87. Молекулярная биология сарком

Введение

Саркомы являются жизнеугрожающими мезенхимальными новообразованиями, на долю которых приходится примерно 1% всех случаев рака человека. Они представляют значительную лечебную проблему; приблизительно 50% пациентов с недавно диагностированной саркомой в конечном счете умирают от болезни. Саркомы также создают значительные диагностические проблемы, поскольку существует более 70 гистологических подтипов с уникальными молекулярными, патологическими, клиническими, прогностическими и лечебными характеристиками.

Саркомы мягких тканей

Генетическая характеристика саркомы мягких тканей позволила улучшить классификацию и разделить саркомы на саркомы с простыми и очень сложными кариотипами (рис. 87.1). Первая группа состоит из сарком с почти диплоидными кариотипами и простыми генетическими изменениями (транслокации, инверсии или специфические активирующие мутации). Транслокация-ассоциированные саркомы обычно наблюдааются у молодых людей с самой высокой частотой в 30-50 лет. Онкогенез в основном является результатом дерегуляции транскрипции, вызванной слияниями генов. Для сарком с аберрантными, очень сложными геномами пик заболеваемости приходится на возрастную группу 50-60 лет. Эти саркомы характеризуются изменениями числа копий при низком мутационным грузом; частыми альтерациями в генах клеточного цикла TP53, CDK4, MDM2, RB1 и INK4a; и дефектами в сигнальных путей факторов роста. Открытие критических подтип-специфических молекулярных альтераций, которые контролируют саркомагенез, позволит разработать целевую терапию. Эта идея иллюстрируется эффективностью иматиниба, ингибирующего ABL, KIT и PDGFRα (platelet-derived growth factor receptor α) тирозинкиназы, в гастроинтестинальных стромальных опухолях, которые имеют активирующие мутации в KIT и PDGFRα (см. Главу 88).

Фигура 87.2 показывает гистологический вид основных подтипов саркомы мягких тканей, а таблица 87.1 описывает диагностические гистологические характеристики и молекулярные и цитогенетические аномалии.

Транслокация-ассоциированные саркомы мягких тканей

Миксоидная/круглоклеточная липосаркома

Миксоидные липосаркомы обычно развиваются в бедре или других глубоких мягких тканях у взрослых (возрастной пик 30-50 лет). Миксоидные липосаркомы обычно имеют характерную морфологию: униформные круглые клетки в окружающем выделяющемся миксоидном матриксе, плексиформная сосудистая сеть и перстне-кольцевидные липобласты. Эти признаки, однако, могут частично теряться в высокозлокачественной форме – округло-клеточной липосаркоме. Почти все миксоидные/круглоклеточные липосаркомы несут сбалансированную транслокацию t(12; 16)(q13; p11), сливающиеся 5′-экзоны FUS (также известные как TLS, кодирующие транскрипционные регуляторные домены, которые взаимодействуют с РНК-полимеразой II) с полностью кодирующей последовательностью DDIT3 (также известной как CHOP или GADD153, транскрипционный фактор лейциновой молнии с ролью в контроле клеточного цикла, дифференцировке адипоцитов и стресс ответе). В редких случаях EWSR1 заменяется его гомологом FUS. Сообщалось о, по меньшей мере, 12 транскрипционных вариантах FUS-DDIT3, и несколько вызывают саркомный фенотип в модельных системах. Онкопротеин слияния связывает кофакторы, включая C/EBP, для дерегулирования экспрессии генов, хотя было подтверждено лишь несколько прямых мишеней. Одним из результатов является активация путей, связанных с ангиогенезом (интерлейкин [IL]-8), PPARγ (early adipose differentiation), сигналингом факторами роста (инсулиноподобный фактор роста [IGF], RET) и контролем клеточного цикла (циклин D, CDK4 ). Другим результатом является репрессия miR-486 и IL-24, которые в противном случае могли бы действовать как опухолевые супрессоры.

Clinically, detection of FUS-DDIT3 translocations by reverse transcription polymerase chain reaction (RT- PCR)8 or fluorescence in situ hybridization (FISH)19 can help confirm the diagnosis, especially in small biopsies dominated by a round cell component. Fusion subtype, however, appears to have little prognostic value independent of stage and grade. In general, the prognostic value of molecular markers in myxoid liposarcoma has been difficult to test, given the difficulty of assembling large series.20 Nevertheless, high levels of p53, IGF1R/IGF2, the receptor tyrosine kinase AXL, and RET may be adverse indicators.14,21,22 In addition, mutations in PIK3CA, which encodes a subunit of phosphatidylinositol 3-kinase, found in 18% of myxoid/round cell liposarcomas, were associated with worse outcome.23 MicroRNA-135b (miR-135b) is highly expressed in the round cell component and promotes myxoid liposarcoma invasion in vitro and metastasis in vivo by directly suppressing thrombospondin 2, which increases the amount of the degradative enzyme matrix metalloproteinase 2 (MMP2).24

Миксоидные липосаркомы имеют густую микроциркуляторную сеть и высокую экспрессию IL-8 и фактора роста эндотелия сосудов (VEGF). Эти характеристики предполагают ценность антиангиогенной терапии и могут лежать в основе наблюдаемой чувствительности к лучевой терапии и трабектедину. Трабектедин может также действовать, нарушая связывание FUS-DDIT3 с промоторами-мишенями и усиливая экспрессию в опухолевых клетках эндогенного ингибитора ангиогенеза тромбоспондина-1 (TSP-1) . Агенты, созданные для таргетинга FUS-DDIT3, пока недоступны.

Саркома Юинга (Ewing)

Ewing sarcomas mostly affect adolescents and young adults and arise more often in the bone compared to soft tissues. A range of aggressive small blue round cell tumors have been subsumed under the term Ewing sarcoma family tumor due to their shared undifferentiated round cell phenotype and recurrent chromosomal translocations. EWSR1, the common 5′ translocation partner, is fused to one of several ETS family transcription factor genes (usually FLI1). In the chimeric protein, EWSR1 provides, at minimum, its N-terminal transcriptional regulatory domain and loses its RNA recognition domain. The ETS factor provides its C-terminal DNA-binding domain and loses its native transactivation domain. The fusion oncoprotein has several validated direct transcriptional targets. Some of these targets are upregulated (PTPL1, PRKCB, DAX1/NR0B1), but more are repressed (FOXO1, TGFBR2, LOX, IGFBP3, and the Let-7 microRNA precursor) with the help of cofactors. These oncogenic gene expression programs are driven at least in part by EWSR1-FLI1 recruitment of the BAF chromatin remodeling complex to tumor-specific enhancers. The net result is enhanced proliferation and cell survival and repression of mesenchymal differentiation.

Фигура 87.1. Изменения нуклеотидов и числа копий в саркоме мягких тканей.

The outer ring indicates chromosomal position. The second to fifth rings represent four subtypes with complex karyotypes (as labeled; MYXF, myxofibrosarcoma; PLEO, pleomorphic liposarcoma; LMS, leiomyosarcoma; DEDIFF, dedifferentiated liposarcoma; GIST, gastrointestinal stromal tumor).

The three inner rings represent subtypes with simple karyotypes (myxoid, myxoid/round cell liposarcoma). The plots show the statistical significance of genomic aberrations, with amplification in red and deletion in blue. Green curves indicate the chromosomal breakpoints of pathognomonic translocations in myxoid/round cell liposarcoma and synovial sarcoma. Genes harboring somatic nucleotide alterations are indicated with green circles whose size is proportional to their frequency of occurrence.

Molecular confirmation of an EWSR1 translocation is critical for patient management because Ewing sarcoma shares many clinical, morphologic, and immunophenotypic features with other osseous round cell tumors, such as mesenchymal chondrosarcoma and small-cell osteosarcoma. Commercially available EWSR1 split-apart FISH probes are valuable ancillary diagnostic tools (see Fig. 87.2).45 RT-PCR is complicated by the many alternative fusion variants but can identify the specific fusion.46 Targeted RNA sequencing tools, such as Archer, have become available and used with success. EWSR1-negative small blue round cell tumors are associated with the alternative fusions CIC-DUX4, BCOR-CCNB3, and other BCOR gene rearrangements in approximately 70%, 13%, and 11% of cases, respectively. CIC-DUX4 sarcomas have a distinct gene signature and immunoprofile48 and a more aggressive clinical course compared with EWSR1-ETS fusion-positive Ewing sarcoma.49 BCOR- CCNB3 Ewing sarcoma–like tumors occur preferentially in young males, and 70% arise from bone.

