Antibody-Based and Cell Therapies for Advanced Mastocytosis: Established and Novel Concepts

Ci a ion: Valen , P.; Akin, C.; A ock, M.; Gleixne , K.V.; G einix, H.; He mine, O.; Ho ny, H.-P.; I ano , D.; O ao, A.; Rabi sch, W.; e al. An ibody-Based and Cell The apies o Ad anced Mas ocy osis: Es ablished and No el Concep s. In . J. Mol. Sci. 2023,24, 15125. h ps:// doi.o g/10.3390/ijms242015125 Academic Edi o : Paolo Colombo Recei ed: 15 Sep embe 2023 Re ised: 3 Oc obe 2023 Accep ed: 3 Oc obe 2023 Published: 12 Oc obe 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). In e na ional Jou nal o Molecula Sciences Re iew An ibody-Based and Cell The apies o Ad anced Mas ocy osis: Es ablished and No el Concep s Pe e Valen 1,2,* , Cem Akin 3, Michel A ock 4, Ka oline V. Gleixne 1,2, Hildega d G einix 5, Oli ie He mine 6, Hans-Pe e Ho ny 7, Daniel I ano 1,2 , Albe o O ao 8, We ne Rabi sch 9, And eas Rei e 10, Axel Schulenbu g 9, Ka l So la 11, Wol gang R. Spe 1,2 and Celale in Us un 12 1Depa men o In e nal Medicine I, Di ision o Hema ology and Hemos aseology, Medical Uni e si y o Vienna, 1090 Vienna, Aus ia 2Ludwig Bol zmann Ins i u e o Hema ology and Oncology, Medical Uni e si y o Vienna, 1090 Vienna, Aus ia 3Di ision o Alle gy and Clinical Immunology, Uni e si y o Michigan, Ann A bo , MI 48106, USA 4Depa men o Hema ological Biology, Pi ié-Salpê iè e Hospi al, So bonne Uni e si y, 75013 Pa is, F ance 5Di ision o Hema ology, Medical Uni e si y o G az, 8010 G az, Aus ia 6 Se ice d’Héma ologie, Imagine Ins i u e Uni e si éde Pa is, INSERM U1163, Cen e Na ional de Ré é ence des Mas ocy oses, Hôpi al Necke , Assis ance Publique Hôpi aux de Pa is, 75015 Pa is, F ance 7Ins i u e o Pa hology, Ludwig-Maximilians Uni e si y, 80539 Munich, Ge many 8Se icio Cen al de Ci ome ia, Cen o de In es igacion del Cance (IBMCC; CSIC/USAL) Ins i u o Biosani a io de Salamanca (IBSAL), CIBERONC and Depa men o Medicine, Uni e si y o Salamanca, 37007 Salamanca, Spain 9Depa men o In e nal Medicine I, S em Cell T ansplan a ion Uni , Medical Uni e si y o Vienna, 1090 Vienna, Aus ia 10 Depa men o Hema ology and Oncology, Uni e si y Hospi al Mannheim, 68135 Mannheim, Ge many 11 Ins i u e o Pa hology, Uni e si y Hospi al Salzbu g, Pa acelsus Medical Uni e si y, 5020 Salzbu g, Aus ia 12 Depa men o Medicine, Di ision o Hema ology, Oncology, and Cell The apy, Coleman Founda ion Blood and Ma ow T ansplan Cen e a Rush Uni e si y Medical Cen e , Chicago, IL 60612, USA *Co espondence: pe e [email p o ec ed]; Tel.: +43-1-40400-54880 o +43-1-40400-60850 Abs ac : Ad anced sys emic mas ocy osis (SM) is a he e ogeneous g oup o myeloid neoplasms cha ac e ized by an uncon olled expansion o mas cells (MC) in one o mo e in e nal o gans, SM-induced issue damage, and poo p ognosis. Ad anced SM can be ca ego ized in o agg essi e SM (ASM), MC leukemia (MCL), and SM wi h an associa ed hema ologic neoplasm (SM–AHN). In a as majo i y o all pa ien s, neoplas ic cells display a KIT mu a ion, mos ly D816V and a ely o he KIT a ian s. Addi ional mu a ions in o he a ge genes, such as SRSF2,ASXL1, o RUNX1, may also be iden i ied, especially when an AHN is p esen . Du ing he pas 10 yea s, imp o ed ea men app oaches ha e led o a be e quali y o li e and su i al in pa ien s wi h ad anced SM. Howe e , despi e he a ailabili y o no el po en inhibi o s o KIT D816V, no all pa ien s en e emission and o he s elapse, o en wi h a mul i-mu a ed and some imes KIT D816V-nega i e disease exhibi ing mul i-d ug esis ance. Fo hese pa ien s, (poly)chemo he apy, an ibody-based he apies, and allogeneic hema opoie ic s em cell ansplan a ion may be iable ea men al e na i es. In his a icle, we discuss ea men op ions o pa ien s wi h d ug- esis an ad anced SM, including no el KIT- a ge ing d ugs, an ibody-based d ugs, and s em cell-e adica ing he apies. Keywo ds: mas ocy osis; s em cell ansplan a ion; CAR-T; CAR- he apy; immuno he apy; mas cell leukemia 1. In oduc ion Sys emic mas ocy osis (SM) is a g oup o s em cell-de i ed, hema opoie ic neoplasms cha ac e ized by uncon olled expansion and accumula ion o issue mas cells (MC) in a ious in e nal o gans, including he bone ma ow (BM), spleen, li e , and gas oin es inal (GI) ac [ 1 – 3 ]. In a subse o pa ien s, he skin may be in ol ed [ 4 ]. Howe e , unlike In . J. Mol. Sci. 2023,24, 15125. h ps://doi.o g/10.3390/ijms242015125 h ps://www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2023,24, 15125 2 o 14 pa ien s wi h ypical indolen SM, some pa ien s wi h ad anced SM may only ha e a ew o no skin lesions. In pa ien s wi h ad anced