Poor Applicability of Currently Available Prognostic Scoring Systems for Prediction of Outcome in KIT D816V-Negative Advanced Systemic Mastocytosis

Simple Summary Most patients (>95%) with systemic mastocytosis (SM) carry a mutation in the KIT gene (KIT D816V). Especially aggressive forms of SM are associated with pronounced clinical symptoms, blood count abnormalities, additional mutations in other genes and a shortened survival. Only a small minority of patients with SM have another mutation on position D816 (e.g., D816H) or do not harbor any mutation in KIT, and data on these small subgroups are scarce. The aim of our study was to characterize these rare subgroups: we examined 7 SM patients with either KIT D816H or KIT D816Y and 12 SM patients without any KIT mutation. We found that (a) both groups frequently appear as mast cell leukemia (the most aggressive SM subgroup), (b) those patients cannot be assessed using conventional risk scores, (c) response to treatment is poor and (d) overall survival is worse than in KIT D816V-positive SM. Abstract Within our nationwide registry, we identified a KIT D816V mutation (KIT D816Vpos.) in 280/299 (94%) patients with advanced systemic mastocytosis (AdvSM). Age, cytopenias and the presence of additional somatic mutations confer inferior overall survival (OS). However, little is known about the characteristics of KIT D816V-negative (D816Vneg.) AdvSM. In 19 D816Vneg. patients, a combination of clinical, morphological and genetic features revealed three subgroups: (a) KIT D816H- or Y-positive SM (KIT D816H/Ypos., n = 7), predominantly presenting as mast cell leukemia (MCL; 6/7 patients), (b) MCL with negative KIT sequencing (KITneg. MCL, n = 7) and (c) KITneg. SM with associated hematologic neoplasm (KITneg. SM-AHN, n = 5). Although >70% of patients in the two MCL cohorts (KIT D816H/Ypos. and KITneg.) were classified as low/intermediate risk according to prognostic scoring systems (PSS), treatment response was poor and median OS was shorter than in a KIT D816Vpos. MCL control cohort (n = 29; 1.7 vs. 0.9 vs. 2.6 years; p < 0.04). The KITneg. SM-AHN phenotype was dominated by the heterogeneous AHN (low mast cell burden, presence of additional mutations) with a better median OS of 4.5 years. We conclude that (i) in MCL, negativity for D816V is a relevant prognostic factor and (ii) PSS fail to correctly classify D816Vneg. patients.


Introduction
Systemic mastocytosis (SM) is a rare myeloid neoplasm with an accumulation of neoplastic mast cells (MC) in various tissues, most often in bone marrow (BM), skin and the gastrointestinal tract.The disease is subcategorized into indolent SM (ISM), smoldering SM (SSM), bone marrow mastocytosis (BMM) and advanced SM (AdvSM), the latter comprising SM with an associated hematologic/myeloid neoplasm (SM-AHN/SM-AMN), aggressive SM (ASM) and mast cell leukemia (MCL) [1].ISM is associated with a normal life expectancy whereas survival in AdvSM is dependent on subtype and ranges between 1.5 and 4 years [1].
Mast cells play an important role in the immune system and are directly involved in allergic reactions, stress and tumor growth and their degranulation may lead to a plethora of different symptoms [2,3].Their pro-and antitumor potential in general is widely discussed, and a complete depletion of mast cells is regarded as a life-threatening condition.The clonal constitutive activation of mast cells in SM may also lead to a multitude of symptoms affecting different organ systems which sometimes makes the diagnosis challenging.