Фигура 87.2. Подтипы саркомы, обсуждаемые в тексте. Верхние панели — парафиновые срезы, окрашенные гематоксилином и эозином. The lower panels are fluorescence in situ hybridization images showing alveolar rhabdomyosarcoma (left) with fusion of probes for PAX3 (red) and FOXO1 (green) and Ewing sarcoma (right) with break apart of probes flanking the Ewing sarcoma breakpoint region, EWSR1. Malign., malignant.

Таблица 87.1. Цитогенетические и молекулярные нарушения в саркомах мягких тканей

Болезнь Диагностическая морфология или иммуногистохимия Цитогенетическая реакция Молекулярная аномалия Молекулярная диагностика a
Михоидная/круглоклеточная Lipoblasts, plexiform vasculature, myxoid matrix t(12;16)(q13;p11)

t(12;22)(q13;q12)

FUS-DDIT3 (>90%)              EWSR1-DDIT3 (<5%) DDIT3 breaks (FISH)
липосаркома
Семейная саркома Юинга

 

Small, blue, round cells; CD99 and FLI1 expression; lack of lymphoid biomarker expression t(11;22)(q24;q12)

t(21;22)(q22;q12)

Alternative events: fusions of 22q12 with  7p22

17q22, 2q33; inv 22q12; t(16;21)

(p11;q22); t(4;19)

(q35;q13) or t(10;19)

(q26;q13); t(X;4)

(p11.4;q31.1)

EWSR1-FLI1 (>80%),    EWSR1-ERG (10%–15%), Other ETS family partners: ETV1, ETV4, FEV, PATZ1 (<5%), FUS-ERG (<1%), CIC-DUX4, BCOR-MAML3 EWSR1 breaks (FISH) or RT-PCR
Десмопластическая мелкоклеточная опухоль Small, blue, round cell islands in dense stroma; positive for keratin, desmin, vimentin, and WT1 t(11;22)(p13;q12) EWSR1-WT1 (>75%) EWSR1 breaks (FISH)
Синовиальная саркома Biphasic histology, positive for TLE177 t(X;18)(p11;q11) (>90%) SYT-SSX1 (66%)

SYT-SSX2 (33%)

SYT-SSX4 (<1%)

SYT breaks (FISH)
Альвеолярная рабдомиосаркома

 

Small, blue cells expressing desmin, myogenin, myoD1 t(2;13)(q35;q14) PAX3-FOXO1 (<80%)

PAX7-FOXO1 (<20%)

PAX3-NCOA1 (<1%)

PAX3-NCOA2 (<1%)

PAX3/7 type-specific FISH or RT-PCR320
t(1;13)(p36;q14)
Альвеолярная мягкая часть саркомы Nested polygonal cells in vascular network; positive for TFE3321 t(X;17)(p11;q25) ASPSCR1-TFE3 (>90%) ASPSCR1-TFE3 RT- PCR322 or TFE3 FISH116
Дерматофибросаркома протуберанс Bland spindle cells, storiform and honeycomb growth in subcutis, positive for CD34 Rings derived from t(17;22) (>75%) t(17;22)(q22; q13.1)126,127,323 (10%) COL1A1-PDGFB
Эмбриональная рабдомиосаркома Spindle cells and rhabdomyoblasts, positive for desmin and myogenin Trisomies 2q, 8, and 20 (>75%) LOH at 11p15 (>75%)
Экстраскелетный миксоид Bland epithelioid cells

arranged in reticular

pattern in myxoid stroma

t(9;22)(q22;q12)

t(9;17)(q22;q11)

t(9;15)(q22;q21)

t(3;9)(q12;q22)

EWSR1-NR4A3 (75%)

TAF15-NR4A3 (<10%)

TCF12-NR4A3 (<10%)

TFG–NR4A3 (<5%)

EWSR1 breaks (FISH); RT-PCR
Хондросаркома Bland spindle cells, positive for CD10 and ER t(7;17)(p15;q21) JAZF1-SUZ12 (30%)
Эндометриальная стромальная опухоль Nested epithelioid cells with clear or amphophilic cytoplasm, positive for S100 and HMB-45 t(12;22)(q13;q12)

t(2;22)(q34;q12)

EWSR1-ATF1 (>75%)

EWSR1-CREB1 (<5%)

EWSR1 breaks (FISH)
Светлоклеточная саркома Monomorphic spindle cells, herringbone pattern t(12;15)(p13;q25) ETV6-NTRK3 (>75%) FISH, RT-PCR
Воспалительная миофибробластическая опухоль Myofibroblastic cells with lymphoplasmacytic infiltrate, positive for ALK t(1;2)(q25;p23), t(2;19)(p23;p13), t(2;17)(p23;q23) ALK-TPM34,                          ALK-TPM                                 ALK-CLTC ALK breaks (FISH)
Солитарная фиброзная опухоль Monomorphic spindle cells, collagenous stroma, and staghorn vasculature; STAT6 expression 12q13 inversion NAB2-STAT6 (>95%) RT-PCR
Гастроинтестинальные стромальные опухоли Spindle (70%), epithelioid (20%), or mixed (10%) morphology; positive for CD117 (KIT), DOG1, and CD34 Monosomies 14 and 22 (>75%) Deletion of 1p (>25) KIT or PDGFRA mutation (>90%) PCR mutation analysis
Десмоидный фиброматоз Bland myofibroblastic-type cells, fascicular growth, nuclear positivity for β-catenin Trisomies 8 and 20 (30%) APC inactivation by mutation/deletion (10%) IHC for β-catenin expression
Мутации CTNNB1 (β-катенин) (85%)
Хорошо дифференцированная/ дедифференцированная липосаркома Atypical multinucleated stromal cells, lipoblasts, positive for MDM2, CDK4 12q13-15 rings and giant markers MDM2 and CDK4 amplification (>85%) MDM2 amplification (FISH)
Плеоморфная липосаркома Pleomorphic spindle and giant cells, pleomorphic lipoblasts Complexb (>90%) Нет
Микофибросаркома и недифференцированная плеоморфная саркома Pleomorphic spindle and giant cells, storiform growth, variable myxoid stroma Complexb (>90%) SKP2 amplification

CCNE1, VGLL3, or

YAP1 amplification

Нет
Леймиосаркома Elongated fusiform cells with eosinophilic cytoplasm, in intersecting fascicles, positive for desmin and smooth muscle actin Complexb (>50%) Deletions of 1p TP53 and RB1 point mutations/deletions Нет
Злокачественная опухоль периферической нервной оболочки Monomorphic spindle cells, high mitotic count, geographic necrosis Complexb (90%) NF1 mutation, loss or deletion (>50%) Нет
Mutations in EED or

SUZ12

Loss of H3K27me3

a Refers to molecular tests that can be run on formalin-fixed, paraffin-embedded material for molecular confirmation of diagnosis: quantitative RT-PCR of transcripts320 or FISH to interphase genomic DNA.327

b Complex karyotypes containing multiple numerical and structural chromosomal aberrations.

FISH, fluorescence in situ hybridization; RT-PCR, reverse transcription polymerase chain reaction; LOH, loss of heterozygosity; IHC, immunohistochemistry; H3K27me3, trimethylation of histone H3 at lysine 27.

Promising therapeutic strategies in Ewing sarcoma that target translocation-induced mechanisms and pathways include inhibitors of insulin-like growth factor 1 receptor (IGF1R) and mammalian target of rapamycin (mTOR) alone and in combination, the combination of small-molecule poly (ADP-ribose) polymerase (PARP) and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, 19S proteasome inhibitors, sirtuin 1 deacetylase, and cyclin-dependent kinase (CDK) inhibitors. Novel therapeutic approaches to inhibit the oncoprotein itself are in active development.