SM, MC in il a ion in a ious o gans is associa ed wi h o gan damage [ 1 – 3 ]. Due o imp o ed diagnosis, he p e alence o (ad anced) SM has inc eased o e he pas ew yea s. The es ima ed p e alence o adul SM is app oxima ely 1 pe 10,000 in he Wes e n wo ld. Acco ding o he classi ica ion o he Wo ld Heal h O ganiza ion (WHO), ad anced SM can be ca ego ized in o agg essi e SM (ASM), SM wi h an associa ed hema ologic neoplasm (SM–AHN), and MC leukemia (MCL) [ 1 – 3 , 5 – 9 ]. ASM can u he be spli in o a non- ans o med a ian and ASM in ans o ma ion o MCL (ASM- ) whe e MC in BM smea s accoun o 5–19% o all nuclea ed cells [ 8 , 10 ]. SM–AHN is u he di ided in o pa ien s wi h indolen SM (ISM–AHN), ASM–AHN, and MCL–AHN [8,10]. Fu he mo e, SM–AHN is also classi ied based on he ype o AHN [1,5–8]. MCL can be di ided in o a leukemic a ian (MC in pe iphe al blood ≥ 10%) and an aleukemic a ian (MC in blood < 10%) [ 5 – 8 , 10 ]. In addi ion, MCL is spli in o a p ima y o m o MCL (de no o MCL) and seconda y MCL a ising om ano he o m o mas ocy osis, such as smolde ing SM (SSM), ASM, SM–AHN, o MC sa coma (MCS) [ 8 , 10 ]. Especially in ASM- and MCS, he a e o ansi ion in o MCL is high [ 10 – 13 ]. The e o e, al hough no an SM as pe SM c i e ia, MCS is a high-g ade MC neoplasm whe e cy o educ i e he apy and o he in ensi e ea men app oaches ha e o be conside ed. In mos pa ien s wi h ad anced SM, neoplas ic cells display ans o ming mu a ions in he KIT oncogene (Table 1) [ 14 – 17 ]. These mu a ions lead o cy okine-independen au onomous g ow h o neoplas ic MC p ogeni o s. The mos p e alen mu an o m o KIT is D816V. This mu a ion is ound in all ca ego ies o ad anced SM, and o en also in he AHN po ion o he disease. Howe e , in some pa ien s wi h ASM, MCL, o MCS, o he KIT-ac i a ing mu a ions may be de ec ed [ 11 – 17 ]. The same holds ue o pedia ic SM, amilial SM, and he well-di e en ia ed sub ypes o SM. Mo eo e , in pa ien s wi h ad anced SM, especially SM–AHN, neoplas ic cells o en exhibi addi ional mu a ions in clinically ele an genes [ 15 – 23 ]. These include mu a ions in SRSF2,ASXL1,RUNX1, o DNMT3A, which may be associa ed wi h disease e olu ion and p og ession as well as d ug esis ance in neoplas ic cells [ 19 – 22 ]. Some imes, mo e malignan subclones, especially AHN- ela ed clones ha a e d ug esis an and lack KIT D816V, de elop ( h ough selec ion) in hese pa ien s, e en i he ini ial dominan sub-clone displayed KIT D816V. Table 1. Es ima ed p e alence o KIT D816V, o he KIT mu a ions, and mu a ions in o he genes in ad anced mas cell neoplasms. Disease Va ian P e alence o KIT D816V O he KIT Mu a ions O he Gene Va ian s * SSM >90% <10% <10% SM–AHN >90% <10% >70% ASM 80–90% 10–20% ** 10–20% Ad SMWD 20% 50–80% ** <10% MCL 70–80% 20–30% ** 10% MCS *** <10% *** <10% 10–30% *** * O he gene ic abno mali ies (apa om KIT mu a ions) a e o en de ec ed in pa ien s wi h SM wi h associa ed hema ologic neoplasm (SM–AHN) and include, among o he s, mu a ions in RUNX1,TET2,SRSF2,RAS, o ASXL1. ** Some o he esul ing KIT mu an o ms a e sensi i e agains ima inib, whe eas KIT D816V con e s esis ance agains ima inib. *** In ue (p ima y) MCS wi hou ea u es o SM, no KIT D816V o o he KIT mu a ions a e ound. Abb e ia ions: SM, sys emic mas ocy osis; SSM, smolde ing SM; ASM, agg essi e SM; Ad SM, ad anced SM; SMWD, well-di e en ia ed SM; MCL, mas cell leukemia; MCS, mas cell sa coma. Ano he majo p oblem in SM is MC ac i a ion. In ac , hese pa ien s o en su e om ecu en , media o -induced symp oms o e en anaphylaxis, especially when a con- comi an , IgE-dependen alle gy is also p esen [ 3 – 8 ]. Media o - ela ed symp oms include, among o he s, lushing, i ching, c amping, dia hea, and hypo ension [ 3 – 8 ]. In many cases, alle gens a e inducing hese symp oms. Howe e , pa ien s wi h indolen o ad anced SM may also de elop hese symp oms upon d ug exposu e. In . J. Mol. Sci. 2023,24, 15125 3 o 14 2. S anda d T ea men Op ions o Pa ien s wi h Ad anced SM In he pas 10 yea s, he ea men o pa ien s wi h ad anced SM has imp o ed sub- s an ially. Fi s , a numbe o KIT D816V- a ge ing y osine kinase inhibi o s (TKI) ha e been de eloped and ha e been applied success ully in pa ien s wi h ad anced SM [ 24 – 33 ]. Two o hese TKI ha e ecen ly been app o ed o he ea men o ad anced ( esis an ) SM by majo heal h au ho i ies in he US and EU, namely midos au in and a ap i inib. Bo h agen s a e able o supp ess disease- ela ed symp oms and he expansion o neoplas ic MC in a conside able numbe o pa ien s [ 26 – 33 ]. A ap i inib is a supe io d ug ha induces hema ologic emission