A Review of the Pathological and Molecular Diagnosis of Primary Myelofibrosis
by Richard Shao 1,Christopher Ryder 2ORCID,Le Wang 3ORCID,Hailing Zhang 2,Lynn Moscinski 2ORCID,Michael Martin 4ORCID,Mac Shebes 4,Julie Y. Li 2,* andJinming Song 2,*
1
College of Medicine, University of Central Florida, 6850 Lake Nona Blvd, Orlando, FL 32827, USA
2
Department of Pathology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA
3
Reading Hospital McGlinn Cancer Institute, Phoenixville Hospital Campus, West Reading, PA 19460, USA
4
College of Medicine, University of South Florida Morsani, Tampa, FL 33612, USA
*
Authors to whom correspondence should be addressed.
Cancers 2026, 18(1), 50; https://doi.org/10.3390/cancers18010050
Submission received: 31 October 2025 / Revised: 9 December 2025 / Accepted: 23 December 2025 / Published: 24 December 2025
(This article belongs to the Special Issue Molecular and Genetic Diagnosis and Targeted Therapy of Myeloproliferative Neoplasms (2nd Edition))
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簡潔な要約
原発性骨髄線維症（PMF）は、異型巨核球および骨髄系細胞のクローン性増殖、骨髄線維症、急性骨髄性白血病（AML）への白血病化リスク上昇を特徴とする骨髄増殖性腫瘍（MPN）である。

本総説では、治療戦略の個別化と患者予後の改善に不可欠な診断精度とリスク層別化を向上させる目的で、その臨床病理学的特徴、遺伝学的・分子生物学的知見、最新の診断基準、鑑別診断を概説する。

抄録
原発性骨髄線維症（PMF）は、フィラデルフィア染色体（Ph）陰性の骨髄増殖性腫瘍（MPN）であり、異型巨核球および骨髄系細胞のクローン性増殖、骨髄線維症、骨髄外造血、ならびに急性骨髄性白血病（AML）への白血病化リスク上昇を特徴とする。

分子研究、特に次世代シーケンシング（NGS）の普及に伴い、PMFの分子病態および特定の遺伝子変異の予後的意義に関する理解が大きく進展した。本総説では、その臨床病理学的特徴、遺伝学的・分子学的知見、更新された診断基準、鑑別診断を概説する。これらの更新は、世界保健機関（WHO）血液リンパ腫分類第5版（WHO-5th）および2022年国際合意分類（ICC）に組み込まれ、治療戦略の個別化と患者転帰の向上に不可欠な診断精度とリスク層別化を向上させている。
キーワード：原発性骨髄線維症；PMF；JAK2；CALR； MPL; 病因; 分子; 診断

1. 序論
原発性骨髄線維症（PMF）は、巨核球系および顆粒球系の異常増殖を特徴とし、進行性骨髄線維症、骨硬化症、骨髄外造血を伴うクローン性造血幹細胞悪性腫瘍である。

その病因は、特にJAK2、MPL、またはCALRの変異によって駆動される恒常的JAK/STATシグナル伝達 (JAK/STAT)シグナル伝達の恒常的活性化、特にJAK2、MPL、またはCALRの変異によって駆動され、サイトカインの過剰産生と線維性骨髄微小環境の発達をもたらす[1]。

PMFは、本態性血小板血症（ET、約1%）や真性多血症（PV、約4%）などの他の骨髄増殖性腫瘍と比較して、急性骨髄性白血病（AML）への移行リスクが高い（約20%） [2]。

PMFの全生存期間中央値は9.2年で、15年生存率32%、20年生存率20%である[3]。主な死因は感染症、血栓症、心不全または肺不全、AMLへの転化である[4]。

PMFと他の骨髄増殖性腫瘍、特にETやPVとの間には臨床的・形態学的特徴において顕著な重複が認められる。WHO第5版およびICCは、形態学的、臨床的、細胞遺伝学的、分子学的特徴を統合した従来のWHO分類の確立された分類体系を踏襲している[5,6]。

NGSや単一細胞シーケンシングなどの技術により、PMFに対する理解は著しく進展した。本総説は、PMFの臨床病理学的および分子学的特徴を包括的に概説するとともに、鑑別診断と予後分子マーカーに関する詳細を追加する。

2. 原発性骨髄線維症の病因と分子所見

PMFおよびその他のMPNにおける体細胞変異は、「ドライバー変異」（JAK2、MPL、CALRなど）と「非ドライバー変異」（ASXL1、SRSF2、U2AF1、EZH2、IDH1、IDH2など）に分類される。

一般的に、ドライバー変異はMPN表現型の確立に必須である一方、非ドライバー変異は疾患進行および白血病化に寄与する[7]。

JAK2、CALR、MPL変異は通常相互排他的（絶対的ではない）であり、PMF症例のそれぞれ約50-60%、30%、5-10%に認められる[8] 。

JAK2変異はSTAT、MAPK、PI3K、Aktを含む下流シグナル伝達経路の活性化を引き起こし、増殖および抗アポトーシス遺伝子の活性化を経て、骨髄増殖とサイトカイン分泌をもたらす[9]。

PMFにおけるJAK2V617Fアレル負荷の低さは、より進行性の臨床経過および白血病フリー生存率の低下と関連することが示されている[10] 。JAK2 V617Fアレル負荷が低いこれらの患者では、クローン進化を起こす傾向がより高いJAK2 V617F陰性クローンが優勢である可能性が推測されており、これはJAK2 V617F変異がMPNのクローン階層において二次的イベントであることを示す証拠が増加していることで裏付けられている。

MPLはトロンボポエチン受容体（TPOR）をコードし、MPL変異体はトロンボポエチン結合に依存せず恒常的に活性型構造をとる。変異TPORは安定な二量体を形成し、関連するJAK2キナーゼを活性化して下流のJAK-STATシグナル伝達を誘発し、その結果として造血幹細胞の自己更新と巨核球形成が起こる[11]。

MPL W515LおよびW515K変異（MPLW515L/K）は、MPLエクソン10変異の中で最も頻度が高い。

変異型カルレチキュリン（CALR）は異常なC末端配列を獲得し、小胞体内でTPORと結合する。この異常な相互作用がリガンド非依存的なTPOR活性化を促進する[12,13]。

CALR変異はタイプ1（52塩基対欠失）とタイプ2（5塩基対挿入）に分類される[14]。タイプ1変異ではCALR C末端の負電荷アミノ酸が全て消失し、タイプ2変異では約半数が消失する。
タイプ1様CALR変異患者はタイプ2様変異患者と比較してOSが有意に改善する[15,16]。JAK2またはMPL変異と比較して、CALR変異はPMFにおいて血栓症リスクの低下および全生存期間の延長と関連するが、JAK2変異とMPL変異の間には有意差はない[13]。

表1は主要なMPNの形態学的・分子的特徴をまとめたものである。
表1. PML、PV、ETにおける主要な検査所見・形態学的所見およびドライバー遺伝子変異

トリプルネガティブ型PMF

PMF症例の約5％は「トリプルネガティブ型」（TN-PMF）であり、JAK2、CALR、MPL変異のいずれにも既知のドライバー変異が認められない[17]。これら3つの主要ドライバー変異が認められない場合、ASXL1、EZH2、TET2、IDH1、IDH2、SRSF2、SF3B1など骨髄性腫瘍に関連する他の変異のスクリーニングが疾患のクローン性を確立するのに有用である[5]。

