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30 January 2024

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

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1
Hematology and Oncology, Medical Faculty Mannheim, Heidelberg University, 68167 Mannheim, Germany
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Institute of Pathology, Ludwig-Maximilian-University, 80337 Munich, Germany
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Department of Oncology, Haematology, Haemostaseology and Stem Cell Transplantation, University Hospital RWTH Aachen, 52074 Aachen, Germany
4
Center for Integrated Oncology (CIO), Aachen, Bonn, Cologne, Düsseldorf (ABCD), 52074 Aachen, Germany
Cancers2024, 16(3), 593;https://doi.org/10.3390/cancers16030593
This article belongs to the Special Issue New Insights of Hematology in Cancer
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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.

1. 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.
Cytopenias (anemia < 10 g/dL, platelets < 100 × 109/L, neutrophils < 1 × 109/L), hepatomegaly with impaired liver function (hypoalbuminemia, elevated alkaline phosphatase, portal hypertension, splenomegaly with hypersplenism, ascites), malabsorption with weight loss and large osteolytic lesions constitute the so-called C-findings indicating organ damage. C-findings are diagnostic criteria for ASM but not for SM-AHN and MCL, but they are in fact identified in the majority of patients with SM-AHN and MCL. Additional somatic mutations are present in 60–80% of patients with AdvSM of which mutations in SRSF2, ASXL1, RUNX1, NRAS and DNMT3A have been associated with poor prognosis [4,5,6]. Several prognostic scoring systems have recently been established variably comprising clinical (age, organomegaly), laboratory (anemia, thrombocytopenia, beta-2-microglobulin and alkaline phosphatase) and genetic (additional somatic mutations) characteristics [4,5,6,7,8].
Depending on the SM subtype, mutations in the receptor tyrosine kinase KIT, typically KIT D816V, are observed in 80% to over 90% of patients. KIT D816Vneg. SM/AdvSM comprises alternative mutations at position 816 (e.g., D816H or Y, KIT D816H/Ypos.) and the absence of any mutation in the coding sequence of KIT (KITneg.) [9,10,11]. Across all SM subtypes, KITneg. SM is identified at a frequency of 5–10%. However, its prevalence is higher in rare subtypes including MCL (10–20%), well-differentiated systemic mastocytosis (WDSM) (approximately 60–70%) [12,13], myelomastocytic leukemia (> 90%) and mast cell sarcoma (MCS, approx. 90%) [14,15]. Due to limited knowledge, we sought to evaluate the clinical, genetic and prognostic characteristics of patients with KIT D816Vneg. AdvSM and MCS.

2. 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.
Table 1. (a) Diagnosis of KITneg. and KIT D816H/Ypos. patients in comparison with a registry-based control group of KIT D816Vpos. AdvSM patients. (b) Clinical, morphological and laboratory parameters of KIT D816H/Ypos. and KITneg. patients in comparison with a registry-based control group of KIT D816Vpos. AdvSM patients.

2.1. Cytomorphology and Histomorphology

All biopsies were evaluated by a reference pathologist (M.R., H.-P.H, K.S.) of the ‘European Competence Network on Mastocytosis’ (ECNM). Mast cell morphology was analyzed in BM aspirate smears stained with May–Grünwald–Giemsa (Carl Roth GmbH, Karlsruhe, Germany) and toluidine blue (Merck/Sigma-Aldrich, Darmstadt, Germany). BM trephine biopsy specimens were decalcified for 8 h, fixed in 4% formalin (Carl Roth GmbH, Karlsruhe, Germany) and paraffin-embedded. Sections were stained with hematoxylin (Carl Roth GmbH, Karlsruhe, Germany) and eosin (Carl Roth GmbH, Karlsruhe, Germany), Giemsa (Carl Roth GmbH, Karlsruhe, Germany), Gömöri´s silver impregnation (Merck/Sigma-Aldrich, Darmstadt, Germany) and naphthol AS-D chloracetate esterase (Merck/Sigma-Aldrich, Darmstadt, Germany).