Patients and Methods
All patients were registered with the "German Registry on Disorders of Eosinophils and Mast cells" (GREM) and gave written informed consent.Detailed information on clinical, morphological and laboratory parameters are presented in Table 1a,b and Appendix A, Table A1.All patients were diagnosed and subtyped as SM according to the 2016 WHO classification.The study design adhered to the tenets of the Declaration of Helsinki and was approved by the responsible institutional review boards.
Prior to manual dissection from the slides, the tissue was deparaffinized and stained with H & E (Carl Roth GmbH, Karlsruhe, Germany).For manual microdissection, compact mast cell infiltrates were identified by a pathologist and the region of interest of the FFPE tissue was scraped into Eppendorf tubes for subsequent DNA/RNA isolation.This method enriched tumor cells, and the tumor cell content was at least 70%.Following the manufacturer's protocol, we prepared a DNA library using a hybrid capture-based TruSight Oncology 500 DNA/RNA NextSeq Kit (Illumina, San Diego, CA, USA).Using the unique molecular identifiers (UMIs) in the TruSight Oncology 500 (TSO 500) (Illumina, San Diego, CA, USA), we determined the unique coverage of each position and reduced background noise.We analyzed sequencing data for genomic alterations, including SNVs, CNVs and fusions.SNVs and small indels with a variant allele frequency (VAF) of less than 2% were excluded.

Mutation Analysis
Quantitative assessment of the expressed allele burden (EAB) at the RNA level was performed by allele-specific reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), as previously described [9].Qualitative assessment of KIT D816H/Y was performed by Sanger sequencing of KIT exon 17 from peripheral blood (PB) or BM according to standard procedures.
For analysis of alternative KIT and additional somatic mutations, Next-Generation Deep Amplicon Sequencing (NGS) by 454 FLX amplicon chemistry (Roche, Penzberg, Germany) or library preparation based on the TruSeq Custom Amplicon Low Input protocol (Illumina, San Diego, CA) followed by sequencing on the MiSeq instrument (Illumina, San Diego, CA, USA) was performed in 18/19 patients.The sequencing panel includes a standard myeloid gene panel covering 18 recurrently mutated genes in myeloid neoplasms.Gene mutations were annotated using the appropriate reference sequence (Ensembl release 85: July 2016).
From 12/12 patients without a mutation in KIT, gDNA isolated from PB and/or BM (including patient #18) was used for NGS analysis of all KIT exons.The mean coverage was 1500 reads (400-8819).In two patients, whole genome and whole transcriptome sequencing was applied.DNA sequencing was performed using library preparation by 'TruSeq DNA PCR-Free' and sequencing by NovaSeq6000 (Illumina, San Diego, CA, USA).RNA sequencing was performed by RNA-Seq (Illumina TruSeq Stranded Total RNA KIT; Unique Dual Indices; Illumina, San Diego, CA, USA).DNA reads were aligned against the human reference genome (hg19); RNA reads were used to detect potential fusion transcripts (Arriba, Manta, STAR-Fusion).

Statistical Analyses
All statistical analyses were performed using IBM ® SPSS statistics (version 25.0, IBM-Corporation, Armonk, NY, USA) or GraphPad Prism (version 8, GraphPad, San Diego, CA, USA).Survival probabilities were calculated by the Kaplan-Meier method and were determined from the date of diagnosis to death or the date of last contact (if alive).Overall survival (OS) was defined as the time from AdvSM diagnosis to death from any cause.In patients undergoing allo-SCT, progression-free survival (PFS) was defined as the time from allogeneic stem cell transplantation (SCT) to disease progression or death from any cause.A p < 0.05 was considered as statistically significant.

Statistical Analyses
All statistical analyses were performed using IBM ® SPSS statistics (version 25.0, IBM-Corporation, Armonk, NY, USA) or GraphPad Prism (version 8, GraphPad, San Diego, CA, USA).Survival probabilities were calculated by the Kaplan-Meier method and were determined from the date of diagnosis to death or the date of last contact (if alive).Overall survival (OS) was defined as the time from AdvSM diagnosis to death from any cause.In patients undergoing allo-SCT, progression-free survival (PFS) was defined as the time from allogeneic stem cell transplantation (SCT) to disease progression or death from any cause.A p < 0.05 was considered as statistically significant.