TN-PMFは予後不良を暗示する。TN-PMF患者は血小板減少症を呈する傾向が強く、脾腫は頻度が低い。TN-PMFでは8番染色体トリソミーの発生率が高く、従来のPMFと比較してASXL1/SRSF2共変異がより頻繁に認められる傾向がある[17]。

ある研究では、生存期間の有意な短縮、より進行性の臨床経過、白血病化率の上昇、およびルクソリチニブに対する奏効期間の短縮が認められた[18]。

RNAスプライシング、エピジェネティック修飾、シグナル伝達（SRSF2、SETBP1、IDH2、CBL、GNAS）に影響を与える変異がより多く見られ、これが進行性の経過を促進し、JAK阻害剤に対する不十分な反応の原因となっている可能性がある[18]。TN-PMFの診断は困難であり、線維化を伴うMDS、二次性MF（ET後/PV後）、CMML、CML（BCR-ABL1陽性）、本態性血小板血症、ならびに急性白血病や骨髄転移などの類似疾患を除外する必要がある。骨髄生検、細胞遺伝学検査、分子検査、免疫組織化学検査を含む包括的な検査が不可欠である。

追加の「非ドライバー」変異（主にエピジェネティック修飾因子やスプライソソーム構成因子の遺伝子に影響）として、ASXL1（21.7%）、TET2（9.7%）、SRSF2（8.5%）、DNMT3A（5.7%）、EZH2（5.1%）、 CBL（4.4%）、IDH1/2（2.6%）など、主にエピジェネティック修飾因子やスプライソソーム構成因子の遺伝子に影響を与える「非ドライバー」変異が頻繁に同定され、予後モデルに組み込まれている[19,20]。

このうち、ASXL1、EZH2、SRSF2、IDHの変異は、早期死亡や白血病化リスクの増加と関連している[19]。したがって、高分子リスク（HMR）変異は、ASXL1、 SRSF2、EZH2、IDH1、IDH2の変異を指す。

一部の予後モデルではTP53およびU2AF1変異もHMRカテゴリーに含まれる[7]。-1 HMR変異の存在は独立して全生存期間（OS）の悪化と関連し、-2 HMR変異は予後不良と関連する[21]。363例のPMF患者コホートにおいて、Loscoccoらは追加変異（CBL、NRAS、KRAS、RUNX1、TP53）の予後的影響を評価した。単変量解析では、CBL、NRAS、KRAS、TP53変異が全生存期間の悪化と有意に関連し、RUNX1は境界域の有意性を示した。しかし、HMR変異（ASXL1、SRSF2、EZH2、IDH1/2、U2AF1）および細胞遺伝学的リスクを含む多変量モデルでは、これらの追加変異は有意な予後的価値を維持しなかった[22]。

Yanらは、PMF患者258例の変異プロファイルを解析し、非ドライバー変異が疾患進行に果たす役割を明らかにした。

ASXL1変異は疾患進行と予後悪化と強く関連していることが判明した [23]。RAS経路の変異もPMF表現型に関連している一方、PVおよびETでは通常、変異アレル頻度（VAF）が低い（<10%）サブクローナルなKRAS/NRAS変異が認められる。これらの知見は、追加の非ドライバー変異が骨髄増殖性腫瘍（MPNs）の表現型に影響を与え、骨髄線維症の発症に寄与する可能性を示唆している [24,25]。


HMR変異の有無は移植判断において常に考慮される。最近の研究では、造血幹細胞移植（HSCT）を受けた原発性および続発性骨髄線維症患者50例を評価し、HMR変異数が移植後の転帰を強く予測することを明らかにした。2つ以上のHMR変異を有する患者では、生存率が有意に低く、非再発死亡率が高かった。個別遺伝子では、DNMT3AおよびEZH2変異が予後不良と関連し、TP53変異は再発リスク上昇を予測した[26]。


3. 原発性骨髄線維症の臨床的特徴
原発性骨髄線維症（PMF）患者の約4分の1から3分の1は無症状であり、こうした症例は原因不明の貧血、脾腫、肝腫大の評価中に発見されることが多い。
原発性骨髄線維症の典型的な初期症状には、脾腫、涙滴状赤血球、血液塗抹標本における白血球・赤芽球変化（有核赤血球および未熟顆粒球の出現）、骨硬化症および膠原線維症を伴う過形成性骨髄が含まれる。本疾患は幅広い血液学的および全身症状を呈する。貧血は頻度が高く、骨髄機能低下による進行性血球減少は感染や出血のリスクを高める。著明な脾腫は腹部不快感、早期満腹感、脾破裂、門脈圧亢進症、および脾臓による血球貯留に伴うさらなる血球減少を引き起こす可能性がある。骨硬化症は骨痛の一因となり、進行期には重度の全身症状と悪液質が顕著となる。一部の患者では特発性血小板血症（ET）に類似した血小板増加症を示すことがある。患者の約10〜20％が、診断時または病程中に血栓塞栓症を発症する。骨髄外造血はあらゆる臓器で発生しうるが、脾臓、肝臓、脊柱、リンパ節に頻発する。

4. 原発性骨髄線維症の組織病理学
PMFの典型的な組織学的特徴は、異型巨核球の増殖と骨髄増殖である（図1）。

PMFの巨核球は、顕著なクラスター形成、裸核、過染色性の球状（「雲状」）核など、異型形態を示す。これはPMFに特徴的な所見であり、ETやPVでは稀にしか観察されず、骨髄異形成症候群の巨核球と区別する必要がある。

骨髄線維症は、レチクリン染色およびトリクローム染色によりMF-0（線維症なし）からMF-3（骨硬化症を伴う緻密なコラーゲン線維症）まで段階分けされる[27]。線維症はレチクリン（コラーゲンIII）の沈着（MF-1）から始まり、続いてコラーゲンIの蓄積（MF-2/3）が進行する。

骨硬化症は通常、PMFの後期段階で発生し、著明な線維化を伴う骨リモデリングおよび骨形成を特徴とする。著明な骨髄線維化と骨硬化症は、PMFの線維化段階においてしばしば洞の拡張および洞内造血の存在をもたらす。

Cancers 18 00050 g001

図1. 原発性骨髄線維症、線維化期の骨髄生検所見。

骨髄には線維化を伴う巨核球過形成が認められる。(A) 巨核球は細胞サイズに多様性を示し、多くの大型で多核分節化および濃染性を呈する（H&E染色、100倍）。(B) 顕著な網状線維線維化（グレード3/3）。

骨髄線維化の程度に基づき、PMFは非線維化期PMF（prePMF）または線維化期PMFにさらに分類される。prePMFは、非定型巨核球性・骨髄性増殖と、最小限または欠如した線維化（-MF-1網状線維線維化）を特徴とする。前線維化期PMFは線維化期PMFと比較して予後が良好であり、通常、高度な脾腫や白血球・赤芽球増加症を伴わない。

線維化期PMFは、高度な線維化（>1 MF-1網状線維線維症）、時に増加または減少した造血細胞密度、および脾腫、貧血、全身症状などの臨床所見によって定義される。線維化期のPMFは予後が最も悪く、BCR::ABL1陰性MPNの中でAMLへの移行率が高い[28,29]。
骨髄外造血（EMH）は、特に肝臓や脾臓で顕著であり、骨髄線維化による造血幹細胞の置換が原因である。

