amplification was used for validation of the NGS method. Sarcoma-specific translocations or gene amplifications were confirmed in 110 out of 113 cases using FISH and/or RT-PCR as gold-standard. MDM2/CDK4 amplification and a total of 25 distinct fusion genes were identified in this cohort of patients using the NGS approach. Overall, the sensitivity of the NGS panel is 97% with a specificity of 100 and 0% failure rate. Targeted NGS appears to be a feasible and cost-effective approach to improve sarcoma subtype diagnosis with the ability to screen for a wide range of genetic aberrations in one test.

Early detection and evolution of preleukemic clones in therapy-related myeloid neoplasms following autologous SCT.

Therapy-related myeloid neoplasms (tMNs) are severe adverse events that can occur after treatment with autologous hematopoietic stem cell transplantation (ASCT). This study aimed to investigate the development of tMNs following ASCT at the molecular level by whole-exome sequencing (WES) and targeted deep sequencing (TDS) in sequential (pre-) tMN samples. WES identified a significantly higher number of mutations in tMNs as compared with de novo myelodysplastic syndrome (MDS) (median 27 vs 12 mutations; P = .001). The mutations found in tMNs did not carry a clear aging-signature, unlike the mutations found in de novo MDS, indicating a different mutational mechanism. In some patients, tMN mutations were identified in both myeloid and T cells, suggesting that tMNs may originate from early hematopoietic stem cells (HSCs). However, the mutational spectra of tMNs and the preceding malignancies did not overlap, excluding common ancestry for these malignancies. By use of TDS, tMN mutations were identified at low variant allele frequencies (VAFs) in transplant material in 70% of the patients with tMNs. Reconstruction of clonal patterns based on VAFs revealed that premalignant clones can be present more than 7 years preceding a tMN diagnosis, a finding that was confirmed by immunohistochemistry on bone marrow biopsies. Our results indicate that tMN development after ASCT originates in HSCs bearing (pre-)tMN mutations that are present years before disease onset and that post-ASCT treatment can influence the selection of these clones. Early detection of premalignant clones and monitoring of their evolutionary trajectory may help to predict the development of tMNs and guide early intervention in the future.

Comprehensive routine diagnostic screening to identify predictive mutations, gene amplifications, and microsatellite instability in FFPE tumor material.

BACKGROUND: Sensitive and reliable molecular diagnostics is needed to guide therapeutic decisions for cancer patients. Although less material becomes available for testing, genetic markers are rapidly expanding. Simultaneous detection of predictive markers, including mutations, gene amplifications and MSI, will save valuable material, time and costs. METHODS: Using a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach, we developed an NGS panel allowing detection of predictive mutations in 33 genes, gene amplifications of 13 genes and microsatellite instability (MSI) by the evaluation of 55 microsatellite markers. The panel was designed to target all clinically relevant single and multiple nucleotide mutations in routinely available lung cancer, colorectal cancer, melanoma, and gastro-intestinal stromal tumor samples, but is useful for a broader set of tumor types. RESULTS: The smMIP-based NGS panel was successfully validated and cut-off values were established for reliable gene amplification analysis (i.e. relative coverage ≥3) and MSI detection (≥30% unstable loci). After validation, 728 routine diagnostic tumor samples including a broad range of tumor types were sequenced with sufficient sensitivity (2.4% drop-out), including samples with low DNA input (< 10 ng; 88% successful), low tumor purity (5-10%; 77% successful), and cytological material (90% successful). 75% of these tumor samples showed ≥1 (likely) pathogenic mutation, including targetable mutations (e.g. EGFR, BRAF, MET, ERBB2, KIT, PDGFRA). Amplifications were observed in 5.5% of the samples, comprising clinically relevant amplifications (e.g. MET, ERBB2, FGFR1). 1.5% of the tumor samples were classified as MSI-high, including both MSI-prone and non-MSI-prone tumors. CONCLUSIONS: We developed a comprehensive workflow for predictive analysis of diagnostic tumor samples. The smMIP-based NGS analysis was shown suitable for limited amounts of histological and cytological material. As smMIP technology allows easy adaptation of panels, this approach can comply with the rapidly expanding molecular markers.

5-Hydroxymethylcytosine: An epigenetic mark frequently deregulated in cancer.

The epigenetic mark 5-hydroxymethylcytosine (5hmC) has gained interest since 2009, when it was discovered that Ten-Eleven-Translocation (TET) proteins catalyze the conversion of 5-methylcytosine (5mC) into 5hmC. This conversion appears to be an intermediate step in the active DNA demethylation pathway. Factors that regulate DNA hydroxymethylation are frequently affected in cancer, leading to deregulated 5hmC levels. In this review, we will discuss the regulation of DNA hydroxymethylation, defects in this pathway in cancer, and novel therapies that may correct deregulated (hydroxy)methylation of DNA.

Characterization of acute myeloid leukemia based on levels of global hydroxymethylation.

Patients with acute myeloid leukemia (AML) frequently harbor mutations in genes involved in the DNA (hydroxy)methylation pathway (DNMT3A, TET2, IDH1, and IDH2). In this study, we measured 5-hydroxymethylcytosine (5hmC) levels in 206 clinically and molecularly well-characterized younger adult AML patients (≤60 years) included in the European Organization for Research and Treatment of Cancer/Gruppo Italiano Malattie Ematologiche dell'Adulto (EORTC/GIMEMA) AML-12 06991 clinical trial and correlated the 5hmC levels with mutational status and overall survival (OS). In healthy control cells, 5hmC levels were confined to a narrow range (1.5-fold difference), whereas in AML cells, a much wider range was detected (15-fold difference). We identified 3 5hmC subpopulations in our patient cohort (low, intermediate, and high). The low 5hmC group consisted almost entirely of patients with TET2 or IDH mutations. As expected, TET2 and IDH mutated patients had significantly lower levels of 5hmC compared with patients without mutated TET2 and IDH1/2 (both P < .001). Interestingly, high 5hmC levels correlated with inferior OS (high vs intermediate 5hmC: P = .047, hazard ratio [HR] = 1.81). Multivariate analysis revealed that high 5hmC is an independent poor prognostic indicator for OS (high vs intermediate 5hmC: P = .01, HR = 2.10). This trial was registered at www.clinicaltrials.gov as NCT00004128.