2.2. Immunohistochemistry

Immunohistochemistry was performed on the fully automated Ventana Benchmark platform (Roche Diagnostics, Mannheim, Germany) using the Ultraview DAB IHC detection kit (Roche Diagnostics, Mannheim, Germany) for visualization. The following antibodies were used: CD117 (Agilent, Santa Clara, CA, USA), CD25 (Leica, Deer Park, TX, USA), CD2 (Leica), MZT (Zytomed, Berlin, Germany), CD30 (Thermo Fisher Scientific, Waltham, MA, USA), CD20 (Agilent, Santa Clara, CA, USA), CD138 (Zytomed, Berlin, Germany), Kappa (Agilent, Santa Clara, CA, USA), Lambda (Agilent, Santa Clara, CA, USA) and CD5 (Leica Biosystems, Nussloch, Germany).

2.3. Microdissection

To exclude false negative KIT mutation analysis in patients with a low mast cell burden (SM-AHN, patients #15–#19), we performed microdissection of mast cell aggregates from paraffin-embedded BM biopsy samples for 4/5 (#15, #16, #17, #19) patients.
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.

2.4. 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).

2.5. Comparison to Control Group

For classification, risk stratification and evaluation of prognostic factors among KITneg. and KIT D816H/Ypos. patients, we utilized a control group of KIT D816Vpos. AdvSM patients (n = 118; SM-AHN, n = 89; MCL-(AHN), n = 29) with known clinical, laboratory and genetic characteristics (Table 1a,b and Table 2).
Table 2. Clinical, morphological and laboratory parameters of KIT D816H/Ypos., KITneg. and KIT D816Vpos. MCL patients from the registry (control group).

2.6. 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.

3. Results

From almost 300 advSM patients collected in the GREM, we identified 19 patients without a KIT D816V mutation. Seven patients carried an alternative mutation on locus KIT D816 (D816H, n = 4; D816Y, n = 3), whereas no mutation in the KIT gene was detected in 12 patients (KITneg.). While almost all (6/7, 86%) patients of the KIT D816H/Ypos. cohort were diagnosed with MCL, the KITneg. subgroup included MCL (7/12, 58%) and SM-AHN (5/12, 42%) patients (Figure 1).
Figure 1. Overview of patient cohorts. Clear dominance of MCL phenotype in KIT D816H/Ypos. and KITneg. patients. MCL patients were subsequently compared to a KIT D816Vpos. 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.

3.1. KIT D816H/Ypos. Patients

Clinical features. The median age of KIT D816H/Ypos. patients (n = 7) was 58 years (range 37–69, m/f: 3/4). The median BM MC infiltration was 50% (range 20–75%); other notable BM features included eosinophilia (6/7, 86%) and fibrosis (4/7, 57%; grade 2, 2/7, grade 3, 2/7). Blood counts revealed mild leukocytosis > 10 and <16 × 109/L (2/7, 29%) and anemia < 10 g/dL (4/7, 57%, one patient transfusion dependent), while thrombocytopenia < 100 G/L was absent in all seven patients. Eosinophilia > 0.4 × 109/L was frequent (5/7, 82%; median 1 × 109/L, range 0–39) while monocytosis > 1 × 109/L was rare (1/7; 14%, median 0.23 × 109/L, range 0.03–1.3). Other C-findings included ascites (5/7, 71%), elevated alkaline phosphatase (4/7, 57%) and hypoalbuminemia (1/7, 14%). Involvement of the gastrointestinal tract and skin (0/7) was rare (1/7, 14%). The median serum tryptase level was 193 µg/L (range 90–1300). Consequently, morphologic subtypes included MCL ± AHN (6/7, 86%; +MCS, 2/7, 29%; with WDSM phenotype, 1/7, 14%) and SM-AHN (1/7, 14%). Clinically relevant anaphylaxis (1/7, 14%) was rare and no other relevant MC-triggered symptoms were noted.

3.1.1. Molecular Findings

Additional somatic high-risk mutations (HRM) in SRSF2, ASXL1, RUNX1 (S/A/R gene panel) [6], NRAS or DNMT3A [4,16] were identified in 2/7 (29%) patients (SRSF2, n = 1; NRAS, n = 1) and other somatic mutations (TET2, JAK2, and CBL) [5] were observed in two patients (29%; CBL/TET2, 1/7; JAK2 V617F, 1/7). Due to the paucity of leukocytosis ≥ 16 × 109/L (0/7), skin involvement (0/7) and HRM (2/7), almost all patients were low/intermediate risk according to current prognostic scoring systems for AdvSM (IPSM, 7/7; MARS, 6/7; MAPS, 5/7; GPS, 6/7) (Figure A1).