Десмопластическая мелко-круглоклеточная опухоль

В десмопластической мелко-круглоклеточной опухоли те же 5′-участки EWSR1, вовлеченные в саркомы Юинга, сливаются с WT1, опухолевым супрессором, делитированным в опухоли Вильмса. Химерный протеин включает последние три ДНК-связывающих домена цинковых пальцев WT1. Несмотря на некоторые сходства с опухолями семейства сарком Юинга, десмопластичные мелко-кругло-клеточные опухоли редко курабельны агрессивной традиционной химиотерапией в сочетании с хирургическим удалением опухоли; их мрачный прогноз требует новых методов лечения. EWSR1-WT1 прямо индуцирует экспрессию PDGFA, что объясняет десмопластический фон и, наряду с гиперэкспрессией VEGFA и VEGF рецептора 2 (VEGFR2), объясняет наблюдаемые частичные ответы на сунитиниб. IL2RB также индуцируется, и его последущие JAK/STAT и AKT/mTOR сигнальные пути являются активными, представляя потенциальные терапевтические мишени. EWSR1-WT1 необходим для роста опухолевых клеток и связывается с промотором ASCL1 для активации программы экспрессии нейрональных генов, которая контролирует частичную нейрональную дифференцировку.

Синовиальная саркома

Synovial sarcoma differs from most translocation-associated sarcomas in that its defining translocation, t(X;18) (p11;q11), fuses epigenetic regulators (SS18 [also known as SYT] and SSX, normally expressed only in testis), not transcription factors. In the fusion oncoprotein, SS18, part of the BAF chromatin remodeling complex, retains all but eight amino acids from its transcriptional activation domain. The SSX partner (SSX1, SSX2, or SSX4) retains only its repressor domain, which confers nuclear localization in association with Polycomb proteins. Expression of SS18-SSX in mesenchymal stem cells75 or mice72,76 recapitulates synovial sarcoma. Neither SS18 nor SSX binds DNA, but the chimeric oncoprotein gains abnormal epigenetic functions, including binding TLE1, a highly expressed diagnostic marker of synovial sarcoma77 that recruits Polycomb repressor complex proteins, and binding the transcription factor ATF2. The net result is Polycomb-mediated epigenetic repression of ATF2 target genes, including the tumor suppressors EGR1 and CDKN2A.78 In addition, when SS18- SSX replaces native SS18 in BAF complexes, the SMARCB1 (SNF5) component is evicted, which increases Polycomb activity and hyperactivates stem cell–associated programs.80,81 Genes and pathways that become activated in synovial sarcoma include histone deacetylases,78 SOX2,79 Wnt/β-catenin,82 the transcription factor TWIST1,83 fibroblast growth factor receptor 2 (FGFR2),84 the apoptosis regulator BCL2,85 the chemokine receptor CXCR4,86 the receptor tyrosine kinase ALK, the hepatocyte growth factor receptor MET,87 and the Akt/mTOR pathway88,89 via IGF2.75,90,91 Therefore, these represent candidates for targeted therapy because no available drugs inhibit SS18-SSX directly. Class I histone deacetylase inhibitors and a lead compound designated SXT1596 have been found to disrupt SS18-SSX’s interaction with TLE1 in synovial sarcoma cells and tissue samples.92

Copy number alterations are more common in adult than in pediatric patients, and both copy number alterations and an expression signature of genes related to mitosis and chromosome function are associated with metastasis.

Альвеолярная рабдомиосаркома

In alveolar rhabdomyosarcoma, an aggressive cancer of older children and adolescents, the transcriptional activation domain of FOXO1 (also known as FKHR) from 13q14 is fused to the DNA-binding domain of paired box transcription factor PAX3 (2q35) or PAX7 (1p36).94,95 Approximately 20% of cases were previously thought to be translocation-negative; such tumors are in fact histologic variants of embryonal rhabdomyosarcoma.96 Translocations involving PAX3 may be associated with a worse prognosis than those involving PAX7,97 so confirming the diagnosis by FISH (see Fig. 87.2) and/or RT-PCR helps optimize treatment plans.98 Either translocation results in high nuclear expression of a chimeric transcription factor that abnormally activates PAX targets, many of which are involved in neurogenesis.99,100 Recently, PAX3-FOXO1 fusion messenger RNA (mRNA) and chimeric protein were found to be expressed transiently during normal fetal muscle development (i.e., in cells without DNA translocations).101 PAX3-FOXO1 direct targets include P-cadherin (CDH3),102 Gremlin 1 (GREM1, a bone morphogenetic protein [BMP] antagonist), death-associated protein kinase 1 (DAPK1), myogenic differentiation 1 (MYOD1),103 JARID2 (a cofactor of polycomb repressor complex 2 [PRC2]),104 and PDGFRα. Inhibitors of PDGFRα are effective in mouse models.105 Targetable kinases expressed in alveolar rhabdomyosarcoma also include IGF1R, ALK,106–108 and PLK1, which phosphorylates and stabilizes the PAX3-FOXO1 fusion protein109 and the tyrosine kinases ephrin receptor B4 (EphB4) and PDGFRβ.110 Another probable direct target of PAX3-FOXO1 is the cell cycle regulator SKP2,111 perhaps helping explain alveolar rhabdomyosarcoma’s response to cytotoxic chemotherapy.

Альвеолярная саркома мягких частей (alveolar soft part sarcoma)

In alveolar soft part sarcoma, which has a similar clinical presentation to other translocation-associated sarcomas, the 5′ half of the widely expressed ASPSCR1 (also known as ASPL) gene on 17q25 is fused to exon 3 or 4 of TFE3 on Xp11, the latter retaining its transcriptional activation, basic helix loop helix, and leucine zipper domains. Similar fusions are present in a subset of renal cell carcinomas, particularly those in younger patients, and in a subset of perivascular epithelioid cell neoplasms. Although alveolar soft part sarcoma has distinctive histology, two useful diagnostic adjuncts are detection of TFE3 rearrangements by FISH116 or RT-PCR and immunohistochemistry for TFE3. Genes transcriptionally activated by the ASPSCR1-TFE3 oncoprotein share a CACGTG motif; validated direct targets include MET and angiogenic mediators. Antiangiogenic therapy is effective in xenograft models. A single-arm phase II study of the VEGFR inhibitor cediranib in metastatic alveolar soft part sarcoma showed a high rate of disease control in association with downregulation of angiogenic genes (including ANGPT2), supporting advanced trials of antiangiogenic agents.

Дерматофибросаркома протуберанс

Отличительным признаком дерматофибросаркомы протуберанцев (DFSP) являются дополнительные кольцевые хромосомы, которые содержат материал из хромосом 17 и 22 или, реже, несбалансированный der (22) t (17; 22) (q21-23; q13). Молекулярным следствием обеих аберраций является гиперэкспрессия субъединицы B PDGF (PDGFB) на хромосоме 22 вследствие слияния с геном коллагена COL1A1 на хромосоме 17. Такое же слияние также наблюдается в двух гистологических вариантах — гигантоклеточной фибробластоме и опухоли Беднара (Bednar) (пигментированная DFSP) , Увеличение числа COL1A1-PDGFB копий связано с фибросаркоматозной трансформацией DFSP, хотя увеличение числа копий не является неизменной чертой этих случаев.

Продукт COL1A1-PDGFB слияния сигнализирует через PDGFR в аутокринной петле. Этот сигналинг может блокироваться ингибиторами тирозинкиназ, действующих на PDGFR, таких как иматиниб. Ряд клинических исследований показал высокий уровень ответа на терапию иматинибом как при локальной, так и при метастатической DFSP. Эти результаты подтверждают концепцию, что DFSP клетки зависят от аберрантной активации PDGF сигналинга для пролиферации и выживания.

Экстраскелетная миксоидная хондросаркома

Большинство экстрасклетных миксоидных хондросарком несут реципрокные транслокации, которые сливают орфанный ядерный рецептор NR4A3 в 9q22-q31.1 с одним из следующих четырех партнеров: EWSR1 в 22q12 (наиболее распространенный), TATA-связывающий протеин-ассоциированный фактор (TAF15) в 17q11, фактор транскрипции 12 (TCF12) в 15q21 или TFG в 3q12. Поскольку эти слитые гены не были описаны ни в одном другом типе опухоли, они представляют собой полезные диагностические маркеры. Четыре разных партнера по слиянию имеют неизвестное прогностическое значение.