PMFを特徴付ける骨髄の進行性線維化は、クローン性造血幹細胞（HSC）と間質微小環境との相互作用の結果である。PMFにおける線維化は、トランスフォーミング成長因子β（TGF-β）、血小板由来成長因子（PDGF）、血管内皮成長因子（VEGF）などの線維化促進性サイトカインの分泌によって促進される[30]。最近の研究では、Gli1+およびLepr+間葉系幹細胞が細胞外マトリックス（ECM）のリモデリングと線維化に大きく寄与することが示されている[31,32]。骨形成に関与する複数のシグナル伝達経路（骨形成タンパク質（BMP）やカノニカルWntシグナル伝達経路など）がTGF-βと協調して骨硬化を促進する可能性がある[33]。骨髄ではVEGF誘導による血管新生が頻繁に亢進する[34]。微小血管密度はPMFにおけるJAK2 V617Fアレル負荷（--55-%変異アレル）と相関する [35]。


5. 原発性骨髄線維症の細胞遺伝学的所見
PMF症例の最大45%で核型異常が認められる。

最も頻度の高い反復性核型異常には、del(20q)、del(13q)、8番染色体トリソミー、9番染色体トリソミーが含まれる[36]。

正常核型、20q欠失、13q欠失、9番染色体三倍体、Y染色体欠失などの良好な核型を有する患者では、全生存期間（OS）の中央値は4.4年と予測される。

+8、-5/5q欠失、7q欠失、複雑核型などの不良核型では、OS中央値は2.9年である。

-7、inv(3)/3q21、i(17q)、12p-、11q-、および+8または+9以外のトリソミーなどの非常に高リスク（VHR）核型は、中央値OS 1.2年と関連している[36]。注目すべきは、これらのOS数値が、前述の研究[3]で報告された一般的なPMF患者の全生存期間中央値9年よりも低いことであり、これは正常核型のみの患者をより多く含んでいる可能性がある。

6. 原発性骨髄線維症の診断基準
WHO第5版および2022年ICCはPMFの明確な診断基準を提供する。前駆PMFまたは顕性PMFの診断には、3つの主要基準すべてと、2回の連続した評価における少なくとも1つの副次基準が必要である。

第1主要基準は前線維化型と顕性線維化型で異なる。

前PMFでは、骨髄生検における巨核球増殖・異型、年齢補正細胞量増加、顆粒球増殖（しばしば赤芽球減少を伴う）、骨髄線維化＜グレード2を含む。線維性PMFでは、グレード2または3のレチクリン線維化および/またはコラーゲン線維化によって定義される。
他の2つの主要基準は両形態で共通である： (2) JAK2、CALR、MPL変異などのクローン性マーカーまたは他のクローン性異常の存在、(3) BCR::ABL1陽性慢性骨髄性白血病、真性多血症（PV）、過多血症（ET）、骨髄異形成症候群（MDS）、その他の骨髄性疾患を含む他の骨髄性腫瘍の除外。

副次基準には以下が含まれる：
(1) 併存疾患で説明できない貧血、
(2) 白血球増加（11×10?/L以上）、
(3) 触知可能な脾腫、
(4) 乳酸脱水素酵素の上昇。線維性PMFでは、白血球・赤芽球増加症が追加の副次基準となる。

線維化前PMFは線維化型PMFより予後が良好であり、ETやPV、その他のMPNと誤診される可能性がある。ETよりも高い血栓リスクを伴う[37]。正確な診断には、臨床症状、検査結果、分子・遺伝学的所見、形態学的特徴の統合が必要である。治療は症状緩和、血栓塞栓症の予防、疾患進行の抑制に重点を置く。ルクソリチニブなどのJAK2阻害剤は一定の効果を示しているが、プレPMFにおけるその役割は現在も検討中である。

PMFは加速期（AP）へ進行することがあり、末梢血または骨髄中の芽球数が10-19%に達し、免疫組織化学で検出可能なCD34陽性芽球のクラスターを形成することが多い。芽球期（BP）への移行、すなわち白血病化は、急性骨髄性白血病と一致する20％以上の芽球数によって定義される。表2はWHO第5版およびICCによるPMF診断基準を概説する。

表2. PMFのWHO第5版およびICC診断基準

7. 他の骨髄性腫瘍との比較と鑑別
7.1. 本態性血小板血症（ET）
PrePMFは、血小板増加症と巨核球増殖を呈するため、臨床的・形態学的にETを模倣することがある。予後に著しい差があるため、PrePMFとETを鑑別することが重要である[38]。PrePMF患者は臨床経過が著しく不良で、全生存率が低く、AMLおよび疾患の線維化段階への進行リスクが高い。ET患者とは対照的に、前PMF患者では白血球増加、LDH値の上昇、循環CD34陽性芽球数の増加、脾腫の頻度上昇がより多く認められる。形態学的特徴を注意深く評価することで、通常は前PMFとETを区別できる。ETにおける巨核球は比較的均一な大きさで、大型の過多葉状「鹿角様」巨核球を呈するが、顕著な過染色性は認められない。疎なクラスターを形成することもあるが、通常は骨髄内により均一に分布する。これに対し、前PMFの巨核球はより顕著なクラスター形成、異常な骨梁傍位置、および小型から大型の過多葉状形態まで多様な細胞サイズを示し、過染色性核が頻繁に認められる。未分化巨核球（典型的な「球根状」核を含む）は前骨髄異形成症候群（prePMF）に極めて特異的である。WHO分類で定義される微小基準である末梢血白血球増加症およびLDH値上昇と併せて、これらの巨核球形態学的特徴は前骨髄異形成症候群（prePMF）の診断確立とETの除外に有用である。
遺伝学的所見はプレPMFとETの鑑別に有用である。JAK2V617Fアレル負荷は一部の症例においてETとプレPMFを区別するのに役立つ[39]。プレPMF患者の約4分の1はJAK2V617Fアレル負荷が50%を超えるのに対し、ET患者では40%未満と低い値を示す。全体として、prePMFはETよりもCALR変異の頻度が高い（35.8% vs. 17.8%）[38]。タイプ1様CALR変異はPMFで有意に多く、タイプ2様変異はETでより一般的である [14,40]。MPL W515L/K変異は、ET（1%）よりもprePMF（5%）でより頻繁に同定される[41]。

7.2. 真性多血症後骨髄線維症および本態性血小板血症後骨髄線維症
PVおよびETはいずれも、疾患の線維化段階として、通常は初診から数年〜数十年後に進行する真性多血症後骨髄線維症（post-PV myelofibrosis （PPV-MF）および後発性ET骨髄線維症（PET-MF）として進行する可能性がある。PPV-MFとPET-MFはいずれも、臨床的・形態学的にPMFの線維化段階から確実に鑑別することはできない。PPV-MFおよびPET-MF患者では通常、脾腫の増大、貧血、末梢血中の白血球・赤芽球増加が認められる。骨髄所見では、異常な大型巨核球が顕著な集簇、過多小葉化、頻発する過染色核を伴い、グレード2-3の網状線維増生が認められる。鑑別診断は臨床的に重要であり、PET-MF患者（中央値73ヶ月）はPMF（45ヶ月）およびPPV-MF（48ヶ月）と比較して全生存期間が長い[42]。PPV-MFおよびPET-MFの診断は、既往のPVまたはET診断歴の記録によってのみ確定できる。JAK2エクソン12変異の同定はPPV-MFの診断を確定可能であり、JAK2エクソン12変異はPVにのみ認められる。表3にPMF、PET-MF、PPV-MFの主要特徴をまとめた。
表3. 原発性骨髄線維症（PMF）、真性多血症後骨髄線維症（PPV-MF）、本態性血小板血症後骨髄線維症（PET-MF）の鑑別診断