in a subs an ial subse o pa ien s wi h ad anced SM [ 30 – 33 ]. Based on hei clinical e icacy, midos au in and a ap i inib a e conside ed i s -line agen s in he ea men o ad anced SM. Howe e , despi e imp essi e esul s, some pa ien s con inue o p og ess o elapse, o en in he o m o a KIT D816V-nega i e disease [ 30 – 33 ]. Fo such cases, al e na i e ea men op ions, including expe imen al d ugs, ha e o be conside ed. Apa om KIT D816V- a ge ing d ugs, also o he an i-neoplas ic agen s a e a ailable o pa ien s wi h ad anced SM. Some o hese pa ien s may espond o clad ibine (2CdA) [ 34 – 38 ]. In o he pa ien s, especially hose wi h apid p og ession o MCL o ano he (acu e) leukemia, poly-chemo he apy o e en allogeneic hema opoie ic s em cell ansplan a ion (alloHSCT, e e ed o as HSCT in his pape ) a e ecommended [ 1 , 2 , 6 ]. Hyd oxyu ea is commonly used as a pallia i e d ug in ad anced SM. Table 2p o ides a summa y o ea men op ions o pa ien s wi h ad anced SM. Table 2. T ea men op ions o pa ien s wi h ad anced mas ocy osis. T ea men Indica ions In e e on-alpha (IFN-A) * T ea men - e ac o y os eopo osis (low dose IFN-A). ASM wi h li e in ol emen and asci es (pa ien no eligible o TKI he apy o 2CdA). IFN-A esponsi e AHN. Clad ibine (2CdA) Pa ien no eligible o KIT TKI he apy o pa ien esis an agains KIT TKI o no KIT TKI a ailable. Ima inib KIT D816V-nega i e ad anced SM, including ASM wi h KIT K509I. Masi inib KIT D816V-nega i e ad anced SM. Midos au in ASM, ASM- , ASM–AHN, MCL, MCL–AHN. ISM–AHN whe e he AHN is a KIT-d i en (KIT D816V+) agg essi e neoplasm. A ap i inib ASM, ASM- , ASM–AHN, MCL, MCL–AHN. ISM–AHN whe e he AHN is a KIT-d i en (KIT D816V+) agg essi e neoplasm. Hyd oxyu ea (HU) Mul i-d ug- esis an ad anced SM, including ASM, MCL and SM–AHN. HU is a s anda d pallia i e d ug. Local adia ion Mas cell sa coma (MCS) o MCS-like p og ession o ASM. Huge splenomegaly: as debulking p io o CT. Skele al disease (huge os eolysis wi h local umo mass). Poly-chemo he apy D ug- esis an ad anced SM. Debulking as p epa a ion o HSCT. SM–CMML wi h p og essing CMML o SM–AML in pa ien s no eligible o HSCT. Mono-chemo he apy: deme hyla ing agen s and o he d ugs, such as ene oclax Pa ien s wi h ad anced, KIT TKI- esis an SM no eligible o poly-chemo he apy o HSCT (and/o esis an agains 2CdA). SM–AHN pa ien s no eligible o HSCT in whom he AHN may be esponsi e: (example: azaci idine in MDS o AML). Allogeneic HSCT D ug esis an ad anced SM, ASM- o MCL. ASM wi h apid p og ession. SM–CMML, SM–AML. * Du ing he ini ial ew weeks o IFN-A, he d ug is o en applied oge he wi h o al p ednisolone (s a ing a 1 mg/kg/day). Abb e ia ions: TKI, y osine kinase inhibi o ; AHN, associa ed hema ologic neoplasm; SM, sys emic mas ocy osis; ASM, agg essi e SM; ASM- , ASM in ans o ma ion; MCL, mas cell leukemia; MDS, myelodysplas ic synd ome; ISM, indolen SM; CT, poly-chemo he apy; HSCT, (allogeneic) hema opoie ic s em cell ansplan a ion; CMML, ch onic myelomonocy ic leukemia; AML, acu e myeloid leukemia. In . J. Mol. Sci. 2023,24, 15125 4 o 14 When conside ing ea men op ions in a pa ien wi h ad anced SM, he i s impo - an ques ions a e whe he he disease is apidly p og essing, whe he an AHN is p esen , and whe he mos o all disease componen s exhibi he KIT D816V mu a ion (Figu e 1). In addi ion, i is impo an o know whe he he pa ien is eligible o in ensi e he apy (Figu e 1). In hose wi h KIT D816V-posi i e ad anced SM, KIT D816V- a ge ing d ugs (mi- dos au in o a ap i inib) a e usually ecommended as i s -line he apy [ 1 , 2 , 25 – 33 ]. When TKI a e no a ailable o he pa ien is in ole an , clad ibine (2CdA) may be conside ed. Howe e , al hough 2CdA is e ec i e, esponse a es a e lowe compa ed o hose seen wi h midos au in o a ap i inib [ 34 – 38 ]. In pa ien s who ha e KIT D816V-nega i e ad anced SM, o he KIT- a ge ing TKI, such as ima inib, may be conside ed, especially when KIT sequencing e eals a sensi i e mu a ion o wild- ype KIT (Figu e 1, Table 2) [ 39 – 42 ]. Mi- dos au in o a ap i inib may also be applied in such pa ien s (Figu e 1). I is impo an o no e in his ega d ha in abou 80% o pa ien s wi h well-di e en ia ed ad anced SM, in abou 20–30% o all cases wi h MCL, and in abou 90% o all pa ien s wi h ue (p ima y) MCS, neoplas ic cells lack KIT D816V (Table 1). In se e al o he pa ien s wi h MCL, MC display o he mu a ions in codon 816, such as D816H o D816Y. I is also impo an o no e ha es ing o he KIT D816V mu a ion should be pe o med wi h a highly sensi i e PCR es in o de o a oid alse-nega i e esul s. In pa ien s wi h well-di e en ia ed SM, a KIT mu