NR4A3, также известный как NOR-1, TEC, MINOR или CHN, экспрессируется повсеместно. T(9;22) сливает трансактивирующий домен EWSR1 с NR4A3 полной длины. Аналогично EWSR1-ETS слиянию, протеин слияния EWSR1-NR4A3 не только показывает сильную транскрипционную активность, но и регулирует РНК сплайсинг.

TAF15 принадлежит тому же семейству протеинов, что и EWSR1 и FUS, и содержит характерный участок распознавания РНК, участвующий в протеин-РНК связывании. N-концевые области EWSR1, FUS и TAF15 содержат дегенерирующие SYGQ повторы и сильно активируют транскрипцию при слиянии с ДНК-связывающими доменами различных факторов транскрипции.

Экстрасклетные миксоидные хондросаркомы представляют собой отдельную геномную форму, в которой активируется несколько генов, включая нейромедин B (NMB, кодирующий секретируемый нейронный пептид), Wnt антагонист DKK1, DNER и CLCN3 (кодирующий потенциал-контролируемый хлоридный канал). NMB высоко экспрессируется при экстрасклетной миксоидной хондросаркоме, но не в других саркомах, что указывает на его потенциальную диагностическую ценность. Тирозинкиназный ингибитор сунитиниб, который ингибирует все три VEGFR1, PDGFRα/β, KIT, RET и FLT3, обладает эффективностью при этой саркоме; 6 из 10 пациентов имели частичные ответы в соответствии с критериями оценки ответа в солидных опухолях (Response Evaluation Criteria in Solid Tumors, RECIST), но 2 других пациента имели стабильное заболевание. Все чувствительные пациенты имели EWSR1-NR4A3 слияние, а 2 пациента с прогрессирующим заболеванием имели TAF15-NR4A3 слияние. RET высоко экспрессировался и фосфорилировался во всех 4 проанализированных образцах опухоли.

Солитарная фиброзная опухоль и гемангиоперицитома

Solitary fibrous tumor (SFT) and hemangiopericytoma share histopathologic features and are now classified as a single entity. Virtually all SFTs share a recurrent NAB2-STAT6 fusion, regardless of anatomic location (pleura, meninges, or soft tissue). In SFT, the adjacent genes NAB2 and STAT6 (on chromosome 12q13) are fused by a chromosomal inversion so that they are transcribed in the same direction (they are normally read in opposing directions).

In contrast to NAB2, which represses early growth response protein 1 (EGR1) activity, the NAB2-STAT6 fusion induces expression of EGR1 target genes. In an RNA sequencing analysis of 7 SFTs and 282 other tumor samples, the SFTs expressed high levels of both EGR1 target genes, including NAB2, NAB1, IGF2, FGF2, and PDGFD, and receptor tyrosine kinases, such as FGFR1 and NTRK1.145 Overexpression of other tyrosine kinase receptor genes, such as DDR1 and ERBB2, has been seen in array-based profiling.

IGF2 is uniformly overexpressed in SFT.146 IGF2 is imprinted on the paternal allele in most adult tissues; IGF2 overexpression in SFTs is related to loss of imprinting. Although IGF2 acts by binding to IGF1R, the receptor is not upregulated in SFTs, so IGF2 may signal through the insulin receptor A pathway.147 Overexpression of IGF2 and consequent activation of the insulin receptor may also explain why a subset of SFT patients present with hypoglycemia. This syndrome, known as Doege-Potter syndrome, has been associated with large tumor size and aggressive clinical behavior and is resolved by surgical resection of the lesion.148

Саркомы мягких тканей простого кариотипа, ассоциированные с мутациями

Десмоидный фиброматоз

Desmoid-type fibromatoses are locally infiltrative, clonal, myofibroblastic proliferations that arise in the deep soft tissues and never metastasize. Although the vast majority result from mutations in adenomatous polyposis coli (APC) or β-catenin (CTNNB1), tumorigenesis may also be influenced by physiologic factors such as pregnancy, trauma, and prior surgery. Desmoids are usually divided into two groups: sporadic desmoids and those in individuals with an inherited APC mutation (inactivation of one copy of APC, usually by point mutation or deletion).149,150 Although germline APC mutations often result in familial adenomatous polyposis,150 some desmoid patients harboring such a mutation have no polyposis.

Among sporadic desmoids, only a minority display APC inactivation, although >95% contain genetic alterations affecting the same signaling pathway (Wnt/β-catenin),151 indicating that these changes drive desmoid initiation in general. A majority (52% to 85%) have an activating point mutation in the β-catenin gene, CTNNB1,152,153 that stabilizes β-catenin, resulting in its overabundance. β-Catenin is negatively regulated by APC, so both APC inactivation and CTNNB1 activating mutations result in upregulation of the Wnt pathway. The specific CTNNB1 mutation may have prognostic significance; patients with S45F-mutant desmoids were reported to have a 5-year recurrence-free survival of only 23%, compared with 57% for those with T41A-mutant tumors and 65% for those with wild-type CTNNB1.152 Another study seemed to confirm that S45F-mutated patients have worse recurrence-free survival,154 but the finding that 27% of the study’s 179 patients were wild type suggests that mutations in CTNNB1 or other Wnt pathway genes were likely missed. Definitive conclusions regarding the relationship between mutations and outcomes will require thorough, sensitive sequencing of relevant genes and careful control for potential confounders such as tumor site.

Given the Wnt pathway mutations in the majority of patients, small-molecule β-catenin antagonists (in preclinical development) would likely provide significant benefit, particularly for patients with advanced disease in whom surgical resection is not feasible. An inhibitor of matrix metalloproteinase, a downstream target of β- catenin, substantially reduced tumor volume and invasion in a transgenic Apc+ /Apc1638N mouse model of aggressive fibromatosis.155

Hedgehog signaling is activated in human and murine desmoid tumors, consistent with their mesenchymal expression profile. Inhibiting hedgehog signaling reduced proliferation and β-catenin protein levels in human desmoid cells and reduced tumor size and number in a murine model,156 suggesting hedgehog antagonists may be a promising therapy for desmoid patients.

Patients with desmoids were found to have elevated levels of PDGF-AA and PDGF-BB, leading to a trial of the tyrosine kinase inhibitor imatinib in patients with advanced disease. Three of 19 (16%) patients had a partial response, and 4 additional patients had stable disease for more than 1 year; overall, the 1-year tumor control rate was 37%.157 The response in these tumors was thought to be mediated by inhibition of PDGFRβ kinase activity. In a more recent study, 7 of 38 (19%) desmoid tumor patients had partial responses at 21 months.158 Sorafenib, a multitargeted tyrosine kinase inhibitor, results in tumor shrinkage in 25% of desmoid patients and stable disease in 70%, along with symptom relief in 70% of patients.159 Cross-talk between the Wnt and Notch signaling pathways provided rationale for a phase I trial of the oral γ-secretase inhibitor PF-03084014, which disrupts Notch signaling, in which 5 of 7 response-evaluable patients with desmoid tumor achieved a partial response (71.4% objective response rate).160

Сложные типы сарком мягких тканей

Хорошо дифференцированная и дедифференцированная липосаркома

Well-differentiated liposarcomas (WDLS) and dedifferentiated liposarcomas (DDLS) represent the most common biologic group of liposarcoma. This group is characterized by amplification of 12q, which usually occurs in double minutes, ring chromosomes, and large marker chromosomes. In addition, the 12q13.2-q23.1 locus often harbors complex rearrangements (see Fig. 87.1). The amplified region generally includes the oncogenes MDM2 (a negative regulator of p53 and p21), the transcriptional regulator HMGA2, and cyclin-dependent kinase 4 (CDK4). Additional driver genes indicated by rearrangements and correlated overexpression include neuron navigator 3 (NAV3), Wnt inhibitory factor 1 (WIF1), MDM1, the proapoptotic kinase DYRK2, the transcription factor ELK3, the phosphatase DUSP6 (a regulator of MAP kinases), the histone acetyltransferase component YEATS4, the NFκB pathway regulator TBK1, and FGFR substrate 2 (FRS2), which are amplified in approximately 14% to 80% of tumors. Aside from 12q aberrations, DDLS contain significant amplifications of 1p (JUN, a component of the early response transcription factor), 1q (miR-214), 5p (TERT, a telomerase subunit), 6q (SASH1), 11q (YAP1, a pro-proliferative transcription factor), and 20q. Chromosomal alterations appear to correlate with prognosis; unsupervised analysis found that 1p-amplified and 5p-amplified DDLS had worse disease-specific survival compared to 6q-amplified DDLS. The same study revealed an association of hypermethylated DDLS containing a low leukocyte fraction with worse disease-specific survival compared to hypomethylated, leukocyte-rich DDLS.1 These results suggest that both copy number alterations and the immune microenvironment drive biologic behavior in DDLS.