7.3. 慢性骨髄性白血病（CML）
CMLは、原発性骨髄線維症（特に骨髄線維症の初期（線維化前）段階） （PMF）−特に血小板増加を伴う場合−またはPMFの線維化期を模倣することがある。これは骨髄線維症や脾腫などの特徴が重複するためである。特に、骨髄線維症はCML症例の約40％で発生し、予後不良と関連するが、チロシンキナーゼ阻害剤（TKI）により可逆的となる可能性がある[43]。これらの類似点にもかかわらず、PMFは形態学的所見および遺伝子プロファイル（BCR-ABL欠如）によりCMLと容易に鑑別できる。CMLでは通常、好塩基球増加が認められ、骨髄には特徴的な低分葉性の「矮小」巨核球が観察される。これに対し、PMFでは大型の多分葉性・高染色性の巨核球および「球状」核を有する異常巨核球が認められる。PCRまたはFISHによるBCR::ABL1融合遺伝子の同定は、CML診断の決定的所見である。
7.4. 骨髄異形成性腫瘍および骨髄異形成性/骨髄増殖性腫瘍
線維化を伴う骨髄異形成性腫瘍（MDS-F）は、貧血と線維化を呈する点でPMFを模倣する[44,45]。MDS-Fでは芽球が増加し、臨床的には血球減少を呈するが、PMFで通常見られる白血球増加や血小板増加は認められない。MDS-Fの巨核球はPMFとは異なり、小粒状の巨核球または微小巨核球が優勢である（図2）。MDS-Fの変異プロファイルはPMFとは異なる。MDS-Fでは通常、ASXL1、SF3B1、TET2、RUNX1などの変異が認められるが、JAK2、CALR、MPL変異は認められない。
Cancers 18 00050 g002
図2. PMF（A-C）とMDS（D-F）における典型的な巨核球形態の比較 PMFの巨核球は通常数が増加し、密なクラスターを形成する（A）。また、濃染性（B）、球状または雲状形態（C）を示す。一方、MDSの巨核球は通常密なクラスターを形成せず（D）、主に小型単葉性の異形成形態（E）または核分離（F）を示す。
慢性骨髄単球性白血病（CMML）も線維化を伴うPMFを模倣し得る[46]。骨髄線維化は、無増悪生存期間の短縮、脾腫、巨核球増加と関連する。一方、PMF患者では病勢進行に伴い単球増加が生じることがある。この鑑別診断は通常、巨核球の形態と変異プロファイルを検討することで解決可能である。CMMLでは典型的に、小形で小葉形成が乏しい形態のMDS型巨核球および微小巨核球が認められる。CMMLではASXL1、TET2、SRSF2、RAS経路遺伝子に特徴的な変異が認められ、MPNドライバー変異は存在しない。特筆すべきは、CMMLとPMFの重なり合う特徴を示す症例が存在し、JAK2またはMPLとASXL1、SRSF2、TET2、NRAS、および/またはKRASの共変異が認められることである[47]。これらの骨髄性腫瘍は、CMMLとPMFの真のグレーゾーンを示す可能性がある。
環状鉄芽球と血小板増加を伴う骨髄異形成/骨髄増殖性腫瘍（MDS/MPN-RS-T）も、PMFと臨床的（白血球増加・血小板増加）および病理学的（JAK2変異、多葉性巨核球、線維化）特徴を共有し得る。しかし、PMF患者では脾腫、CALRまたはMPL変異、過染色核を有する巨核球がより多く認められる一方、MDS/MPN-RS-T患者ではSF3B1変異、著明に増加した環状シデロブラスト、多系統性異形成が認められる傾向がある。
7.5. 全身性肥満細胞症（SM）
SMでは、骨梁周囲および骨梁傍の肥満細胞集塊に伴う線維化が頻繁に認められる[48]。分子生物学的手法によるCD2、CD25および/またはCD30の異常発現、ならびにKIT D816V変異を有する腫瘍性肥満細胞の集塊または結節の同定により、診断は容易である。SMでは巨核球形態の評価が重要である。巨大・多房性および／または濃色性の巨核球が認められた場合、NGSまたはPCRによるJAK2、CALR、MPL変異の有無を速やかに評価すべきである。なぜなら、PMFがSMと関連血液腫瘍（SM-AHN）として併存する可能性があるからである[49]。
7.6. 転移性癌
骨は様々な転移性癌の好発部位である。前立腺癌や乳癌などの転移性癌は骨髄に線維化を頻発させる[50]。このような症例では線維化が広範に及ぶことがあり、転移性癌の存在を覆い隠し、PMFの線維化段階を模倣することがある。このような症例では、染色パネルにサイトケラチンの免疫組織化学染色を含めるべきであり、これにより転移性癌の診断が確定される。
(臨床情報も大事)

7.7. 自己免疫性骨髄線維症（AIMF）
AIMFは、線維症、白血球・赤芽球増加症、脾腫、巨核球異型性を伴う症例群においてPMFを模倣することがある [51,52]。

AIMFは全身性自己免疫疾患の有無にかかわらず発生する。

患者は通常、血球減少症と骨髄線維症を示し、骨髄はしばしば過形成を呈し、良性リンパ球性集合体を含み、時に巨大で異常な小葉構造または過染色性を示す巨核球を含む。

PMFとは異なり、AIMFは通常、抗核抗体、抗二本鎖DNA抗体、抗リン脂質抗体/ループス抗凝固因子、リウマチ因子などの自己免疫抗体と関連し、JAK2、CALR、MPLのドライバー変異は認められない。

51. Gangat, N.; Reichard, K.; Orazi, A.; Tefferi, A. Autoimmune myelofibrosis: A Mayo Clinic series of 22 patients. Br. J. Haematol. 2024, 205, 956-960.
52. Piatek, C.I.; Vergara-Lluri, M.E.; Pullarkat, V.; Siddiqi, I.N.; O’Connell, C.; Brynes, R.K.; Feinstein, D.I. Autoimmune Myelofibrosis: Clinical Features, Course, and Outcome. Acta Haematol. 2017, 138, 129-137. 