Cytomorphology and Histomorphology
The BM aspirates of patients with KIT neg.MCL exhibited atypical MC with round nuclei rather than a spindle-shaped morphology (Figure 3A).Two cases presented a well-Cancers 2024, 16, 593 10 of 18 differentiated phenotype with prominent cytoplasmic granulation, and only a few neoplastic MCs displayed aberrant expression of CD25.In all BM biopsies, multifocal dense infiltrates with aggregates of ≥15 MC were detected, often localized paratrabecularly.Reticulin fibers were increased, consistent with a myelofibrosis grade 1 (MF1) in the majority of cases.Of the five cases diagnosed with KIT neg.SM-AHN, two patients were classified as ISM with associated MDS and multilineage dysplasia (MDS-MLD), one patient was diagnosed with SM-acute myeloid leukemia (AML) and one patient fulfilled criteria for each associated myelofibrosis (SM-MF) and MDS/MPN, respectively.Striking features included: (a) WDSM with a solitary dense infiltrate, diffuse interstitial MC hyperplasia and normal immunophenotype in one patient (case 15) and (b) atypical, spindle-shaped MC exhibiting aberrant CD25 expression without formation of dense clusters in one patient (case 18) (Figure 3B).n.d., not done; n.k., not known; SM-AHN systemic mastocytosis with an associated hematological neoplasm; MCL, mast cell leukemia; M, male; F, female; +, positive; -, negative.

Cytomorphology and Histomorphology
The BM aspirates of patients with KIT neg.MCL exhibited atypical MC with round nuclei rather than a spindle-shaped morphology (Figure 3A).Two cases presented a welldifferentiated phenotype with prominent cytoplasmic granulation, and only a few neoplastic MCs displayed aberrant expression of CD25.In all BM biopsies, multifocal dense infiltrates with aggregates of ≥15 MC were detected, often localized paratrabecularly.Reticulin fibers were increased, consistent with a myelofibrosis grade 1 (MF1) in the majority of cases.Of the five cases diagnosed with KIT neg.SM-AHN, two patients were classified as ISM with associated MDS and multilineage dysplasia (MDS-MLD), one patient was diagnosed with SM-acute myeloid leukemia (AML) and one patient fulfilled criteria for each associated myelofibrosis (SM-MF) and MDS/MPN, respectively.Striking features included: a) WDSM with a solitary dense infiltrate, diffuse interstitial MC hyperplasia and normal immunophenotype in one patient (case 15) and b) atypical, spindle-shaped MC exhibiting aberrant CD25 expression without formation of dense clusters in one patient (case 18) (Figure 3B).
were no significant differences regarding BM MC infiltration, serum tryptase levels and blood counts (Table 2 and Figure 5).

Discussion
The pathogenetic driver mutation KIT D816V is detected in >90% of SM patients and is therefore of utmost relevance for the diagnosis of this heterogeneous disease.Its presence and variant allele frequency (VAF) in BM and PB are of paramount importance in assessing the phenotype and overall burden of involvement of MC, but often also non-MC lineages in SM-AHN.Currently available PCR assays are capable of detecting KIT D816V VAF with a sensitivity of up to 0.003%, allowing quantification in patients with very low disease burden at diagnosis and also monitoring response and residual KIT D816V mutational burden during or after treatment with KIT inhibitors, chemotherapy or allogeneic SCT [9,17].