3.1.2. Treatment

Treatment comprised midostaurin (5/7, 71%) with or without sequential cladribine (3/7, 43%) and supportive care only (2/7, 29%; G-CSF, local radiation). The response to treatment was only partially effective and of short duration. Allogeneic SCT was performed in 2/7 (29%) patients who relapsed early and deceased at months +10 and +12 after transplant (Figure 2). Despite a predominantly low/intermediate risk profile, the median OS was only 22 months (range 12–49).
Figure 2. Individual course of KIT D816H/Ypos. patients (A) and KITneg. patients (B) Detailed information on treatment sequences for KIT D816H/Ypos. and KITneg. patients. KITneg. 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.

3.2. KITneg. Patients

Due to significant clinical and morphological differences, two subgroups were considered (Table 3): MCL ± AHN (7/12, 58%) and SM-AHN (5/12, 42%).
Table 3. Morphological differences between KITneg. MCL ± AHN and SM-AHN patients.

3.2.1. Cytomorphology and Histomorphology

The BM aspirates of patients with KITneg. MCL exhibited atypical MC with round nuclei rather than a spindle-shaped morphology (Figure 3A). Two cases presented a well-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 KITneg. 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).
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.

3.2.2. Molecular Findings

Additional somatic high-risk mutations (HRM) in the S/A/R gene panel [6], NRAS or DNMT3A [4,16] were identified in 4/12 (33%) patients (ASXL1, n = 2; SRSF2, n = 1; DNMT3A, n = 1; RUNX1, n = 1). Other somatic mutations (TET2, JAK2, KRAS, CSF3R, U2AF1, CBL, FLT3-ITD) [5] were observed in 6/12 (50%) patients.

3.2.3. Treatment

Treatment comprised TKI (MCL, 6/7 and SM-AHN, 2/5; midostaurin 6/7 and 1/5; imatinib, 3/7 and 1/5) with or without sequential cladribine (6/7 and 0/5) and intensive chemotherapy (0 and 1/5) without significant or durable response. Following allogeneic SCT (3/7 and 1/5), all patients died (months +5, +6, +7, +24) due to progressive disease (Figure 2). According to risk scoring systems of AdvSM, the majority of patients were low or intermediate (IPSM, 2/7 and 5/5; MARS, 5/7 and 5/5; MAPS, 5/7 and 4/5; GPS, 4/7 and 5/5) (Figure A1). The median OS was significantly different (0.9 versus 4.5 years, p = 0.037).

3.3. Commonalities and Differences between the Two Cohorts of KITneg. AdvSM

The median age (56/62 years, range 27–69) and the m/f ratio were not different (5/7 and 3/5). Significant differences were identified for BM MC infiltration (median 80% versus 20%, p = 0.0013), serum tryptase levels (median 451 µg/L versus 34 µg/L, p = 0.0025), hemoglobin < 10 g/dL (57% versus 0%; median 9.3 g/dL versus 13 g/dL, p = 0.01) and additional somatic mutations (43% and 100%: HRM 2/7 and 2/5; other somatic mutations 2/7 and 4/5). See Figure 4 and Appendix A, Table A2 for detailed molecular features. Although present in some patients, no significant differences between the two subtypes were found for leukocytosis, monocytosis, eosinophilia, alkaline phosphatase (AP), albumin and ascites.
Figure 4. Mutation profile of individual patients. Most additional somatic mutations were detected in KIT D816V-negative SM-AHN patients. Mutations detected in genes were involved in epigenetics (TET2, ASXL1 and DNMT3A), cell signaling (KRAS, NRAS, CBL, JAK2 and CSF3R), transcription regulation (RUNX1) and mRNA splicing (SRSF2 and U2AF1). Compared to other chronic myeloid neoplasms, mutations in high-risk genes (e.g., SRSF2, RUNX1, ASXL1) were less common. Abbreviations: SM, systemic mastocytosis; MCL, mast cell leukemia; AHN, associated hematologic neoplasm.