7.8. 炎症性および感染性原因
HIVや結核などの感染症、あるいは肉芽腫性炎症は骨髄線維症を誘発する可能性がある[53]。これらの病態ではPMFに特徴的な異型巨核球は認められない。診断には血清学検査、PCR、組織の特殊染色（例：結核の抗酸染色）などの検査が必要である。

8. 結論
PMF研究分野は、WHO/ICC統合分類基準の導入により大きく進展した。これにより予後モデルの適用や、最適な治療のためのPMF患者の層別化が容易となった。NGSによる形態学的・分子マーカーは、PMFを他のMPN、MDS、反応性病変から鑑別するのに有用であり、分子マーカー（例：ASXL1、SRSF2、TP53）は個別化治療の指針となり得る。
中間-2および高リスクMFに承認されたJAK阻害剤は、TN-PMFを含む全サブグループで有効である。COMFORT-I試験ではJAK2状態に関わらず59%が脾臓体積35%以上減少を達成。COMFORT-II試験では変異状態に関わらず48週時点で28%減少と症状・QOL改善が報告された。これらの結果は、MF病態におけるJAK-STAT異常調節の中心的役割を強調している。ただし、JAK阻害剤は疾患進行や生存率を著しく変化させない。
今後の研究では、TN-PMFを含む全てのMFサブタイプにおける転帰改善を目指し、疾患修飾療法に焦点を当てるべきである[54]。

Simple Summary
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that features clonal proliferation of atypical megakaryocytes and myeloid cells, fibrosis of the bone marrow, and increased risk of leukemic transformation to acute myeloid leukemia (AML). In this review, we summarize its clinicopathologic features, genetic and molecular findings, updated diagnostic criteria, and differential diagnosis, in an aim to improve diagnostic accuracy and risk stratification, which are essential for tailoring treatment strategies and enhancing patient outcomes.

Abstract
Primary myelofibrosis (PMF) is a Philadelphia chromosome (Ph)-negative myeloproliferative neoplasm (MPN) that features clonal proliferation of atypical megakaryocytes and myeloid cells, fibrosis of the bone marrow, extramedullary hematopoiesis, and increased risk of leukemic transformation to acute myeloid leukemia (AML). With the widespread application of molecular studies, especially next generation sequencing (NGS), significant advances have reshaped our understanding of the molecular pathogenesis of PMF and the prognostic relevance of specific gene mutations. In this review, we summarize its clinicopathologic features, genetic and molecular findings, updated diagnostic criteria, and differential diagnosis. These updates have been incorporated into the 5th edition of the World Health Organization classification of Hematolymphoid Tumors (WHO-5th) and the 2022 International Consensus Classification (ICC), thereby improving diagnostic accuracy and risk stratification, both of which are essential for tailoring treatment strategies and enhancing patient outcomes.
Keywords: primary myelofibrosis; PMF; JAK2; CALR; MPL; pathogenesis; molecular; diagnosis
1. Introduction
Primary myelofibrosis (PMF) is a clonal hematopoietic stem cell malignancy characterized by abnormal proliferation of megakaryocytic and granulocytic lineages accompanied by progressive bone marrow fibrosis, osteosclerosis and extramedullary hematopoiesis. Its pathogenesis results from constitutive Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling, especially driven by mutations in JAK2, MPL, or CALR, leading to cytokine overproduction and the development of a fibrotic bone marrow microenvironment [1].
PMF carries a high risk of transformation to AML (~20%), compared to other myeloproliferative neoplasms such as essential thrombocythemia (ET, ~1%) and polycythemia vera (PV, ~4%) [2]. The overall median survival time in PMF is 9.2 years, with 15- and 20-year survival rates of 32% and 20% [3]. The principal causes of death include infection, thrombosis, cardiac or pulmonary failure, and transformation to AML [4].
There are significant overlaps in the clinical and morphologic features between PMF and other myeloproliferative neoplasms, particularly ET and PV. WHO-5th and ICC followed the proven classification scheme of the previous WHO classification that integrated morphological, clinical, cytogenetic and molecular features for diagnosis [5,6]. Our understanding of PMF has evolved remarkably through technologies like NGS and single-cell sequencing. This review provides a comprehensive overview of the clinicopathologic and molecular features of PMF, with additional detail on differential diagnosis and prognostic molecular markers.
2. Pathogenesis and Molecular Findings of Primary Myelofibrosis
Somatic mutations in PMF and other MPNs are categorized as “driver” mutations, such as JAK2, MPL, and CALR, and “non-driver” mutations, including ASXL1, SRSF2, U2AF1, EZH2, IDH1, IDH2, and others. In general, driver mutations are essential for establishing the MPN phenotype, whereas non-driver mutations contribute to disease progression and leukemic transformation [7]. JAK2, CALR, and MPL mutations are usually mutually but not absolutely exclusive, occurring in ~50-60%, 30%, and 5-10% of PMF cases, respectively [8]. JAK2 mutation leads to downstream activation of signal transduction pathways including STAT, MAPK, PI3K, and Akt with subsequent activation of proliferation and anti-apoptotic genes, which lead to myeloproliferation and cytokine secretion [9]. It has been shown that low JAK2V617F allele burden in PMF is associated with a more aggressive clinical course and inferior overall with leukemia-free survival [10]. It was speculated that these patients with low JAK2 V617F allele burden might have a more dominant JAK2 V617F negative clone with higher propensity to undergo clonal evolution, which is supported by increasing evidence that points to JAK2 V617F mutation as a secondary event in the clonal hierarchy of MPNs.
MPL encodes the thrombopoietin receptor (TPOR), and MPL mutants cause the receptor to adopt a constitutively active conformation independent of thrombopoietin binding. Mutant TPOR forms a stable dimer that activates the associated JAK2 kinase, triggering downstream JAK-STAT signaling, with resultant hematopoietic stem cell self-renewal and megakaryopoiesis [11]. MPL W515L, and W515K mutations (MPLW515L/K) are the most common MPL exon 10 mutations.
Mutant calreticulin (CALR) acquires an abnormal C-terminal sequence that allows it to bind TPOR in the endoplasmic reticulum. This aberrant interaction promotes ligand-independent activation of TPOR [12,13]. CALR mutations are divided into type 1 (52-bp deletion) and type 2 (5-bp insertion) mutations [14]. The type 1 mutations eliminate all negatively charged amino acids in the CALR C terminus, and the type 2 mutations eliminate about half of the negatively charged amino acids. Patients with type 1-like CALR mutations have significantly improved OS compared with patients with type 2-like mutations patients [15,16]. Compared with JAK2 or MPL mutations, CALR mutation is associated with a decreased risk of thrombosis and longer overall survival in PMF, while there is no significant difference between JAK2 or MPL mutations [13]. Table 1 summarizes the major morphologic and molecular features of major MPNs.
Table 1. Major laboratory and morphologic findings and driver gene mutations in PML, PV and ET.