Discussion
The pathogenetic driver mutation KIT D816V is detected in >90% of SM patients and is therefore of utmost relevance for the diagnosis of this heterogeneous disease.Its presence and variant allele frequency (VAF) in BM and PB are of paramount importance in assessing the phenotype and overall burden of involvement of MC, but often also non-MC lineages in SM-AHN.Currently available PCR assays are capable of detecting KIT D816V VAF with a sensitivity of up to 0.003%, allowing quantification in patients with very low disease burden at diagnosis and also monitoring response and residual KIT D816V mutational burden during or after treatment with KIT inhibitors, chemotherapy or allogeneic SCT [9,17].
Of 298 patients with AdvSM included in the GREM, we identified 19 KIT D816V neg.patients, corresponding to a 94% prevalence of KIT D816V in AdvSM.Approximately 35% of KIT D816V neg.patients were positive for alternative KIT D816 mutations.In seven patients, we identified only D816H and D816Y, which are known recurrent mutations, whereas D816G, D816I, D816T and D816N were identified only in single cases [18].Similar to what has been described in the literature [18], all but one of the seven patients in our series had an MCL phenotype.Of note, despite the low prevalence of poor prognostic markers such as cytopenias, other C-findings and additional somatic mutations that placed the vast majority of patients in low/medium risk categories in most available prognostic scoring systems, prognosis was poor with a median survival of only 1.8 years.Application of the IPSM risk score delivered the best results: here, the majority of patients were classified into the AdvSM-3 risk group.According to Sperr et al. [8], patients within the AdvSM-3 and AdvSM-4 exhibit a significantly worse OS compared to AdvSM-1 and AdvSM-2 patients.However, none of the patients were classified into the AdvSM-4 subgroup.These findings suggest a potentially better discrimination of non-molecular scoring systems such as the IPSM but also show that patients with a lack of cytopenia(s) are rarely stratified into the high-risk subgroups of molecular PSS and or AdvSM-4, which overall show the worst OS.
In the remaining 12 patients without a mutation at position 816, no alternative mutations were detected in the complete coding sequences of KIT.From a clinical perspective, two distinct subgroups emerged.Patients with MCL had a very high MC burden and a poor prognosis despite the absence of additional somatic mutations and a predominantly good/intermediate prognostic risk score using molecular annotated risk scores.Again, IPSM was the best discriminator of OS in this subgroup (Appendix A, Figure A1).All MCL patients were aleukemic with three of seven patients presenting with an immature MC morphology, which was predictive of an inferior OS in multivariate analysis in a retrospective study by Pardanani and colleagues [28].
In contrast, patients in the second cohort were diagnosed with SM and an associated hematological neoplasm (SM-AHN) according to the WHO classification or an associated myeloid neoplasm (SM-AMN) according to the ICC.Interestingly, no cases of chronic myelomonocytic leukemia were diagnosed, despite it being the most common SM-associated AHN based on previous studies [29,30].Patients from our cohort mainly presented with MDS, MDS/MPN overlap or MPN as associated AHN.In two cases, the diagnosis could only be made through a thorough integration of clinical data and elevated tryptase levels, as either a major histomorphological criterion was lacking or only one minor criterion was present.This highlights the necessity for meticulous morphological analysis supplemented by additional immunohistochemistry and interdisciplinary collaboration when faced with KIT neg.SM cases.All patients had a dominating AHN with only a low MC burden and all patients exhibited additional somatic mutations.Nevertheless, the prognosis was significantly better than in patients with the MCL phenotype.
The diagnosis of all subtypes of SM can be notably challenging when the fraction of diagnostic neoplastic cells in the BM is low.Consequently, the application of sensitive diagnostic methods becomes imperative, often necessitating a comprehensive approach involving multiple techniques, such as thorough clinical evaluation, histomorphology, immunohistochemistry and molecular genetics.The updated WHO classification and ICC require the fulfillment of one major criterion and two minor criteria, or alternatively, three minor criteria for the diagnosis of SM [14,15].One of the minor criteria involves proving the clonality of the MC through the detection of a KIT mutation, making the diagnosis challenging in cases lacking this mutation.In an unknown proportion of patients, the presence of two clonally independent diseases seems possible, similar to the recently reported cohort of patients with recurrent concurrent presence of KIT D816V pos.SM and JAK2 V617F pos.MPN in the same individual [31].
In a large series of 92 MCL patients of the ECNM, 73% were KIT D816V pos., 11% exhibited alternative KIT mutations and 17% were KIT neg.[32].In other series, positivity for KIT D816V ranged from 23% to 68% [19,33,34].We compared the two KIT D816H/Y pos./KIT neg.MCL groups with a larger cohort of 29 registry patients with KIT D816V pos.MCL.Although patients with KIT D816V pos.MCL were median >10 years older and had a significantly higher incidence of HRM, median OS was significantly better.This suggests that yet unknown mechanisms beyond MC burden, phenotype and genetic profile contribute to the poor prognosis of the different MCL subtypes.The 13 KIT neg./KIT D816H/Y pos.MCL patients did not show a significant and durable response to midostaurin or imatinib, and all five allogeneic transplanted patients died within two years after transplantation.KIT D816V neg.MCL is therefore a high-risk condition regardless of established prognostic scoring systems, especially molecular annotated risk scores.There are no data yet on the efficacy of newly developed tyrosine kinase inhibitors (TKIs) such as avapritinib or bezuclastinib in KIT D816V neg.patients.
Important limitations of our analyses include that elaborate statistical analyses and validations in independent control cohorts could not be performed due to the rarity but also heterogeneity of KIT D816V neg.AdvSM.Notwithstanding, most of the few available case reports or small series in the literature lack a comparable thorough clinical, morphological and molecular work-up.The data clearly show that KIT D816V neg.AdvSM is similarly heterogeneous to KIT D816V pos.AdvSM and that a distinct differentiation between the various subtypes, e.g., MCL and SM-AHN, is of utmost relevance for prognostication and treatment decisions.