Амплификация JUN (на 1р32) предполагается в качестве объяснения блока в дифференцировке адипоцитов в DDLS. Тем не менее, амплификация или гиперэкспрессируется JUN наблюдается только приблизительно в 40% этих видов рака. Другие гены, которые ингибируют дифференцировку адипоцитов, включают DDIT3, дифференцировка-регулирующую фосфатазу PTPRQ, YAP1 и C/EBPα. C/EBPα слабо экспрессируется во многих DDLS тканях и клеточных линиях. Экзогенно экспрессируемый C/EBPα ведет к 50% снижению пролиферации, остановке G2 / M, апоптозу и восстановлению способности индуцировать маркеры раннего адипогенеза. Экспрессия C / EBPα независимо предсказывает отдаленную безрецидивную выживаемость у пациентов с первичной липосаркомой, а потеря 19q регина, включаюющего C/EBPα, связана с плохими исходами в первичной ретроперитонеальной DDLS. C/EBPα downregulation may result from epigenetic defects; 24% of DDLSs had C/EBPα promoter methylation, and 8.3% had mutations in histone deacetylase 1 (HDAC1). Treating DDLS cells with a demethylating agent and the histone deacetylase inhibitor vorinostat increased C/EBPα expression 19-fold, decreased proliferation, induced apoptosis, and reduced xenograft tumor growth by 50% to 70%.Taken together, these results suggest that C/EBPα acts as a tumor suppressor in DDLS and that its loss may explain the undifferentiated state of DDLS.

In WDLS and DDLS, cell cycle and checkpoint pathways are activated by upregulation of CDK4; MDM2; CDK1; CDC7; TOP2A; PRC1; PLK1; and cyclins B1, B2, and E2, as shown by microarray analysis.174,175 Therefore, these pathways may be useful as therapeutic targets. In fact, nutlin-3a, a selective MDM2 antagonist, induces apoptosis and inhibits proliferation of DDLS cell lines at concentrations that do not affect normal adipose- derived stem cells. Furthermore, PD0332991, a selective CDK4/CDK6 inhibitor, inhibits proliferation by inducing G1 cell cycle arrest and senescence in DDLS cell lines and xenografts.23 In a phase II trial of PD0332991 (now named palbociclib) in 30 patients with advanced CDK4-amplified liposarcoma, 66% of patients were progression free at 12 weeks, and a subset had a radiographic response and prolonged stable disease.176 In an expanded cohort of 30 additional patients, progression-free survival was 57% at 12 weeks, median progression- free survival was 17.9 weeks, and there was one complete response.177 Loss of MDM2 protein was associated with response to palbociclib by induction of a DDLS cell senescence program that requires ATRX.178

These results provide a rationale for use of MDM2 antagonists and CDK4 inhibitors in patients with well- differentiated liposarcoma and DDLS.

Плеоморфная липосаркома

Pleomorphic liposarcoma, accounting for 5% of all liposarcomas, is the least common subtype. It is characterized by high chromosome counts and complex rearrangements, with many unidentifiable marker chromosomes and nonclonal alterations. A high-resolution single nucleotide polymorphism (SNP) array analysis has revealed multiple regions of significant copy number amplification and deletion.179 The most common alteration, found in approximately 60% of tumors, was a deletion of 13q14.2-q14.3 that includes the RB1 tumor suppressor. The next most common alteration was loss of 17p13.1, including TP53. Both RB1 and TP53 deletions were a mixture of hemizygous loss and, less frequently, homozygous deletion. In addition, TP53 point mutations were found in 17% of tumors.23 In TP53-mutant cells, antagonism of MDM2 by nutlin-3a enhances chemosensitivity,180 suggesting the potential therapeutic utility of combining nutlin-3a with chemotherapy in TP53-mutant pleomorphic liposarcoma.

A third genetic alteration identified in a SNP analysis was the deletion of 17q11.2, including the tumor suppressor NF1. Among 24 pleomorphic liposarcomas, 9 (38%) had NF1 loss, including 1 case of a homozygous deletion and 2 cases of a mutation of the nondeleted allele.23 Because loss of NF1 function appears to activate the RAS and mTOR pathways, the frequent NF1 aberrations suggest that MEK or mTOR inhibitors may have clinical utility.

Миксофибросаркома (myxofibrosarcoma) и недифференцированная плеоморфная саркома (злокачественная фиброзная гистиоцитома)

Pathologists now regard myxofibrosarcoma (MFS) as a distinct tumor type with clearly defined criteria for diagnosis. Undifferentiated pleomorphic sarcoma (UPS), however, is less well defined, and it remains controversial whether it represents either (1) a pleomorphic sarcoma showing fibroblastic or myofibroblastic differentiation, and thus sharing a common set of genomic alterations with MFS, or (2) an end-stage undifferentiated morphologic pattern with genomic alterations distinct from those of MFS. In favor of the former conclusion, a large-scale multiplatform The Cancer Genome Atlas (TCGA) genomic analysis has shown that UPS and MFS are largely indistinguishable across multiple platforms.

Миксофибросаркома

MFS, also known as myxoid variant of malignant fibrous histiocytoma, is a malignant fibroblastic lesion with variably myxoid stroma (at least 10%) composed of hyaluronic acid and solid sheets of spindled and pleomorphic tumor cells. Karyotypes tend to be highly complex, often with multiple numerical and structural rearrangements and with chromosome numbers in the triploid or tetraploid range.183–185 No consistent chromosomal aberration has emerged. In general, karyotype complexity is greater in high-grade lesions and in recurrences.

In a SNP array analysis of 38 MFSs, approximately 55% harbored amplification of chromosome 5p,23 which contains RICTOR (a binding partner of mTOR), CDH9, and LIFR. Other amplified regions included several discontinuous loci on 1p and 1q spanning PI4KB (phosphatidylinositol 4-kinase beta), ETV3 (a transcriptional repressor), and MCL1. MCL1, an antiapoptotic gene, was overexpressed in these tumors. MFS also harbored deletions of tumor suppressors including CDKN2A (encoding the p16INK4A and p14ARF cell cycle inhibitory proteins) and CDKN2B (encoding the cell cycle inhibitor p15Ink4b), RB1, TP53, NF1, and the phosphatase PTEN, leading to extensive loss of function.23 A recent gene expression study of 64 primary high-grade MFSs identified the critical role of integrin-α10 for MFS growth and survival.186 High expression of the corresponding gene, ITGA10, was most significantly associated with worse outcomes among all genes in the poor-prognosis signature. Integrin-α10 promotes cell growth and migration by activating TRIO and RICTOR, which are coamplified and overexpressed in approximately 50% of MFSs. Accordingly, inhibitors of the complexes those proteins regulate (RAC and mTOR) inhibit growth of MFS in vitro and in vivo, with the combination having greater efficacy than either drug alone.186 This work informed the phase II Alliance trial of the mTOR inhibitor MLN0128 in patients with MFS and UPS.

Недифференцированная плеоморфная саркома (злокачественная фиброзная гистиоцитома)

More than 50% of soft tissue sarcomas occurring in older adults are histologically pleomorphic and high-grade. Most have traditionally been classified as malignant fibrous histiocytoma (MFH).187,188 MFH was originally defined as a malignant pleomorphic spindle cell neoplasm showing fibroblastic and histiocytic differentiation. More recently, pathologists have accepted that this morphology may be shared by a wide range of malignant neoplasms.189 In many sarcomas previously classified as pleomorphic MFH, careful immunohistochemical and histopathologic analyses revealed specific lines of differentiation allowing reclassification as MFS (30%), myogenic sarcoma (30%), liposarcoma (4%), malignant peripheral nerve sheath tumor (2%), or soft tissue osteosarcoma (3%), whereas approximately 30% had no specific differentiation or were myofibroblastic.181 The term undifferentiated pleomorphic sarcoma is now reserved for pleomorphic sarcomas that show no definable line of differentiation by current technology.