Approximately 5% of cases of PMF are “triple negative” (TN-PMF) without known driver mutations in JAK2, CALR, or MPL mutations [17]. In the absence of these three major driver mutations, screening for other mutations associated with myeloid neoplasms, such as ASXL1, EZH2, TET2, IDH1, IDH2, SRSF2, and SF3B1, can help establish the clonal nature of the disease [5]. TN-PMF portends a poor prognosis. Patients with TN-PMF more likely have thrombocytopenia and less frequent splenomegaly. TN-PMF tends to show increased incidence of trisomy 8 and more frequent ASXL1/SRSF2 co-mutations than conventional PMF [17]. One study observed significantly decreased survival and more aggressive clinical behavior with higher rates of leukemic transformation and shorter duration of response to ruxolitinib [18]. Mutations affecting RNA splicing, epigenetic modification, and signaling (SRSF2, SETBP1, IDH2, CBL, and GNAS) are more common, which likely drive its aggressive course and may account for suboptimal responses to JAK inhibition [18]. TN-PMF diagnosis is challenging and requires exclusion of mimicking conditions such as MDS with fibrosis, secondary MF (post-ET/post-PV), CMML, CML (BCR-ABL1+), and essential thrombocythemia, as well as acute leukemias and marrow metastases. A comprehensive workup-including bone marrow biopsy, cytogenetics, molecular testing, and immunohistochemistry-is essential.
Additional “non-driver” mutations, mostly affecting genes of epigenetic modifiers or spliceosome components, such as ASXL1 (21.7%), TET2 (9.7%), SRSF2 (8.5%), DNMT3A (5.7%), EZH2 (5.1%), CBL (4.4%), and IDH1/2 (2.6%) are frequently identified in PMF by NGS studies and have been incorporated into prognostic models [19,20]. Of these, mutations in ASXL1, EZH2, SRSF2, and IDH are associated with increased risk of premature death or leukemic transformation [19]. Accordingly, high-molecular risk (HMR) mutations are defined as mutations in ASXL1, SRSF2, EZH2, IDH1, and IDH2. TP53 and U2AF1 mutations have been included in the HMR category in some prognostic models [7]. The presence of -1 HMR mutations is independently associated with inferior overall survival (OS) while leukemia-free survival (LFS), and -2 HMR mutations are associated with a dismal outcome [21]. In a cohort of 363 PMF patients, Loscocco et al. assessed the prognostic impact of additional mutations (CBL, NRAS, KRAS, RUNX1, TP53). Univariate analysis showed that CBL, NRAS, KRAS, and TP53 mutations were significantly associated with inferior overall survival, while RUNX1 had borderline significance. However, in multivariate models that included HMR mutations (ASXL1, SRSF2, EZH2, IDH1/2, U2AF1) and cytogenetic risk, these additional mutations did not retain significant prognostic value [22]. Yan et al. analyzed the mutational landscape in 258 patients with PMF to identify the role of non-driver mutations in disease progression. ASXL1 mutations were found to be strongly associated with disease advancement and worse prognosis [23]. Mutations in the RAS pathway have also been linked to the PMF phenotype, whereas PV and ET typically harbor subclonal KRAS/NRAS mutations with low variant allele frequencies (VAFs, <10%). These findings suggest that additional non-driver mutations may affect the phenotype of myeloproliferative neoplasms (MPNs) and contribute to the development of myelofibrosis [24,25].
The presence of HMR mutations is regularly considered for transplant decision making. A recent study evaluated 50 patients with primary and secondary myelofibrosis undergoing hematopoietic stem cell transplant (HSCT) and found the number of HMR mutations is a strong predictor of post-transplant outcomes. Patients with two and more HMR mutations had significantly worse survival and higher non-relapse mortality. Among individual genes, DNMT3A and EZH2 mutations were associated with poor outcomes, and TP53 mutations predicted higher relapse risk [26].
3. Clinical Features of Primary Myelofibrosis
About one fourth to one third of patients with PMF are asymptomatic, and such cases are often discovered during the evaluation of unexplained anemia, splenomegaly, or hepatomegaly.
The classic presenting features of primary myelofibrosis include splenomegaly, teardrop-shaped red blood cells, and leukoerythroblastic changes (appearance of nucleated red blood cells and immature granulocytes) on the blood smear and a hypercellular bone marrow with osteosclerosis and collagen fibrosis. The disease encompasses a wide range of hematologic and systemic manifestations. Anemia is common, and progressive cytopenias, due to declining marrow function, increase the risk of infection and bleeding. Massive splenomegaly may cause abdominal discomfort, early satiety, splenic rupture, portal hypertension, and further cytopenias due to sequestration. Osteosclerosis contributes to bone pain, while advanced stages are marked by severe constitutional symptoms and cachexia. A subset of patients may show thrombocytosis resembling ET. Around 10-20% of patients develop thromboembolic events at diagnosis or over the course of the disease. Extramedullary hematopoiesis can develop in any organ, but more frequently occurs in spleen, liver, vertebral column, and lymph nodes.
4. Histopathology of Primary Myelofibrosis
The typical histologic feature of PMF is the proliferation of atypical megakaryocytes and myeloid proliferation (Figure 1). Megakaryocytes in PMF display atypical morphology, including prominent clustering, naked nuclei, and hyperchromatic, bulbous (“cloud-like”) nuclei-a feature characteristic of PMF and rarely observed in ET or PV, and need to be distinguished from those in myelodysplastic syndrome. Bone marrow fibrosis is graded between MF-0 (absent) and MF-3 (osteosclerosis with dense collagen fibrosis) on reticulin and trichrome stain [27]. The fibrosis starts with the deposition of reticulin (collagen III) (MF-1), followed by accumulation of collagen I (MF-2/3). Osteosclerosis typically occurs in late-stage PMF and is characterized by bone remodeling and bone formation along with marked fibrosis. Marked bone marrow fibrosis and osteosclerosis often result in dilated sinuses and the presence of intrasinusoidal hematopoiesis in the fibrotic stage of PMF.
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Figure 1. Bone marrow biopsy of primary myelofibrosis, fibrotic stage. The marrow shows megakaryocytic hyperplasia with associated fibrosis. (A) The megakaryocytes range in cell size with many large hyperlobulated and hyperchromatic forms (H&E stain, 100×). (B) Marked reticulin fibrosis (grade 3 of 3).
Based on the level of bone marrow fibrosis, PMF is further subclassified as prefibrotic PMF (prePMF) or fibrotic stage of PMF. The prePMF is characterized by atypical megakaryocytic and myeloid proliferation and minimal or absent fibrosis (-MF-1 reticulin fibrosis). The prePMF has a favorable prognosis when compared with the fibrotic stage of PMF and usually lacks advanced splenomegaly or leukoerythroblastosis. The fibrotic stage of PMF is defined by the presence of advanced fibrosis (>1 MF-1 reticulin fibrosis), sometimes increased and sometimes reduced hematopoietic cellularity, and clinical findings like splenomegaly, anemia, and constitutional symptoms. The fibrotic stage PMF has the worst prognosis with a higher rate of AML transformation among BCR::ABL1 negative MPNs [28,29]. Extramedullary hematopoiesis (EMH), most notably in the liver and spleen, is a consequence of marrow fibrosis displacement of hematopoietic stem cells.
The progressive fibrosis of the bone marrow that characterizes PMF is a consequence of interactions between clonal hematopoietic stem cells (HSCs) and the stromal microenvironment. Fibrosis in PMF is driven by the secretion of profibrotic cytokines, including transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) [30]. Recent studies showed that Gli1+ and Lepr+ mesenchymal stem cells are significant contributors to remodeling of the extracellular matrix (ECM) and fibrosis [31,32]. A number of signaling pathways involved in bone development, such as bone morphogenetic protein (BMP) and canonical Wnt signaling pathway, may cooperate with TGF-β to promote osteosclerosis [33]. The bone marrow frequently shows increased angiogenesis, which is induced by VEGF [34]. The microvessel density correlates with a high JAK2 V617F allele burden (--55-% mutant alleles) in PMF [35].
5. Cytogenetic Findings of Primary Myelofibrosis
Karyotypic abnormalities can be seen in up to 45% of PMF cases. The most common recurrent karyotypic abnormalities include del(20q), del(13q), trisomy 8, and trisomy 9 [36]. Patients with favorable karyotypes, such as normal karyotype, 20q-, 13q-, trisomy 9, and Y-, expect a median OS of 4.4 years. Unfavorable karyotypes, including +8, -5/5q-, 7q-, complex karyotypes, have a median OS of 2.9 years. Very high-risk (VHR) karyotypes, such as -7, inv(3)/3q21, i(17q), 12p-, 11q-, and trisomy’s other than +8 or +9, are associated with a median OS of 1.2 years [36]. It is of note that these OS numbers are lower than the median overall survival of 9 years in the general PMF patients as reported in the previously described study [3], which might include more patients with only normal karyotype.
6. Diagnostic Criteria of Primary Myelofibrosis
WHO-5th and the 2022 ICC provide defined diagnostic criteria of PMF. Diagnosis of prePMF or overt PMF requires all three major criteria and at least one minor criterion on two consecutive assessments.
The first major criterion differs between the prefibrotic and overtly fibrotic forms. In prePMF, it includes megakaryocytic proliferation and atypia on bone marrow biopsy, increased age-adjusted cellularity, granulocytic proliferation, often with decreased erythropoiesis, and bone marrow fibrosis < grade 2. In fibrotic PMF, it is defined by reticulin and/or collagen fibrosis of grade 2 or 3. The other two major criteria are the same for both forms: (2) presence of a clonal marker such as JAK2, CALR, or MPL mutation, or another clonal abnormality, and (3) exclusion of other myeloid neoplasms, including BCR::ABL1-positive chronic myeloid leukemia, PV, ET, myelodysplastic syndromes, or other myeloid disorders.
The minor criteria include the following: (1) anemia not explained by comorbid conditions, (2) leukocytosis -11 × 109/L, (3) palpable splenomegaly, and (4) elevated lactate dehydrogenase. Fibrotic PMF also includes leukoerythroblastosis as an additional minor criterion.
Prefibrotic PMF has a more favorable prognosis than fibrotic PMF and can be mis-diagnosed as ET or PV or other MPNs. It is associated with higher thrombotic risk than ET [37]. Its accurate diagnosis requires the integration of clinical presentation, lab results, molecular and genetic findings, and morphological features. Its management focuses on reducing symptoms and thromboembolic events and preventing disease progression. JAK2 inhibitors like ruxolitinib have shown some effects but their role in prePMF is still under investigation.
PMF can progress to an accelerated phase (AP), characterized by a blast count of 10-19% in the peripheral blood or bone marrow, often forming clusters of CD34-positive blasts detectable by immunohistochemistry. Transformation to the blast phase (BP), or leukemic transformation, is defined by a blast count of -20%, consistent with acute myeloid leukemia. Table 2 outlines the PMF diagnostic criteria as defined by the WHO-5th and the ICC.
Table 2. WHO-5th and ICC diagnostic criteria for PMF.