3.4. Analysis of Three MCL Cohorts

Based on registry data, the two MCL cohorts (KIT D816H/Ypos., n = 6; KITneg. MCL ± AHN, n = 7) were compared with a KIT D816Vpos. MCL cohort (n = 29). The most notable significant differences (KIT D816Vpos. versus KIT D816H/Ypos. versus KITneg. MCL ± AHN) included age (median 68 versus 55 versus 56 years, p < 0.02), vitamin B12 levels (median 1597 versus 300 versus 3091 µg/L, p < 0.04), HRM (66% versus 33% versus 29%, p = 0.03) and survival (2.6 versus 1.7 versus 0.9 years; p = 0.04). One patient (#3) had chronic MCL. There were no significant differences regarding BM MC infiltration, serum tryptase levels and blood counts (Table 2 and 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.

4. 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].
In contrast, very little is known about the clinical phenotype of adult KIT D816Vneg. SM. It has been primarily associated with MCL, WDSM and MCS [18]. Alternative mutations in KIT are recurrently identified at position 816, e.g., D816H, D816N or D816Y [12,19]. In isolated cases, other acquired imatinib-resistant mutations have been identified in coding regions of TK domains 1 and 2 (exons 13 to 18), e.g., I817V in WDSM, D820G in ASM and N822K in SM-AHN [18]. Potentially imatinib-sensitive mutations between exons 8 and 10 may correspond to germline mutations, e.g., S451C, K509I or F522C, which often show a familial aggregation pattern [20,21,22,23,24,25,26,27].
Of 298 patients with AdvSM included in the GREM, we identified 19 KIT D816Vneg. patients, corresponding to a 94% prevalence of KIT D816V in AdvSM. Approximately 35% of KIT D816Vneg. 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 KITneg. 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 D816Vpos. SM and JAK2 V617Fpos. MPN in the same individual [31].
In a large series of 92 MCL patients of the ECNM, 73% were KIT D816Vpos., 11% exhibited alternative KIT mutations and 17% were KITneg. [32]. In other series, positivity for KIT D816V ranged from 23% to 68% [19,33,34]. We compared the two KIT D816H/Ypos./KITneg. MCL groups with a larger cohort of 29 registry patients with KIT D816Vpos. MCL. Although patients with KIT D816Vpos. 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 KITneg./KIT D816H/Ypos. 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 D816Vneg. 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 D816Vneg. 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 D816Vneg. 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 D816Vneg. AdvSM is similarly heterogeneous to KIT D816Vpos. 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.

5. Conclusions

In conclusion, the diagnosis of KITneg. 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. KITneg. 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) KITneg. MCL with a very poor prognosis despite the absence of C-findings and HRM (iii) KITneg. 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 KITneg. MCL.

Author Contributions

Conceptualization, N.N., M.R., J.S. and A.R.; methodology, N.N., J.L., M.R., K.S., G.M. and J.B.; formal analysis, A.F., N.C.P.C. and W.-K.H.; investigation, N.N. and J.S.; data curation, N.N. and J.S.; writing—original draft preparation, N.N., J.S., M.R and A.R.; writing—review and editing, H.-P.H., A.F., W.-K.H., G.M., D.C., N.C.P.C. and J.P.; visualization, M.R., J.L. and N.N.; supervision, A.R. and J.S.; project administration, N.N. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of the Medical Faculty of Mannheim, Heidelberg University (Heidelberg, Germany) (protocol code 2020-593N, 15 September 2020).

Data Availability Statement

The data presented in this study are available on request from the corresponding author (accurately indicate status).

Acknowledgments

The technical advice by Helga Kleiner and Susanne Brendel is acknowledged.

Conflicts of Interest

J.S. received honoraria and research support from Novartis Pharma and Blueprint; A.R. received consultancy honoraria, travel reimbursement and research support from Novartis Pharma, Blueprint, Incyte and Deciphera; H.-P.H. received consultancy honoraria from Novartis, Deciphera and Blueprint; K.S. received honoraria and travel support from Novartis; W.-K.H. received research funding from Novartis; N.C.P.C. received honoraria from Novartis, Incyte and Ascentage and research support from Novartis. The other authors declare no conflict of interest.