This change in diagnostic criteria complicates evaluation of the genetic basis of UPS. Among the more than 60 cases in the Mitelman Database of Chromosome Alterations in Cancer described as storiform or pleomorphic MFH or MFH not otherwise specified, the karyotypes are highly complex. Most have chromosome numbers in the triploid or tetraploid range, but a few are near haploid.190–194 Telomeric associations, ring chromosomes, and dicentric chromosomes are common. A comparative genomic hybridization (CGH) study of 33 tumors, 25 of which were UPSs as currently defined, found numerous copy number changes. The most frequent (found in 50% to 65% of tumors) were gains in chromosome 1p (1p33-p32, 1p31, and 1p21), 1q21, and 20q13 and losses in 1q41, 2q36-q37, 10q25-q26, 13q13-q14, 13q14-q21, and 16q12.195 Mutations and/or deletions of TP53, RB1, and INK4a have been suggested to be drivers of oncogenesis. High-level amplification of cyclin E1 CCNE1, the transcription factor VGLL3, or YAP1 has been detected in approximately 10% of UPSs and MFSs, correlating with significant overexpression of these genes compared to other high-grade sarcoma types.1,201 VGLL3 and YAP1 are members of the Hippo signaling pathway that drive proliferation, so this pathway may represent a promising therapeutic target in these sarcomas.

The stemlike tumor-initiating cells isolated from UPS show activation of both the Hedgehog and Notch pathways. Inhibition of these pathways in UPS xenograft models decreased the proportion of stemlike cells and suppressed tumor self-renewal. Thus, targeting signaling pathways activated in a small subpopulation of tumor- initiating cells may be a promising approach for these undifferentiated tumors.

A recent multicenter phase II trial of the anti–programmed cell death protein 1 (PD-1) antibody pembrolizumab found a 40% response rate in patients with advanced UPS. This efficacy makes sense in light of the correlation between dendritic cell numbers and better outcomes revealed by a TCGA analysis using mRNA gene signatures. Similarly, UPS and MFS had the highest median expression of the known druggable immune microenvironment markers B7-H3, TGFB1, and TIM3 compared to other complex sarcoma types.1 Future studies should examine whether the nature of the immune infiltrate in UPS can serve as a biomarker for response to anti–PD-1 therapy.

Лейомиосаркома

Leiomyosarcoma is defined as a malignant tumor with evidence of smooth muscle differentiation. Karyotypes tend to be complex, with amplifications, gains, and losses involving multiple chromosomes.204–206 Frequently observed aberrations include losses of 1p12-pter, 2p, 13q14-q21 (including RB1),207 10q (including PTEN),208 and 16q and gains of 17p, 8q, and 1q21-31; these have been associated with aggressive clinical behavior. The myocardin (MYOCD) gene on 17p, associated with smooth muscle differentiation, is significantly amplified and overexpressed in retroperitoneal and extremity tumors.1,209 Knockdown of MYOCD in leiomyosarcoma cell lines harboring this amplification decreases smooth muscle differentiation and inhibits cell migration.210 In an analysis of copy number alterations in 27 leiomyosarcomas,23 deletions, which were more common than amplifications, encompassed tumor suppressors such as TP53, RB1, BRCA2, and FANCA (the latter two genes’ products are both involved in DNA damage repair). The most prominent changes were chromosome 10 deletions (50% to 70% of cases) (see Fig. 87.1). The TCGA analysis confirmed these findings; mutations of TP53, RB1, and PTEN were detected in 50%, 15%, and 5% of samples, respectively. Other shared features of leiomyosarcoma were elevated miR-143 and miR-145 expression, low mRNA expression of inflammatory response genes, low leukocyte fraction as revealed by methylation analysis, and high AKT pathway scores by reverse phase protein array (RRPA) analysis.1

Indeed, genetic inactivation of PTEN (human 10q23.21) in smooth muscle in mice recapitulates human leiomyosarcoma, suggesting that 10q loss is an early tumorigenic event. Moreover, partial inactivation of PTEN and TP53 in the smooth muscle lineage in mice results in the development of high-grade pleomorphic sarcomas and leiomyosarcomas with complex karyotypes.212 The sarcomas deficient in both PTEN and TP53 showed upregulated Notch signaling and a greater metastatic potential, which could be attenuated by a γ-secretase inhibitor. In addition to PTEN inactivation, we identified homozygous deletions in MTOR. Because PTEN is a repressor of Akt, both of these events suggest a critical role for aberrant Akt-mTOR signaling in leiomyosarcoma. mTOR inhibitors such as everolimus (RAD001) and temsirolimus have shown some efficacy in patients with leiomyosarcoma in clinical trials.

RB1 deletion is common in leiomyosarcomas, with 70% harboring heterozygous deletions and 8% harboring homozygous deletions. A role for RB1 deletion in leiomyosarcoma fits with the high incidence of leiomyosarcoma in individuals with hereditary retinoblastoma.

Злокачественная опухоль периферической нервной оболочки

Злокачественные опухоли оболочки периферических нервов (MPNST) представляют собой высокоагрессивные саркомы мягких тканей, которые могут возникать спорадически в общей популяции, но гораздо чаще встречаются у пациентов с нейрофиброматозом 1 типа (NF1), синдромом наследственной предрасположенности к опухоли, вызванным гетерозиготными мутациями NF1 гена или у пациентов после лучевой терапии. MPNST у NF1 пациентов обычно происходят из нейрофибром. Пожизненный риск развития MPNST у NF1 пациентов составляет 8-13%, в отличие от 0,001% риска в общей популяции.

NF1 ген вовлечен как в спорадическую, так и в NF1-ассоциированную MPNST. Приблизительно 70% спорадических и NF1-ассоциированных MPNST демонстрируют моноаллельные или двуаллельные потери в NF1 локусе в 17q. NF1 кодирует протеин нейрофибромин, который ускоряет Ras-GTP гидролиз и, таким образом, негативно регулирует Ras. У индивидумов с NF1 нейрофибромы развиваются, когда неизвестный тип клеток в линии шванновских клеток теряет оставшийся функциональный NF1 ген, что ведет к потере нейрофибромина и последующей активации Ras сигналинга. Однако мало что известно о генетических изменениях, которые ведут к прогрессированию нейрофибром в MPNST у NF1 пациентов, или о молекулярных реакциях, которые контролируют саркомагенез в спорадических и радиация-ассоциированных MPNST.

Both sporadic and NF1-associated MPNSTs display complex karyotypes and clonal chromosomal aberrations. In CGH analyses of MPNSTs, the most frequent minimal regions of gain were 1q24.1-q24.2, 1q24.3-q25.1, 8p23.1-p12, 9q34.11-q34.13, and 17q23.2-q25.3.228 The 17q gain was associated with poor survival and overexpression of genes previously implicated in cancer, including topoisomerase 2α (TOP2A), ETV4, ERBB2, and survivin (BIRC5).228–230 Other frequent alterations include rearrangement or loss of 9p21 and 13q14, inactivating CDKN2A and RB1, respectively. In a high-resolution CGH analysis of NF1-associated MPNST, the most frequently deleted locus (33% of cases) encompassed the CDKN2A, CDKN2B, and MTAP genes on 9p21.3.224 A recent comprehensive genomic analysis of 15 MPNSTs from 12 patients identified somatic alterations of CDKN2A in 81% of all MPNSTs. These studies implicate the p16INK4A-RB1 pathway in MPNST pathogenesis.