7. Comparison and Differentiation with Other Myeloid Neoplasms
7.1. Essential Thrombocythemia (ET)
PrePMF can mimic ET clinically and morphologically with presentation of thrombocytosis and megakaryocyte proliferation. It is important to differentiate prePMF from ET as they have significant difference in prognosis [38]. Patients with prePMF have a significantly worse clinical course, with lower overall survival and increased risk of progression to AML and the fibrotic stage of the disease. In contrast to patients with ET, patients with prePMF are more likely to have leukocytosis, higher LDH value, higher number of circulating CD34-positive blasts, and more frequent splenomegaly. Careful evaluation of morphologic features usually can distinguish prePMF from ET. The megakaryocytes in ET are relatively uniform in size with large hyperlobulated “staghorn-like” megakaryocytes but without significant hyperchromatic. They may form loose clusters but usually are more evenly distributed in the marrow. In contrast, the megakaryocytes in the prePMF show more prominent clustering, abnormal paratrabecular location, and variable cell sizes, ranging from small to large, hyperlobulated forms with frequent hyperchromatic nuclei. Megakaryocytes with dysmaturation, including the typical “bulbous” nuclei, are highly specific for prePMF. These morphologic features of megakaryocytes along with peripheral blood leukocytosis and increased LDH level as defined in the WHO classification as minor criteria can help establish the diagnosis of prePMF and rule out ET.
Genetic findings can help differentiate prePMF from ET. JAK2V617F allele burden can help discriminate ET from prePMF in a subset of cases [39], as approximately a quarter of prePMF patients have JAK2V617F allele burden of more than 50%, while ET patients show lower JAK2V617F allele burden of less than 40%. Overall, prePMF has more frequent CALR mutations than ET (35・8% vs. 17・8%) [38]. Type 1-like CALR mutations are significantly more frequent in PMF, while type 2-like mutations are more common in ET [14,40]. MPL W515L/K mutations are more frequently identified in prePMF than ET (5% vs. 1%) [41].
7.2. Post-Polycythemia Vera Myelofibrosis and Post-Essential Thrombocythemia Myelofibrosis
Both PV and ET can develop into fibrotic stage of the disease as post-PV myelofibrosis (PPV-MF) and post-ET myelofibrosis (PET-MF) with disease progression, usually years or decades after initial diagnosis. Both PPV-MF and PET-MF cannot be reliably differentiated from the fibrotic stage of PMF both clinically and morphologically. Patients with PPV-MF and PET-MF usually show increasing splenomegaly, anemia, and leukoerythroblastosis in the peripheral blood. Their bone marrow shows abnormal large megakaryocytes with more prominent clustering, hyperlobulation, and frequent hyperchromatic nuclei, as well as grade 2-3 reticulin fibrosis. The differential diagnosis is clinically relevant, as the median overall survival is longer in patients with PET-MF (73 months) versus PMF (45 months) and PPV-MF (48 months) [42]. The diagnosis of PPV-MF and PET-MF can only be confirmed by documented history of prior diagnosis of PV or ET. The identification of JAK2 exon 12 mutations can confirm the diagnosis of PPV-MF, as JAK2 exon 12 mutations are exclusively seen in PV. Table 3 summarizes the major features of PMF, PET-MF, and PPV-MF.
Table 3. Differential diagnosis of PMF, post-polycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis.