an o m ha is esponsi e o ima inib, may be de ec ed [ 39 – 43 ]. In mos pa ien s wi h ue MCS, no KIT mu a ions a e ound, and neoplas ic cells a e usually esis an agains KIT- a ge ing and con en ional d ugs, including chemo he apy [11–13]. In .J.Mol.Sci.2023,24,xFORPEERREVIEW5o 15    Figu e1.T ea men algo i hm o pa ien swi had ancedsys emicmas ocy osis(SM).A e ha ing es ablished hediagnosiso ad ancedSM, he a ian o disease, heclinicalbeha io (agg essi e wi h apidp og essiono indolen ),and hep esenceand ypeo AHNneed obede e minedusing WHOc i e iaand/o c i e iap o idedby hein e na ionalconsensusclassi ica ion(ICC).Inaddi- ion, hep esenceand ypeo molecula  a ge sneed obede ined.Inpa ien swi hKITD816V+ ad ancedSMo o he simila mu a ionsa codon816(KIT816-m), y osinekinaseinhibi o s(TKI) di ec edagains KITD816Va eusuallyappliedas i s -line he apy.TwosuchTKIha ebeenap- p o edby heFDAandEMA:midos au inanda ap i inib.Inpa ien swhoha ea apidlyp o- g essingASMo MCLo SM–AHNwhe e heAHNisanacu emyeloidleukemia(AML)o  apidly p og essingch onicmyelomonocy icleukemia(CMML),in ensi epoly-chemo he apy(CT)should beconside edasanal e na i e i s -line ea men op ion.Inpa ien swhoa eeligibleand espond oTKIo CT,subsequen allogeneichema opoie ics emcell ansplan a ionshould henalsobe conside ed.Inaddi ion,pa ien swho elapsea e d ug he apyshouldbeconside ed o CTand subsequen HSCT.Howe e ,suchpa ien smayalsobecandida es o expe imen ald ugso palli- a i ed ugs.Inpa ien swi had ancedSMwhoha enoKITmu a iono amu an  o msensi i e o ima inib,ima inibmaybeconside edas i s line he apy.Finally,inpa ien swi hSM–AHNin whom heAHNisanagg essi ediseaseexhibi ingce ainmolecula  a ge s, a ge edd ug he a- piesa eusually ecommended.Thes anda dpallia i ed ug o pa ien swi hd ug- esis an disease who elapseda e CTo HSCT, emainshyd oxyu ea(HU).Abb e ia ions:SM,sys emicmas ocy- osis;ASM,agg essi eSM;SM–AHN,SMwi hanassocia edhema ologicneoplasm;MCL,mas  cellleukemia;CCR,con inuouscomple e emission;WHO,wo ldheal ho ganiza ion;FDA,Food andD ugAdminis a ion;EMA;Eu opeanMedicinesAgency. Fo pa ien swi h apidlyp og essingASMo MCL,poly-chemo he apywi ho  wi hou subsequen HSCTiso en ecommended(Figu e1)[1,2,44–47].Fo  hosewhoa e eligible o HSCT, heop imalway op oceedmaybe os a debulking(byaTKIand/o  chemo he apy)and oin oduceHSCTasea lyaspossible[44–46].Thisalsoholds ue o pa ien swi hMCS,asmanyo  hesecases ans o m oMCL[11–13,48]. 3.SpecialConside a ions o  heT ea men o Pa ien swi hSM–AHN Inpa ien swi hSM–AHN, ea men o  heAHNiso en equi ed.Indeed,insuch cases, heAHNmaybeanagg essi emalignancy.In hesepa ien s,i isessen ial oknow Figu e 1. T ea men algo i hm o pa ien s wi h ad anced sys emic mas ocy osis (SM). A e ha ing es ablished he diagnosis o ad anced SM, he a ian o disease, he clinical beha io (agg essi e wi h apid p og ession o indolen ), and he p esence and ype o AHN need o be de e mined using WHO c i e ia and/o c i e ia p o ided by he in e na ional consensus classi ica ion (ICC). In addi ion, he p esence and ype o molecula a ge s need o be de ined. In pa ien s wi h KIT D816V+ ad anced SM o o he simila mu a ions a codon 816 (KIT 816-m), y osine kinase inhibi o s (TKI) di ec ed agains KIT D816V a e usually applied as i s -line he apy. Two such TKI ha e been app o ed by he FDA and EMA: midos au in and a ap i inib. In pa ien s who ha e a apidly p og essing ASM o MCL o SM–AHN whe e he AHN is an acu e myeloid leukemia (AML) o apidly p og essing ch onic myelomonocy ic leukemia (CMML), in ensi e poly-chemo he apy (CT) should be conside ed In . J. Mol. Sci. 2023,24, 15125 5 o 14 as an al e na i e i s -line ea men op ion. In pa ien s who a e eligible and espond o TKI o CT, subsequen allogeneic hema opoie ic s em cell ansplan a ion should hen also be conside ed. In addi ion, pa ien s who elapse a e d ug he apy should be conside ed o CT and subsequen HSCT. Howe e , such pa ien s may also be candida es o expe imen al d ugs o pallia i e d ugs. In pa ien s wi h ad anced SM who ha e no KIT mu a ion o a mu an o m sensi i e o ima inib, ima inib may be conside ed as i s line he apy. Finally, in pa ien s wi h SM–AHN in whom he AHN is an agg essi e disease exhibi ing ce ain molecula a ge s, a ge ed d ug he apies a e usually ecommended. The s anda d pallia i e d ug o pa ien s wi h d ug- esis an disease who elapsed a e CT o HSCT, emains hyd oxyu ea (HU). Abb e ia ions: SM, sys emic mas ocy osis; ASM, agg essi e SM; SM– AHN, SM wi h an associa ed hema ologic neoplasm; MCL, mas cell leukemia; CCR, con inuous comple e emission; WHO, wo ld heal h o ganiza ion; FDA, Food and D ug Adminis a ion; EMA; Eu opean Medicines Agency. Fo pa ien s wi h apidly p og essing ASM o MCL, poly-chemo he apy wi h o wi hou subsequen HSCT is o en ecommended (Figu e 1) [ 1 , 2 , 44 – 47 ]. Fo hose who a e eligible o HSCT, he op imal way o p oceed may be o s a debulking (by a TKI and/o chemo he apy) and o in oduce HSCT as ea ly as possible [ 44 – 46 ]. This also holds ue o pa ien s wi h MCS, as many o hese cases ans o m o MCL [11–13,48]. 