Other studies have implicated the p19ARF-MDM2-p53 and epidermal growth factor receptor (EGFR) pathways in MPNST oncogenesis. TP53 (on 17p13) is frequently mutated or deleted in MPNST. The mentioned comprehensive analysis also revealed mutually exclusive loss-of-function somatic alterations in EED or SUZ12 in 80% of MPNST, with similar rates across subtypes.231 EED and SUZ12 are core components of the Polycomb repressive complex 2 (PRC2), which cooperates with EZH1 and EZH2 to regulate methylation of K27 of histone H3.235 MPNSTs with homozygous alterations in EED or SUZ12 showed complete loss of trimethylation of histone H3 at lysine 27 (H3K27me3) and aberrant activation of multiple PRC2-repressed homeobox master regulators.231 H3K27me3 loss was associated with progression of neurofibroma to MPNST, suggesting that complete loss of PRC2 function and inactivation of CDKN2A in addition to NF1 loss may be critical cooperative events required to drive MPNST sarcomagenesis. Immunohistochemical staining for H3K27me3 serves as a powerful ancillary method for the diagnosis of all subtypes of MPNST.

Building on these observations, MPNST driver genes were sought in a Sleeping Beauty (SB) transposon-based somatic mutagenesis screen in mice bearing transgenes that confer a somatic loss of p53 function and/or overexpression of human EGFR.237 EGFR overexpression and p53 mutation cooperated to significantly increase neurofibroma and MPNST formation, effects that were enhanced by SB mutagenesis. The mutations found in the highest percentage of MPNSTs in this genetic screen were in PTEN and NF1, suggesting that these mutations cooperate to drive MPNST development. In addition, the researchers found that Forkhead box R2 (FOXR2) acts as a protooncogene by promoting anchorage-independent MPNST cell growth and tumorigenicity.237

NF1-deficient Schwann cells derived from human neurofibromas show activation of mTOR, even in the absence of growth factors. Furthermore, these cells, transformed mouse cells in which NF1 is knocked down, and tumors in a genetic mouse model of NF1 are highly sensitive to the mTOR inhibitor rapamycin,238 which reduces activation of the mTOR target cyclin D1. These results demonstrate that mTOR inhibitors may be an effective targeted therapy for patients with neurofibromatosis and MPNST.

The chemokine receptor CXCR4 is highly expressed in NF1-associated MPNST, and CXCR4, along with its ligand CXCL12, promotes MPNST growth by stimulating cyclin D1 expression. The highly specific CXCR4 antagonist AMD3100 decreased growth of MPNST cell lines, allografts, and tumors in transgenic mouse models of spontaneous MPNST. These results suggest that targeting autocrine cell cycle progression regulated by CXCR4/CXCL12 may represent a promising therapy for MPNST.239

Inactivation of NF1 and the consequent activation of the Ras/Raf/MAPK pathway in most MPNSTs support targeting B-Raf with the B-Raf tyrosine kinase inhibitor sorafenib. MPNST cell lines are sensitive to sorafenib at nanomolar concentrations, mediated by suppression of cyclin D1 and hypophosphorylation of RB1, resulting in G1 cell cycle arrest.240 A phase II trial of sorafenib in patients with metastatic MPNST was recently completed. Although none of the 12 patients with MPNST had RECIST responses, 3 had stable disease and 2 had regression or cystification of metastatic disease.

Ангиосаркома

Angiosarcomas are rare vascular malignancies of endothelial differentiation that arise either de novo or secondary to radiation therapy or chronic lymphedema. Angiosarcomas are characterized by upregulation of vascular- specific receptor tyrosine kinases, including TIE1, KDR (VEGFR2), TEK (TIE2), and FLT1 (VEGFR1).242 Full sequencing of these genes identified mutations in KDR in 10% of angiosarcoma patients, all of whom had tumors in the breast, with or without prior radiation.243 KDR mutations were associated with strong KDR protein expression, although no gains in KDR copy number were detected. KDR mutants expressed in COS-7 cells showed ligand-independent activation of the kinase, which was inhibited with specific KDR inhibitors.242 In contrast with other sarcoma types, angiosarcoma showed downregulation of VEGF ligand expression (VEGFA and VEGFB), in keeping with the constitutive activation of KDR independent of exogenous VEGF.242 Whole- genome, whole-exome, and targeted sequencing of 39 de novo and radiation- and lymphedema-associated angiosarcomas found recurrent mutations in PTPRB and PLCG, two angiogenesis signaling genes, in 38% of tumor samples.244 These results provide a basis for the activity of VEGFR-directed therapy in angiosarcoma.

Despite their similar morphology, de novo angiosarcoma and radiation- or lymphedema-associated angiosarcoma appear to be genetically distinct. An array-CGH study identified a set of recurrent genetic abnormalities in radiation- and lymphedema-associated but not de novo angiosarcomas.245,246 The most frequent recurrent changes were high-level amplifications on chromosome 8q24.21 (MYC, 50%), followed by amplification on 10p12.33 (33%) and 5q35.3 (FLT4, 11%). That high-level amplification of MYC is a distinctive feature of radiation- and lymphedema-associated angiosarcomas was confirmed by FISH247,248 but was not found to predispose patients to higher grade morphology or increased proliferation. Recurrent rearrangements and mutations in the transcriptional repressor CIC were found to occur preferentially in de novo soft tissue and visceral angiosarcomas of young adults and were associated with worse survival.243 In addition, a recurrent novel gene fusion of the nucleoporin 160 (NUP160)gene with the nucleobase transporter SLC43A3, was detected in 9 of 25 de novo scalp angiosarcoma specimens and associated with more rapid locoregional tumor progression. The NUP160-SLC43A3 fusion was found to be oncogenic, as transfected fibroblast cell lines produced angiosarcoma-like tumors in mouse xenografts.

Опухоли костей и хрящей

Опухоли хрящей

Cartilaginous tumors, which are the most common primary bone tumors, all produce chondroid matrix, at least focally.144 The most common benign tumors are enchondromas and osteochondromas; they may represent precursors to chondrosarcoma.

Энхондрома

Enchondromas may occur as solitary lesions in the metaphysis of bone or as multiple lesions, as is found in Ollier disease or Maffucci syndrome. Enchondromas have constitutively active hedgehog signaling, which blocks normal chondrocyte differentiation and drives proliferation. Heterozygous somatic mutations in PTHR1, which encodes the receptor for parathyroid hormone–like hormone (PTHLH), have been found in approximately 15% of patients with Ollier disease. PTHR1 mutation disrupts the normal Indian hedgehog–PTHLH feedback loop, leading to activated hedgehog signaling and presumably to pathogenesis of Ollier disease in some patients. The genetic deficit in Maffucci syndrome is unknown.

Остеохондрома

Osteochondroma is a cartilage-capped bony outgrowth from the metaphysis of bone. Osteochondromas can be solitary or multiple, as in multiple osteochondroma syndrome (MO), which is caused by dominant germline mutations in the tumor suppressor exostosin genes EXT1 (located on 8q) and EXT2 (located on 11p).255,256 The remaining wild-type allele is lost in approximately 38% of sporadic osteochondromas and in 25% of hereditary osteochondromas.257,258 Biallelic inactivation of EXT1 recapitulates the morphology of human MO in mice.259,260 EXT1 and EXT2 encode glycosyltransferases that catalyze elongation of heparan sulfate on proteoglycans.261 Defective heparan sulfate synthesis affects the diffusion of hedgehog ligands in the extracellular space, which in turn enables growth plate chondrocyte growth in the wrong direction262 and interferes with ossification of the perichondrium to facilitate osteochondromagenesis.

Хондросаркома

Chondrosarcoma is a malignant cartilaginous matrix–producing tumor with diverse morphologic features. It tends to occur in older patients with a peak incidence at ages 40 to 70 years. Low-grade chondrosarcomas rarely metastasize but may progress to high-grade chondrosarcomas, which metastasize in approximately 70% of patients. Some chondrosarcomas arise from benign lesions (enchondromas or osteochondromas); these are termed secondary chondrosarcomas.144

A prominent genetic alteration in cartilaginous tumors is somatic mutation of isocitrate dehydrogenase (IDH) genes. IDH1 and IDH2 mutations are found in approximately 60% of cartilaginous tumors (56%).Among cartilaginous tumors, IDH mutations appear to be confined to enchondromas, periosteal chondrosarcomas, and central (intramedullary) chondrosarcomas of conventional or dedifferentiated histology. IDH mutations have not been found in secondary peripheral chondrosarcomas, which instead share molecular characteristics with osteochondromas, or in osteochondromas and osteosarcomas, including chondroblastic osteosarcomas. Thus, mutation detection may aid diagnosis.