Conclusions
In conclusion, the diagnosis of KIT neg.AdvSM demands a high level of expertise and close collaboration between hematologists and hematopathologists to avoid diagnostic pitfalls and ensure optimal patient therapy.The integration of multiple diagnostic modalities and molecular studies is essential to achieve accurate SM diagnosis and provide patients with the most effective treatment options.KIT neg.AdvSM can be subdivided into (i) patients with other mutations at codon 816 such as KIT D816H/N/Y, most frequently associated with an MCL phenotype, (ii) KIT neg.MCL with a very poor prognosis despite the absence of C-findings and HRM (iii) KIT neg.SM-AHN with low MC burden and dominating AHN, making SM-specific treatment dispensable and prognosis mainly triggered by AHN.Our data highlight the poor applicability of the majority of current prognostic scoring systems for KIT neg.MCL.
For classification, risk stratification and evaluation of prognostic factors among KIT neg.and KIT D816H/Y pos.patients, we utilized a control group of KIT D816V pos.AdvSM patients (n = 118; SM-AHN, n = 89; MCL-(AHN), n = 29) with known clinical, laboratory and genetic characteristics (Tables 1a,b and 2).

Figure 1 .
Figure 1.Overview of patient cohorts.Clear dominance of MCL phenotype in KIT D816H/Y pos.and KIT neg.patients.MCL patients were subsequently compared to a KIT D816V pos.MCL control cohort from the registry (see red box).Abbreviations: MCL, mast cell leukemia; AHN, associated hematologic neoplasm; SM, systemic mastocytosis; neg., negative; pos., positive.

Figure 1 .
Figure 1.Overview of patient cohorts.Clear dominance of MCL phenotype in KIT D816H/Y pos.and KIT neg.patients.MCL patients were subsequently compared to a KIT D816V pos.MCL control cohort from the registry (see red box).Abbreviations: MCL, mast cell leukemia; AHN, associated hematologic neoplasm; SM, systemic mastocytosis; neg., negative; pos., positive.

Figure 2 .
Figure 2. Individual course of KIT D816H/Y pos.patients (A) and KIT neg.patients (B) Detailed information on treatment sequences for KIT D816H/Y pos.and KIT neg.patients.KIT neg.patients were further subdivided into MCL and SM-AHN phenotypes.Treatment sequences are depicted in different colors.Six patients underwent allogeneic stem cell transplantation with rapid relapse.Inappropriate treatment response led to death in 14/19 patients.Abbreviations: MCL, mast cell leukemia; AHN, associated hematologic neoplasm; SM, systemic mastocytosis †, death.

Figure 2 .
Figure 2. Individual course of KIT D816H/Y pos.patients (A) and KIT neg.patients (B) Detailed information on treatment sequences for KIT D816H/Y pos.and KIT neg.patients.KIT neg.patients were further subdivided into MCL and SM-AHN phenotypes.Treatment sequences are depicted in different colors.Six patients underwent allogeneic stem cell transplantation with rapid relapse.Inappropriate treatment response led to death in 14/19 patients.Abbreviations: MCL, mast cell leukemia; AHN, associated hematologic neoplasm; SM, systemic mastocytosis †, death.