3. Special Conside a ions o he T ea men o Pa ien s wi h SM–AHN In pa ien s wi h SM–AHN, ea men o he AHN is o en equi ed. Indeed, in such cases, he AHN may be an agg essi e malignancy. In hese pa ien s, i is essen- ial o know whe he mos AHN cells (subclones) ca y KIT D816V. Likewise, in SM wi h ch onic myelomonocy ic leukemia (SM–CMML), neoplas ic monocy es a e o en KIT D816V- posi i e, which may a o ea men wi h a KIT D816V- a ge ing d ug [ 49 , 50 ]. By con as , in acu e myeloid leukemia (AML), some o e en mos AML subclones may lack KIT D816V, so ha ea men wi h a KIT D816V- a ge ing d ug alone may e en lead o selec ion o mo e KIT D816V-nega i e sub-clones [ 51 – 53 ]. In gene al, pa ien s wi h SM–AHN should be ea ed o hei AHN as i no SM was diagnosed, and he SM po ion o he disease should be ea ed as i no AHN was ound [ 1 – 3 , 5 – 7 , 45 , 47 ]. This s a egy implies ha se e al o hese pa ien s ecei e combina ions o an i-neoplas ic d ugs o sequen ial d ug he apies. Fo example, in a pa ien wi h ASM–AML exhibi ing KIT D816V and a FLT3-ITD mu a ion, he disease may be ea ed wi h midos au in, oge he wi h chemo he apy (di ec combina- ion). Thus, i is impo an ha bo h he SM and he AHN po ion o he disease a e ea ed in a a ge -speci ic manne . The e o e, i is also o c ucial impo ance o es ablish he co ec inal diagnosis in each case, and o de ine bo h disease componen s by adding he molecula signa u e in he epo [ 1 – 3 , 5 – 7 , 45 , 47 ]. Fo example, in a pa ien wi h ISM exp essing KIT D816V wi h an associa ed JAK2 V617F-posi i e p ima y myelo ib osis (PMF), he inal diagnosis is ISM–PMF exhibi ing KIT D816V (in SM cells) and JAK2 V617F (in MPN cells). In such pa ien , no KIT D816V- a ge ing d ug is equi ed, bu when PMF- ela ed symp oms occu , he pa ien may be a candida e o ea men wi h a JAK2 V617F- a ge ing d ug. In some pa ien s wi h SM–AHN, i may be di icul o in e p e po en ial C- indings (like cy openia), especially when bo h he SM po ion and he AHN could cause cy openia, o example, ASM wi h associa ed myelodysplas ic synd omes/neoplasm (MDS). In hese pa ien s, he AHN may be an ad anced KIT D816V-nega i e MDS (o seconda y AML) equi ing ea men wi h 5-azaci idine (AZA) o AZA plus ene oclax. In such cases, he AHN po ion o he disease may o may no espond o hese s anda d ea men s in he same way as in pa ien s wi hou SM [ 54 – 56 ]. Midos au in and a ap i inib may also be e ec i e in hese cases, especially when mos o all AHN cells a e KIT mu a ed, o a e d i en by KIT-induced oncogenic pa hways [ 57 , 58 ]. Howe e , in SM–AML, some o e en mos AML-subclones may lack KIT D816V [ 51 , 52 ]. The same holds ue o pa ien s wi h MCS and some wi h MCL. O e all, i seems as i mo e ad anced, p og essi e SM is associa ed wi h he de elopmen o mul iple d ug- esis an subclones, and some o e en mos o hese subclones lack KIT mu an o ms including KIT D816V. In . J. Mol. Sci. 2023,24, 15125 6 o 14 In pa ien s in whom he KIT D816V mu a ion s a us is unknown o no KIT mu a ion was ound, a KIT inhibi o may s ill be a he apeu ic op ion. Indeed, midos au in and a ap i inib supp ess he ac i i y o wild- ype KIT, and bo h may exe an i-neoplas ic e ec s in SM ega dless o he KIT mu a ion s a us. Some imes, KIT D816V is no de ec ed in neoplas ic cells based on he low sensi i i y o he assay. The e o e, we ecommend es ing o KIT D816V wi h a highly sensi i e PCR assay be o e s a ing he apy in all pa ien s [15–17,59]. As men ioned, when bo h he SM and he AHN equi e speci ic an i-neoplas ic he a- pies, d ug combina ions ha e o be conside ed. In pa ien s wi h an agg essi e malignancy, such as MCL and/o AML, in ensi e he apy (plus TKI he apy) is in oduced o achie e emission be o e he pa ien is p epa ed o allogeneic HSCT [ 1 , 2 , 45 – 47 , 58 ]. In hose who a e no eligible o HSCT, con inuous ea men wi h a ap i inib may be an al e na i e ea men op ion. 4. T ea men Op ions o Pa ien s wi h D ug-Resis an Ad anced SM The p ognosis o pa ien s wi h ad anced SM who a e esis an agains KIT- a ge ing d ugs and/o o he an i-neoplas ic d ugs, including 2CdA, is usually dismal. Some o hese pa ien s may no ha e ye been ea ed wi