The common IDH mutations in chondrosarcoma affect IDH1 R132 (approximately 90% of IDH-mutant cases) and IDH2 at the homologous position, R172 (approximately 10%). These mutations are also common in glioma and acute myeloid leukemia. The mutations disrupt the enzymes’ ability to convert isocitrate to α-ketoglutarate, which in turn increases levels of HIF1A, a subunit of a transcription factor that facilitates tumor growth in hypoxic environments. HIF1A is highly expressed in high-grade central chondrosarcoma.

IDH1 R132 and IDH2 R172 mutations confer on the enzymes a new ability to convert α-ketoglutarate to (R)-2-hydroxyglutarate (2HG), resulting in markedly elevated levels of 2HG. 2HG itself appears to be oncogenic. 2HG induces CpG island DNA hypermethylation in low-grade gliomas, acute myeloid leukemia, and chondrosarcoma containing IDH1 and IDH2 mutations. Across all these cancer types, increased production of 2HG is associated with inhibition of DNA demethylation, leading to a hypermethylation phenotype that affects genes of the retinoic acid receptor activation pathway and, in chondrosarcoma, genes implicated in stem cell maintenance and differentiation. Expression of an IDH2 mutant in mesenchymal progenitor cells is oncogenic in vitro and in vivo and leads to DNA hypermethylation and a differentiation block, which is reversible with DNA demethylating agents. Thus, for patients with IDH-mutant chondrosarcomas, there may be therapeutic potential for demethylating agents or for selective inhibitors of the mutant IDH proteins, such as an IDH1 R132 mutant inhibitor currently in development.

A whole-exome sequencing study278 showed that 37% of chondrosarcomas have insertions, deletions, or rearrangements of COL2A1, which encodes the alpha chain of type II collagen fibers—the major collagen constituent of articular cartilage. Such mutations may interfere with the production of mature collagen fibrils. They also identified mutations in IDH1/2 (59%), TP53 (20%), and genes of the RB1 pathway (33%) and hedgehog pathway (18%).

Other potentially targetable abnormalities in chondrosarcoma include the hedgehog pathway, IGF pathway, CDK4, MDM2, phosphatidylinositol 3-kinase (PI3K)/mTOR, and SRC.279 Hedgehog signaling in primary central chondrosarcoma is constitutively activated; activation is thought to occur early in tumorigenesis and maintains chondrocytes in a proliferative state. Hedgehog inhibitors such as cyclopamine and triparanol inhibit chondrosarcoma cell growth in vitro and in xenografts to varying degrees. These results suggest that patients with conventional chondrosarcoma may benefit from hedgehog pathway inhibitors, such as the selective smoothened inhibitor vismodegib (GDC-0449, recently approved for basal cell carcinoma). Unlike primary central chondrosarcoma, secondary peripheral chondrosarcoma actually has decreased hedgehog signaling,284 suggesting that hedgehog blockade might not be effective for the peripheral subtype. GLI family zinc finger 2 (GLI2) overexpression in a mouse model induces benign cartilage tumors, whereas GLI2 overexpression combined with p53 (TRP53) deficiency results in the development of chondrosarcoma-like tumors by negatively regulating apoptosis through activated IGF signaling.285 Thus, inhibition of IGF signaling may represent an attractive therapeutic target. Kinome profiling has demonstrated SRC pathway activation in chondrosarcoma cell lines, and indeed, the SRC inhibitor dasatinib decreased chondrosarcoma cell viability.286

Остеосаркома

Остеосаркома является первичным злокачественным новообразованием кости, которое чаще всего возникает в длинных костях из остеоид-продуцирующих клеток, прилегающих к ростовым пластинам, обычно у детей и подростков. Остеосаркомы характеризуются сложными альтерациями числа ДНК копий с небольшим количеством рекурентными альтерациями и высоким уровнем генетической нестабильности. Большинство остеосарком являются спорадическими. Факторы риска включают предшествующую лучевую терапию и химиотерапию. Семейные синдромы, ассоциированные с остеосаркомой, включают синдром Ротмунда-Томсона (Rothmund-Thomson), синдром Ли-Фраумени (Li-Fraumeni) (ассоциированный с мутацией TP53) и наследственную ретинобластому.

Children with hereditary retinoblastoma are up to 1,000 times more likely to develop osteosarcoma compared with the general population. Osteosarcoma appears to nearly universally arise from G1/S deregulation by RB1 loss (found in up to 80% of primary osteosarcomas), CDK4 amplification, or CDKN2A (encoding p16INK4A) loss. The latter alterations are collectively observed in approximately 20% of osteosarcomas; RB1 and CDKN2A alterations are mutually exclusive.

Another gene significantly associated with osteosarcoma is TP53. The frequency of somatic TP53 mutations in osteosarcomas ranges from 19% to 38%,295,296 and TP53 mutations are associated with high levels of genomic instability.296 An additional 5% to 10% of osteosarcomas harbor amplification of MDM2.297 TP53 mutations in osteosarcomas are frequently associated with hypermethylation of HIC1 (hypermethylated in cancer 1). Specifically, HIC1 was hypermethylated in 12 of 29 (41%) human osteosarcomas with TP53 mutations compared to 2 of 24 (8%) tumors without TP53 mutations (P = .007).298 Experiments in mice with heterozygous deletion of both HIC1 and TP53 have demonstrated cooperation between these two genes in osteosarcomagenesis.298 All these results suggest that loss of HIC1 function may complement TP53 mutations in the development of a subset of human osteosarcomas.

CGH of conventional osteosarcoma, which accounts for 75% of osteosarcomas, has shown that 1p36, 6p21, 8q24, 16p13, 17p11, and 19p13 are recurrently gained or amplified and 2q, 6p, 8p, 10p, and 17p13 are recurrently lost or deleted. High copy gains and/or amplification of chromosome arms 6p, 8q, and 17p are frequently reported and are believed to confer a more aggressive disease course.304 High copy number gain of the MYC oncogene at 8q24 was found in 43% of osteosarcomas. Other potential oncogenes on 8q24 include RECQL4 and EXT1. Germline RECQL4 helicase-inactivating mutations lead to the Rothmund-Thomson syndrome306 and EXT1 mutations to multiple exostoses307; both syndromes include strong predisposition to osteosarcoma.

Будущие направления: секвенирование следующего поколения и функциональные скрининги

New targeted therapies are desperately needed for the approximately 5,800 patients who die of sarcoma each year in the United States. A key challenge will be to identify the alterations that drive sarcomagenesis for each subtype. Once these driver alterations are identified, new small molecules to target them can be sought through functional screens, high-throughput compound screens, combinatorial chemistry, and structural biology.

Next-generation sequencing can vastly expand our knowledge of the mutations, translocations, epigenetic alterations, and aberrant signaling pathways associated with specific sarcoma types and subtypes. Concurrent massively parallel sequencing and integrative analysis now enable incredibly deep analysis of the sarcoma genome, including copy number alterations, structural rearrangements, expressed coding mutations, alternative splice forms, digital expression, chimeric/fusion transcripts, and DNA methylation status. For example, on a single tumor sample, it is now possible to resequence all of the protein-coding regions of the genome, generate detailed transcriptome profiles (RNA-seq), and perform genome-wide profiling of epigenetic marks and chromatin structure (using ChIP-seq, methyl-seq, or DNase-seq). In gene expression studies, microarrays are being replaced by RNA-seq, which provides far more precision on transcript levels, alternative splicing, and sequence variation in identified genes and can identify rare transcripts without prior knowledge of a particular gene. Integrating seq-based methods with high-throughput RNA interference screens in cell lines harboring the genetic alterations found in human sarcoma samples will substantially enhance our ability to identify and target the signaling pathways and proteins driving sarcomagenesis.

“Smart” compounds, reflecting the three-dimensional structure of the targeted protein, may then be designed using high-throughput biochemical screens capable of identifying low-affinity compounds, together with sensitive biophysical techniques such as nuclear magnetic resonance, x-ray diffraction, and protein-ligand cocrystallography. The resulting physicochemical data should facilitate virtual screening of library structures for their three-dimensional fit with pharmacophores and speed the discovery of new selective small- molecule inhibitors targeting the signaling pathways essential for sarcoma growth and survival.

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