7.3. Chronic Myeloid Leukemia (CML)
CML can mimic either the early (prefibrotic) phase of primary myelofibrosis (PMF)-particularly when presenting with thrombocytosis or the fibrotic phase of PMF, due to overlapping features such as bone marrow fibrosis and splenomegaly. Notably, bone marrow fibrosis may occur in approximately 40% of cases of CML and is associated with a poor prognosis, though it may be reversible via tyrosine-kinase inhibitors (TKIs) [43]. Despite these similarities, PMF can be easily distinguished from CML by morphology and genetic profile (lack of BCR-ABL). CML usually shows basophilia and the bone marrow shows characteristic hypolobulated “dwarf” megakaryocytes, in contrast to the large hyperlobulated hyperchromatic megakaryocytes and abnormal megakaryocytes with “bulbous” nuclei in PMF. The identification of BCR::ABL1 fusion by PCR or FISH is the hallmark for CML diagnosis.
7.4. Myelodysplastic Neoplasms and Myelodysplastic/Myeloproliferative Neoplasms
Myelodysplastic neoplasm with fibrosis (MDS-F) mimics PMF with anemia and fibrosis [44,45]. MDS-F shows increased blasts and clinically presents with cytopenia and no leukocytosis or thrombocytosis as usually seen in PMF. The megakaryocytes in MDS-F differ from that of PMF with predominance of small, hypolobulated megakaryocytes or micro-megakaryocytes (Figure 2). The mutation profile of MDS-F is distinct from that of PMF. MDS-F usually show mutations in ASXL1, SF3B1, TET2, RUNX1, etc., and no JAK2, CALR, MPL mutations.
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Figure 2. Comparison of the typical megakaryocyte morphology of PMF (A-C) and MDS (D-F). The megakaryocytes in PMF are usually increased in number and form dense clusters (A), and show hyperchromatic (B), bulbous or cloud-like morphology (C); while the megakaryocytes in MDS usually do not form dense clusters (D), and show mostly small monolobated dysplastic forms (E) or nuclear separation (F).
Chronic myelomonocytic leukemia (CMML) can also mimic PMF with fibrosis [46]. Bone marrow fibrosis is associated with shorter progression-free survival, splenomegaly, and increased megakaryocytes. On the other side, monocytosis can develop in patients with PMF during disease progression. This differential diagnosis can usually be resolved by reviewing the morphology of the megakaryocytes and mutation profiles. CMML typically shows MDS-type megakaryocytes with small, hypolobulated forms and micro-megakaryocytes. CMML shows characteristic mutations in ASXL1, TET2, SRSF2, and RAS pathway genes, and no MPN driver mutations. Of note, there are some cases with overlapping features between CMML and PMF, which show co-mutations involving JAK2 or MPL and ASXL1, SRSF2, TET2, NRAS, and/or KRAS [47]. These myeloid neoplasms may represent a true gray zone between CMML and PMF.
Myelodysplastic/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis (MDS/MPN-RS-T) can also share clinical (leukocytosis and thrombocytosis) and pathological (JAK2 mutation, hyperlobated megakaryocytes, fibrosis) with PMF. However, patients with PMF are more likely to have splenomegaly, CALR or MPL mutations, and megakaryocytes with hyperchromatic nuclei, while patients with MDS/MPN-RS-T tend to have SF3B1 mutations, a much more increased number of ring-sideroblasts, and multilineage dysplasia.
7.5. Systemic Mastocytosis (SM)
SM frequently shows fibrosis associated with peri-trabecular and paratrabecular aggregates of mast cells [48]. The diagnosis is straightforward with identification of aggregates or nodules of neoplastic mast cells with abnormal expression of CD2, CD25 and/or CD30, and KIT D816V by molecular methods. It is important to evaluate megakaryocyte morphology in SM. Presence of large, hyperlobated and/or hyperchromatic megakaryocytes should prompt evaluation of the presence of JAK2, CALR or MPL mutations by NGS or PCR, as PMF may coexist with SM as an associated hematological neoplasm (SM-AHN) [49].
7.6. Metastatic Carcinoma
Bone is a frequent site of involvement for a variety of metastatic carcinomas. Metastatic carcinomas like prostate carcinoma and breast cancer frequently induce fibrosis in the marrow [50]. Fibrosis in such cases may be extensive and mask the presence of metastatic carcinoma and mimic the fibrotic stage of PMF. Immunohistochemical staining for cytokeratins should be included in the panel of stains in such cases and will confirm the diagnosis of metastatic carcinoma.
7.7. Autoimmune Myelofibrosis (AIMF)
AIMF can mimic PMF with fibrosis, leukoerythroblastosis, splenomegaly, and megakaryocyte atypia in a subset of cases [51,52]. AIMF occurs in the presence or the absence of systemic autoimmune disease. Patients typically exhibit cytopenias and bone marrow fibrosis, with the marrow often hypercellular and containing benign lymphoid aggregates and occasionally large, abnormally lobulated or hyperchromatic megakaryocytes. Unlike PMF, AIMF is usually associated with autoimmune antibodies, such as anti-nuclear, anti-double stranded DNA, anti-phospholipid antibody/lupus anticoagulant antibodies and rheumatoid factor, and no driver mutations in JAK2, CALR, MPL.
7.8. Inflammatory and Infectious Causes
Infections including HIV and tuberculosis or granulomatous inflammation may induce bone marrow fibrosis [53]. These conditions lack the atypical megakaryocytes characteristic of PMF. Lab tests, such as serology, PCR, and special stains on the tissue (for example, Acid-Fast Bacilli stain for Tuberculosis) are necessary for the diagnosis.
8. Conclusions
The area of PMF research has come a long way with integrated WHO/ICC classification criteria, which facilitate the application of prognostic models and the stratification of patients with PMF for optimal treatment. Morphologic and molecular markers by NGS help to distinguish PMF from other MPNs, MDS, and reactive processes, while molecular markers (e.g., ASXL1, SRSF2, TP53) can additionally guide individualized therapy.
JAK inhibitors, approved for intermediate-2 and high-risk MF, are effective across all subgroups, including TN-PMF. In COMFORT-I, 59% achieved -35% spleen volume reduction regardless of JAK2 status; COMFORT-II reported a 28% reduction at 48 weeks with symptoms and quality-of-life improvements regardless of mutational status. These results highlight the central role of JAK-STAT dysregulation in MF pathogenesis. However, JAK inhibitors do not significantly alter disease progression or survival.
Future research should focus on disease-modifying therapies to improve outcomes across all MF subtypes, including TN-PMF [54].
Author Contributions
Conceptualization-R.S. and J.S. and J.Y.L.; writing-original draft preparation, C.R., L.W., H.Z., L.M., M.M., and M.S.; supervision, J.S.; All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Data Availability Statement
No new data were created.
Conflicts of Interest
The authors declare no conflict of interest.
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MDPI and ACS Style
Shao, R.; Ryder, C.; Wang, L.; Zhang, H.; Moscinski, L.; Martin, M.; Shebes, M.; Li, J.Y.; Song, J. A Review of the Pathological and Molecular Diagnosis of Primary Myelofibrosis. Cancers 2026, 18, 50. https://doi.org/10.3390/cancers18010050

AMA Style
Shao R, Ryder C, Wang L, Zhang H, Moscinski L, Martin M, Shebes M, Li JY, Song J. A Review of the Pathological and Molecular Diagnosis of Primary Myelofibrosis. Cancers. 2026; 18(1):50. https://doi.org/10.3390/cancers18010050

Chicago/Turabian Style
Shao, Richard, Christopher Ryder, Le Wang, Hailing Zhang, Lynn Moscinski, Michael Martin, Mac Shebes, Julie Y. Li, and Jinming Song. 2026. "A Review of the Pathological and Molecular Diagnosis of Primary Myelofibrosis" Cancers 18, no. 1: 50. https://doi.org/10.3390/cancers18010050

APA Style
Shao, R., Ryder, C., Wang, L., Zhang, H., Moscinski, L., Martin, M., Shebes, M., Li, J. Y., & Song, J. (2026). A Review of the Pathological and Molecular Diagnosis of Primary Myelofibrosis. Cancers, 18(1), 50. https://doi.org/10.3390/cancers18010050