h a ap i inib because he d ug was no a ailable. These pa ien s may bene i om a ap i inib he apy [ 32 , 33 ]. O he pa ien s may bene i om expe imen al d ugs o d ug combina ions using TKI (midos au in, a ap i inib) and o he a ge ed d ugs such as ene oclax, o TKI and con en ional an ineoplas ic d ugs such as 2CdA. Howe e , mos esponses a e only ansien and a e ollowed by a elapse. The e o e, when eligible, hese pa ien s a e p epa ed o HSCT. I HSCT is no a iable op ion, pa ien s a e ea ed wi h expe imen al chemo he apy o pallia i e d ugs such as hyd oxyu ea (HU) [ 1 , 2 , 45 ]. In hose wi h d ug- esis an SM–AML who a e no eligible o HSCT, chemo he apy egimens con aining AZA, 2CdA, ene oclax, o o he d ugs may be o e ed [1,2,45,60]. Howe e , mos o hese pa ien s ha e a sho su i al ime. 5. Allogeneic HSCT HSCT emains he only po en ially cu a i e ea men o pa ien s wi h ad anced SM who a e esis an agains mul iple d ug he apies. Especially young and i pa ien s wi h mul i- esis an ad anced SM may bene i om HSCT [ 44 – 46 , 56 , 58 , 61 – 64 ]. In hese pa ien s, g a e sus mas ocy osis e ec s ha e been documen ed [ 44 , 62 , 63 ]. Howe e , so a , no con olled clinical s udies explo ing he de ini i e alue o HSCT in pa ien s wi h ad anced, d ug- esis an SM ha e been pe o med. In one s udy, pa ien s wi h ad anced SM who we e ea ed wi h HSCT (ei he i s -line o a e d ug he apy) we e examined in a e ospec i e mul i-cen e s udy [ 44 ]. In his, s udy, pa ien s wi h ASM, MCL, and SM–AHN we e included. The ou comes a e HSCT conce ning su i al and elapse- ee su i al we e ound o be be e o hose who had ASM o SM–AHN compa ed o hose wi h MCL [ 44 ]. The ou comes a e HSCT we e also ound o be mo e a o able when myeloabla i e condi ioning was pe o med compa ed o dose- educed (non-myeloabla i e) condi ioning [ 44 ]. The e a e addi ional ac o s ha may impac on ou come and p ognosis o HSCT in pa ien s wi h ad anced SM. Fo example, he ou come may be be e when success ul debulking can be pe o med p io o HSCT. Indeed, in pa ien s in whom de- bulking wi h a ge ed d ugs o chemo he apy ailed, HSCT is di icul o pe o m, and pos -HSCT ou comes a e usually poo . The e o e, mos expe s ecommend ea ly HSCT and ea ly p e-HSCT he apy in eligible pa ien s wi h ad anced SM. The ype o debulking he apy depends on he ype o AHN, he p esence o KIT D816V, and o he molecula a ge s displayed by neoplas ic cells. In hose wi h a high a ian allele equency o KIT D816V, a ap i inib may be app op ia e and su icien o induce debulking [ 58 ]. In he case o SM–AML, poly-chemo he apy should be conside ed as he mos e ec i e and mos p omising app oach o achie e subs an ial debulking be o e HSCT. The e is also a g owing discussion abou he use o KIT- a ge ing d ugs (midos au- in, a ap i inib) a e success ul HSCT. Indeed, based on case epo obse a ions, such In . J. Mol. Sci. 2023,24, 15125 7 o 14 ea men , when in oduced a e hema ologic egene a ion ollowing HSCT ( o example om day +100), may keep he neoplas ic disease p ocess unde con ol [ 28 , 64 ]. Howe e , no esul s om con olled clinical ials a e a ailable, and i emains unknown whe he such pos -HSCT he apy exe s majo e ec s on esidual neoplas ic s em cells (NSC) as a use ul main enance he apy. In addi ion, i emains unce ain how long such pos -HSCT he apy wi h KIT- a ge ing d ugs should be pe o med. In mos cen e s, such pa ien s ha e been ea ed wi h midos au in o 1 o 2 yea s pos -HSCT. In pa ien s wi h measu - able minimal esidual disease (by KIT D816V PCR), longe ea men wi h KIT- a ge ing d ugs may be conside ed. A ap i inib has ecen ly been applied pos -HSCT in pa ien s wi h RUNX1-RUNX1T1-posi i e KIT-mu a ed AML wi h minimal esidual disease who ailed immuno he apy wi h in e e on alpha o dono lymphocy e in usions [ 65 ]. In hese pa ien s, cy openia occu ed equen ly. Howe e , no clinical s udies using a ap i inib pos -HSCT in ad anced SM ha e been conduc ed o da e. 6. An ibody-Based T ea men o D ug-Resis an Ad anced SM Al hough an ibody-based d ug he apies a e a ailable o pa ien s wi h high- isk AML and he apy- e ac o y AML, no an ibody-based he apy o pa ien s wi h MCL o o he o ms o ad anced SM is a ailable. Since mos MC and mos NSC, including AHN- ela ed NSC, display CD33 in mos SM pa ien s (Table 3) [ 66 – 69 ], he CD33- a ge ed d ug conjuga e gem uzumab ozogamicin (GO) has been es ed in p eclinical s udies. Based on i s an i-neoplas ic e ec s on neoplas ic MC and NSC in in i o s udies [ 67 , 68 ], GO has been p oposed o he ea men o ad anced SM. Howe e , only a ew anecdo al epo s ha e been published so a . In one