Figure 3 .
Figure 3. Histomorphology (2.5× and 40× magnification) of a patient with MCL (A) and SM-AHN (B).(A) Bone marrow aspirate shows an increase of atypical mast cells (≥20%); in bone marrow core biopsies dense infiltrates of hypogranular mast cells with positivity of CD25 were present.(B) Bone

Figure 3 .
Figure 3. Histomorphology (2.5× and 40× magnification) of a patient with MCL (A) and SM-AHN (B).(A) Bone marrow aspirate shows an increase of atypical mast cells (≥20%); in bone marrow core biopsies dense infiltrates of hypogranular mast cells with positivity of CD25 were present.(B) Bone marrow core biopsies showed diagnostic compact mast cell infiltrates of spindle-shaped mast cells with expression of mast cell tryptase and aberrant expression of CD25.Abbreviations: MCL, mast cell leukemia; AHN, associated hematologic neoplasm; SM, systemic mastocytosis.

Figure 5 .
Figure 5. Kaplan-Meier overall survival of patients with mast cell leukemia and presence of KIT D816V, KIT D816H/Y or absence of a KIT mutation.KIT D816H/Y-positive MCL patients are associated with the worst outcome.KIT D816V-positive MCL patients have the worst prognosis within all D816V-positive SM subgroups, but still have a better overall survival than KIT D816V-negative MCL patients.Abbreviations: MCL, mast cell leukemia; AHN, associated hematologic neoplasm.

Figure 5 .
Figure 5. Kaplan-Meier overall survival of patients with mast cell leukemia and presence of KIT D816V, KIT D816H/Y or absence of a KIT mutation.KIT D816H/Y-positive MCL patients are associated with the worst outcome.KIT D816V-positive MCL patients have the worst prognosis within all D816V-positive SM subgroups, but still have a better overall survival than KIT D816V-negative MCL patients.Abbreviations: MCL, mast cell leukemia; AHN, associated hematologic neoplasm.

Figure A1 .
Figure A1.Assignment of risk scores for individual patients.The majority of patients were grouped as low-or intermediate-risk within the molecular risk scores for advanced systemic mastocytosis (MARS, MAPS, GPS).IPSM was the most effective score in discriminating between low risk (AdvSM-1/2) and higher risk profile (AdvSM-3/4).Abbreviations: IPSM, International Prognostic Scoring System for Mastocytosis; GPS, Global Prognostic Score for Mastocytosis; MAPS, Mayo Alliance Prognostic System; MARS, Mutation-Adjusted Risk Score for Advanced Mastocytosis, MCL, mast cell leukemia; SM, systemic mastocytosis; int., intermediate; *, SM-AHN.

Figure A1 .
Figure A1.Assignment of risk scores for individual patients.The majority of patients were grouped as low-or intermediate-risk within the molecular risk scores for advanced systemic mastocytosis (MARS, MAPS, GPS).IPSM was the most effective score in discriminating between low risk (AdvSM-1/2) and higher risk profile (AdvSM-3/4).Abbreviations: IPSM, International Prognostic Scoring System for Mastocytosis; GPS, Global Prognostic Score for Mastocytosis; MAPS, Mayo Alliance Prognostic System; MARS, Mutation-Adjusted Risk Score for Advanced Mastocytosis, MCL, mast cell leukemia; SM, systemic mastocytosis; int., intermediate; *, SM-AHN.

Table 1 .
(a) Diagnosis of KIT neg.and KIT D816H/Y pos.patients in comparison with a registrybased control group of KIT D816V pos.AdvSM patients.(b) Clinical, morphological and laboratory parameters of KIT D816H/Y pos.and KIT neg.patients in comparison with a registry-based control group of KIT D816V pos.AdvSM patients.

Table 2 .
Clinical, morphological and laboratory parameters of KIT D816H/Y pos., KIT neg.and KIT D816V pos.MCL patients from the registry (control group).

Table A2 .
Detailed localization of additional mutations on cDNA and protein level.