Appendix A

Table A1. Summary of relevant individual patient data.
Table A1. Summary of relevant individual patient data.
#Pat.DiagnosisAge
(Years)		SexDate
of Dx		KIT
Status		Additional
Mutations		Tryptase
(µg/L)		BM
Infiltration (%)		A/TM/EAP
(U/L)		Alb
(g/L)		Death
#1SM-B-NHL66M2017D816HNone9020+/−−/−48nkAlive
#2MCL61F2011D816YNone130050−/−−/+16236Death
#3MCL58M2013D816HNone19350−/+−/−8345Death
#4MCL-CEL45F2015D816HNRAS15775+/−−/+12232Death
#5MCL-CMML69F2011D816YTET2, CBL, SRSF235450+/−+/−24639Death
#6MCL-CEL51F2015D816HJAK213550−/−−/+11735Death
#7MCL37M2018D816YNone22560+/−−/−10636Death
#8MCL-CLL54M2018NegativeNone88590−/−−/−8141Death
#9MCL71M2015NegativeNone115050+/+−/−9536Death
#10MCL-MM79M2013NegativeTET218050−/+−/−5939Death
#11MCL63M2014NegativeNone45180+/+−/−15935Death
#12MCL28F2018NegativeNone20240+/+−/−13120Death
#13MCL27M2021NegativeNone11380−/−−/−43733Death
#14MCL-MDS/MPN56F2021NegativeJAK2, DNMT3A, CSF3R63190+/+−/+21031Death
#15SM-MDS64F2017NegativeTET29010−/+−/−6941Alive
#16SM-AML57F2013NegativeRUNX19620−/−−/−6239Death
#17SM-MF59M2015NegativeJAK2, KRAS, TET23420−/−+/−37743Alive
#18SM-MDS62M2022NegativeASXL1, CSF3R, SRSF23135−/−−/−10738Alive
#19SM-MDS/MPN82M2022NegativeFLT3-ITD, U2AF1, CBL, TET21810−/−+/−4536Alive
Pat., patient number; M, male; F, female; Dx, diagnosis; BM, bone marrow; AP, alkaline phosphatase (normal value: 40-130U/L); Alb, albumin (normal value: 35–48 g/L); SM, systemic mastocytosis; MCL, mast cell leukemia; CMML, chronic myelomonocytic leukemia; CEL, chronic eosinophilic leukemia; B-NHL, B-cell non-Hodgkin lymphoma; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm; CLL, chronic lymphocytic leukemia; MM, multiple myeloma; AML, acute myeloid leukemia; MF, myelofibrosis; nk, not known; A/T: anemia Hb ≤ 10.0 g/dL (+), Hb > 10.0 g/dL (−), platelets < 100 × 109/L (+), platelets ≥ 100 × 109/L (−); M/E: monocytosis > 1 × 109/L (+), ≤1 × 109/L (−), eosinophilia > 1 × 109/L (+), ≤1 × 109/L (−), tryptase (normal value: <20 µg/L).
Table A2. Detailed localization of additional mutations on cDNA and protein level.
Table A2. Detailed localization of additional mutations on cDNA and protein level.
#PatientGeneReference SequencecDNA VariantProtein Variant
#4NRASNM_002524.5codon 61codon 61
#5TET2NM_001127208.3c.4210C>Tp.Arg1404*
CBLNM_005188.4c.1169A>Tp.Asp390Val
SRSF2NM_001195427.2c.284C>Ap.Pro95His
#6JAK2NM_004972.4c.1849G>Tp.Val617Phe
#10TET2NM_001127208.3c.2681_2687delp.Ser894Tyrfs*25
#14JAK2NM_004972.4c.1849G>Tp.Val617Phe
DNMT3ANM_022552.4c.2645G>Ap.Arg882His
CSF3RNM_000760.4not availablenot available
#15TET2NM_001127208.3c.1795C>Tp.Gln599*
TET2NM_001127208.3c.2428C>Tp.Gln810*
#16RUNX1ENST00000344691c.877C>Tp.Arg293*
#17JAK2NM_004972.4c.1849G>Tp.Val617Phe
TET2NM_001127208.3c.4546C>Tp.Arg1516*
TET2NM_001127208.3c.4621C>Tp.Gln1541*
KRASNM_004985.5c.108A>Gp.Ile36Met
#18SRSF2NM_001195427.2c.284C>Tp.Pro95Leu
ASXL1NM_015338.6c.3514delp.Ala1172Leufs*2
CSF3RNM_000760.4c.2221C>Tp.Gln741*
#19TET2NM_001127208.3c.1526C>Gp.Ser509*
TET2NM_001127208.3c.5688G>Tp.Arg1896Ser
CBLNM_005188.4c.1259G>Tp.Arg420Leu
U2AF1NM_006758.3c.101C>Tp.Ser34Phe
Cancers 16 00593 g0a1
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.

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