epo , ea men o a pa ien wi h d ug- esis an SM–AHN esul ed in disease debulking and sus ained emission [ 70 ]. In o he pa ien s wi h ad anced SM, ea men wi h GO and poly-chemo he apy esul ed in a subs an ial dec ease in he numbe s o NSC [ 68 ]. The e a e also o he su ace a ge s ha a e exp essed on neoplas ic MC and/o NSC, and may he e o e se e as molecula a ge s in ad anced SM (see below). Table 3. Cell su ace a ge exp ession p o iles o neoplas ic mas cells and neoplas ic s em cells in pa ien s wi h ad anced SM. CD An igen, Ta ge Cell Su ace Exp ession De ec ed by Flow Cy ome y * on Neoplas ic Mas Cells CD34+/CD38−S em Cells ASM SM–AHN MCL ASM SM–AHN MCL CD2 LFA-2 + + +/− − − − CD13 A-Pep -N + + + + + + CD25 IL-2RA + + + +/−+/- +/- CD30 Ki-1 +/−+/−+/− −/+ −/+ −/+ CD33 Siglec-3 + + + + + + CD44 He mes + + + + + + CD47 IAP + + + + + + CD52 Campa h-1 + + + −/+ −/+ −/+ CD117 KIT + + + + + + CD123 IL-3RA +/−+/−+/−+++ CD184 CXCR4 + + + + + + CD274 PD-L1 + + + n.k. n.k. n.k. CD327 Siglec-6 + + + −−− * Su ace exp essions o a ge s we e analyzed wi h luo och ome-labeled an ibodies and mul i-colo low cy ome- y. Exp essions o a ge s on KIT ++ mas cells and CD34 + /CD38 − s em cells we e quan i ied using he ollowing sco e: +, clea ly exp essed on >75% o all cells; +/ − , exp essed weakly o only on a subse (10–50%) o all cells; − /+, exp essed only in ace amoun s o in a e y small sub- ac ion o cells (<10%). Abb e ia ions: SM, sys emic mas ocy osis; ASM, agg essi e SM; SM–AHN, SM wi h an associa ed hema ologic neoplasm; MCL, mas cell leukemia; A-Pep -N, aminopep idase N; IL-2RA, in e leukin-2 ecep o alpha chain; IAP-1, in eg in-associa ed p o ein; IL-3RA, in e leukin-3 ecep o alpha chain; PD-L1, p og ammed dea h ligand-1; n.k., no known. In . J. Mol. Sci. 2023,24, 15125 8 o 14 7. Ta ge Exp ession P o iles o NSC in Ad anced SM Recen da a sugges ha NSC in ad anced SM (including ASM, SM–AHN, and MCL) eside in a small CD34 + /CD38 − ac ion o he malignan clone [ 68 ]. These cells ha e a selec i e po en ial o ini ia e and p opaga e he disease in i o in NSG mice exhibi ing hu- man memb ane-bound s em cell ac o (NSG hSCF mice) [ 68 ]. Al hough no all obse a ions can be ansla ed om mouse models o he human sys em, hese NSC may also p opaga e he disease in pa ien s wi h ad anced SM o unlimi ed ime pe iods. By con as , he mo e ma u e cells in he same disease, including CD34 + /CD38 + p ogeni o cells and he bulk o neoplas ic MC, a e unable o ini ia e and p opaga e he malignancy in i o [ 68 ]. As a consequence, any he apy can only ac as a cu a i e app oach when elimina ing mos o all CD34 + /CD38 − NSC in a gi en pa ien . In mos pa ien s wi h ad anced SM, CD34 + /CD38 − NSC exhibi he key s em cell ma ke s CD13 (aminopep idase-N), CD123 (IL-3 ecep o alpha), and CD133 (AC133) [ 68 ]. These an igens a e also exp essed on neoplas ic MC in SM, and o en also on AHN cells. In addi ion, NSC as well as MC exp ess a numbe o cell su ace a ge s, such as CD33 (Siglec-3), CD44 (He mes), and CD117 (KIT) (Figu e 2, Table 3) [ 67 – 75 ]. In con as , o he immunological a ge s, such as CD30 (Ki-1) o CD327 (Siglec-6), a e only de ec able on neoplas ic MC, bu a e no de ec able (o only ound in ace amoun s) on NSC in ad anced SM (Table 3) [ 76 – 78 ]. These a ge s may be less a ac i e, since he apies di ec ed agains hese an igens would only lead o an e adica ion o MC, bu no o an elimina ion o NSC. Ano he impo an aspec is ha almos all su ace a ge s iden i ied on NSC in ad anced SM a e also exp essed on no mal hema opoie ic s em cells. This holds ue o CD33, CD44, CD117, and CD123. As a consequence, a ge ed ea men app oaches can lead o he e adica ion o no mal hema opoie ic s em cells, and hus p olonged cy openia. This is a majo issue when conside ing he de elopmen o an ibody-based he apies o cell-based he apies such as CAR-T o CAR-NK cell he apies. On he o he hand, se e al o hese a ge s a e exp essed on NSC a much highe le els compa ed o no mal s em cells, so ha ea men wi h an ibody-based a ge ed d ugs may be a easible app oach because o he he apeu ic window. This may hold ue o CD33, CD44, and CD123. Indeed, he ea men o AML wi h he CD33-based oxin conjuga e GO a ecommended doses is o en associa ed wi h p olonged cy openia, bu usually does no lead o i e e sible aplasia [ 79 ]. Whe he his is also he case wi h an ibody-based d ugs di ec ed agains CD117 o CD123 emains unknown. Fo example, no use ul he apeu ic window has been iden i ied o CD117 on neoplas ic MC o NSC in SM, as he su ace exp ession is some imes e en lowe on NSC compa ed o no mal s em cells [ 68 ]. Howe e , he e is s ill some hope ha CD117- a ge ed d ug he apies may no lead o comple e s em cell exhaus ion. In ac , in pa ien s wi h Ph+ ch onic myeloid leukemia, long- e m ea men wi h s ong inhibi o s o KIT such as ima inib does no lead o se e e aplasia, e en when he pa ien s a e ea ed o se e al decades [ 80 ]. Finally, some o he cell-based he apies, such as CAR-T cell he apies, may be combined wi h HSCT in o de o sol e he p oblem o e adica ion o no mal s em cells. In . J. Mol. Sci. 2023,24, 15125 9 o 14 In .J.Mol.Sci.2023,24,xFORPEERREVIEW9o 15   since he apiesdi ec edagains  hesean igenswouldonlylead oane adica iono MC, bu no  oanelimina iono NSC.Ano he impo an aspec is ha almos allsu ace a - ge siden i iedonNSCinad ancedSMa ealsoexp essedonno malhema opoie ics em cells.Thisholds ue o CD33,CD44,CD117,andCD123.Asaconsequence, a ge ed ea men app oachescanlead o hee adica iono no malhema opoie ics emcells,and husp olongedcy openia.Thisisamajo issuewhenconside ing hede elopmen o an- ibody-based he apieso cell-based he apiessuchasCAR-To CAR-NKcell he apies. On heo he hand,se e alo  hese a ge sa eexp essedonNSCa muchhighe le els compa ed ono mals emcells,so ha  ea men wi han ibody-based a ge edd ugsmay bea easibleapp oachbecauseo  he he apeu icwindow.Thismayhold ue o CD33, CD44,andCD123.Indeed, he ea men o AMLwi h heCD33-based oxinconjuga e GOa  ecommendeddosesiso enassocia edwi hp olongedcy openia,bu usuallydoes no lead oi e e sibleaplasia[79].Whe he  hisisalso hecasewi han ibody-based d ugsdi ec edagains CD117o CD123 emainsunknown.Fo example,nouse ul he a- peu icwindowhasbeeniden i ied o CD117onneoplas icMCo NSCinSM,as he su aceexp essionissome imese enlowe onNSCcompa ed ono mals emcells[68]. Howe e , he eiss illsomehope ha CD117- a ge edd ug he apiesmayno lead o comple es emcellexhaus ion.In ac ,inpa ien swi hPh+ch onicmyeloidleukemia, long- e m ea men wi hs onginhibi o so KITsuchasima inibdoesno lead ose e e aplasia,e enwhen hepa ien sa e ea ed o se e aldecades[80].Finally,someo  he cell-based he apies,suchasCAR-Tcell he apies,maybecombinedwi hHSCTino de  osol e hep oblemo e adica iono no mals emcells.  Figu e2.Exp essionso cellsu acema ke sonneoplas ics emcellsinSM.Bonema owmononu- clea cellswe eob ained omapa ien wi hsys emicmas ocy osis(SM)wi hanassocia edhema- Figu e 2. Exp essions o cell su ace ma ke s on neoplas ic s em cells in SM. Bone ma ow mononu- clea cells we e ob ained om a pa ien wi h sys emic mas ocy osis (SM) wi h an associa ed hema o- logic neoplasm (SM–AHN), and s ained wi h phycoe y h in (PE)-conjuga ed monoclonal an ibodies di ec ed agains CD2, CD25, CD30, CD33, CD44, and CD52, as well as luo och ome-labeled an i- bodies agains CD34 and CD38. Exp essions o a ge an igens on CD34 + /CD38 − s em cells we e assessed ia mul i-colo low cy ome y, and a e indica ed by he blue his og ams. The iso ype- ma ched con ol an ibody is also shown (black open his og ams). Rep esen a i e his og ams show exp essions o CD25, CD33, and CD44 on neoplas ic s em cells, whe eas hese cells exp essed only ace amoun s o CD30 and CD52, and s ained nega i e o CD2. S em cells also displayed CD123 (no shown), con i ming he neoplas ic na u e o hese cells. 8. No el App oaches o Ta ge NSC in Ad anced SM A numbe o he apeu ic app oaches di ec ed agains ce ain su ace an igens ( a ge s) exp essed on NSC ha e been de eloped. An ibody-based he apies include an ibody– oxin conjuga es, bi-speci ic o i-speci ic linke -cons uc s, o an ibodies ha kill a ge cells h ough complemen -dependen mechanisms [ 68 , 77 , 81 ]. Whe eas he an ibody– oxin conjuga e GO has been applied in a ew cases, bi-speci ic o i-speci ic linke -cons uc s ha e so a no been applied in clinical p ac ice in pa ien s wi h ad anced SM. Recen ly, howe e , a i-speci ic kille engage CD16xIL15xCD33 ha induces NK cell ac i a ion and cy o oxici y agains neoplas ic MC has been p esen ed [ 81 ]. Whe he such engage cons uc s will be able o elimina e neoplas ic MC and NSC in pa ien s wi h ad anced SM emains a p esen unknown. In addi ion, i emains unknown whe he CAR-T cell o CAR-NK cell he apies will be de eloped a enough o each clinical applica ion in in e en ional ials. Cu en ly, majo e o s a e being unde aken o es ablish CAR-T cell app oaches di ec ed agains CD33, KIT, and a ew o he a ge s, wi h he aim o elimina e NSC in pa ien s wi h ad anced SM, including SM–AHN. One speci ic aspec o conside wi h hese he apies is ha an ibodies and CAR cells (CAR-T o CAR-NK) a acking MC may no only induce a umo lysis synd ome, bu also a MC media o elease synd ome,