Clinical and biological impact of TET2 mutations and expression in younger adult AML patients treated within the EORTC/GIMEMA AML-12 clinical trial.

We assessed the prognostic impact of TET2 mutations and mRNA expression in a prospective cohort of 357 adult AML patients < 60 years of age enrolled in the European Organization For Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of TET2 mutations were investigated. TET2 mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (p = 0.024). TET2 and IDH1/2 mutations strongly associated with aberrations in the DNA methyltransferase DNMT3A. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant TET2 forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function TET2 mutations predicted poor outcome, we questioned whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival.

Read the clonotype: Next-generation sequencing-based lymphocyte clonality analysis and perspectives for application in pathology.

Clonality assessment using the unique rearrangements of immunoglobulin (IG) and T-cell receptor (TR) genes in lymphocytes is a widely applied supplementary test for the diagnosis of B-cell and T-cell lymphoma. To enable a more sensitive detection and a more precise comparison of clones compared with conventional clonality analysis based on fragment analysis, the EuroClonality NGS Working Group developed and validated a next-generation sequencing (NGS)-based clonality assay for detection of the IG heavy and kappa light chain and TR gene rearrangements for formalin-fixed and paraffin-embedded tissues. We outline the features and advantages of NGS-based clonality detection and discuss potential applications for NGS-based clonality testing in pathology, including site specific lymphoproliferations, immunodeficiency and autoimmune disease and primary and relapsed lymphomas. Also, we briefly discuss the role of T-cell repertoire of reactive lymphocytic infiltrations in solid tumors and B-lymphoma.

Cross-Resistance between Platinum-Based Chemotherapy and PARP Inhibitors in Castration-Resistant Prostate Cancer.

Patients with metastatic castration-resistant prostate cancer (mCRPC) harbouring homologous recombination repair-related gene aberrations (HRRm) can derive meaningful benefits from both platinum-based chemotherapy (PlCh) and PARP inhibitors (PARPi). Cross-resistance between these agents is well-recognised in other tumour types but data on prostate cancer is lacking. In this retrospective pre-planned study, we assessed 28 HRRm mCRPC patients who received PlCh and PARPi. Progression-free survival (PFS) on initial therapy was longer than on subsequent therapy (median 5.3 vs. 3.4 months, p = 0.016). The median PFS of PlCh was influenced by the order of agents, with 3.6 months shorter PFS after PARPi than when administered first. The median PFS of PARPi was less influenced, with 0.9 months shorter PFS after PlCh than before. In the PARPi-first subgroup, six out of 16 evaluable patients (37.5%) had a >50% PSA decline to PlCh, and two of eight (25.0%) had a radiographic response to PlCh. In the PlCh-first subgroup, 6/10 (60.0%) had a >50% PSA decline, and 5/9 (55.6%) had a radiographic response to PARPi. These data show >40% of the cohort is sensitive to a subsequent HRR-targeting agent. PlCh appears to induce less cross-resistance than PARPi. Additional data on resistance mechanisms will be crucial in defining an optimal treatment sequence in HRRm mCRPC patients.

Establishment and characterization of the first patient-derived radiation-induced angiosarcoma xenograft model (RT-AS5).

Angiosarcomas are a heterogeneous group of rare endothelial malignancies with a complex, not completely unravelled biology. They encompass primary (sporadically occurring) angiosarcomas of several origins and secondary angiosarcomas, which often arise due to DNA damaging factors including radiotherapy or ultraviolet light exposure. The optimal treatment of metastatic angiosarcomas is unclear and the prognosis is poor. In order to discover novel treatment strategies for angiosarcomas it is important to take the heterogeneity of these tumors into account. For this reason it is also important to have preclinical models available for the different clinical subtypes. Owing to the rarity of angiosarcomas, models are scarce. So far, only five human cell lines of angiosarcomas (all of the scalp after UV exposure) are available worldwide. In this paper we describe a novel established patient-derived xenograft model of a radiotherapy-induced angiosarcoma of the breast. The tumor was characterized by a MYC amplification, CD31 and ERG immunohistochemical positivity and was further characterized by using next generation sequencing (TruSight Oncology 500) in combination with the R-package XenofilteR to separate mouse from human sequence reads.

PAX5 P80R-mutated B-cell acute lymphoblastic leukemia with transformation to histiocytic sarcoma: clonal evolution assessment using NGS-based immunoglobulin clonality and mutation analysis.

Clonality assessment by the detection of immunoglobulin (IG) gene rearrangements is an important method to determine whether two concurrent or subsequent lymphoid malignancies in one patient are clonally related. Here, we report the detailed clonality analysis in a patient with a diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) followed by a histiocytic sarcoma (HS), in which we were able to study clonal evolution by applying next generation sequencing (NGS) to identify IG rearrangements and gene mutations. Using the sequence information of the NGS-based IG clonality analysis, multiple related subclones could be distinguished in the PAX5 P80R-mutated B-ALL. Notably, only one of these subclones evolved into HS after acquiring a RAF1 mutation. This case demonstrates that NGS-based IG clonality assessment and mutation analysis provide clear added value for clonal comparison and thereby improves clinicobiological understanding.