ACRT Journal

NF-1 associated MPNST

Malignant peripheral nerve sheath tumors (MPNSTs) are rare sarcomas, encompassing only 2% to 4% of all soft tissue sarcomas [16, 17]. The incidence of these tumors is 1:100000 in the general population. MPNST is a malignant tumor frequently associated with NF1; the risk of developing it during life is 8-13% in NF1 patients [16, 18, 19]. A slight predominance in males has been reported [20-23]. The peak incidence of these tumors differs between NF1-related tumors and sporadic tumors. NF1 patients have an incidence peak in the third and fourth decades, while sporadic tumors are usually diagnosed in the sixth decade. Few data are available on prognostic factors and on the best therapy for this kind of tumor. Still, maximal safe resection is crucial in both adult and pediatric cases. Reports on pediatric intracranial sarcomas, and in particular, MPNST, are

limited to single institutional case reports or small, single institutional case series, and in all of these, a complete tumor resection promotes the chances of patient survival. Furthermore, the vast majority of long-term survivors have received multimodal treatment to achieve local tumor control with radiotherapy and ifosfamide, carboplatin, and etoposide chemotherapy [24-26]. Various small targeted molecules were tested in small cohorts of MPNST patients, but trials have not yet progressed in treating this disease [8]. The survival of patients with metastatic or unresectable disease is poor, with a median progression-free survival of approximately four months and an overall survival of 13 months [9].

NF-1 associated MPNSTs Target Therapy

NF1 gene mutations are used as a diagnostic marker for neurofibromatosis type 1. Genetic testing can identify these mutations, aiding in early diagnosis and management. Understanding the pathways and mechanisms involving neurofibromin can help in developing targeted therapies for conditions resulting from its dysfunction, particularly tumors associated with NF1. Currently, no systemic drugs are specifically approved for patients with advanced MPNST, and the existing drugs used in soft-tissue sarcoma generally have a modest activity in this specific histologic subtype. Consequently, in those patients, the prognosis continues to be poor. NF1 individuals carry mutations in the NF1 gene, which are responsible for functional alterations in Neurofibromin 1 and consequently in the MAPK pathway. As detailed before, these subjects are more predisposed to develop neoplasms. With the advent of next-generation sequencing, other kinds of mutations in many genes that cooperate with the NF1 gene have been identified [27]. In particular, the loss of the tumor suppressor genes CDKN2A and/or PTEN, which control cell cycle progression [10, 27]; but, also oncogene activation, important for tumor progression, like PDGFRA, MET, c-kit, and epidermal growth factor receptor (EGFR) is frequently observed in MPNST [28, 29]. Another tumor suppressor gene involved in MPNST is TP53, although there are conflicting opinions regarding the frequency of mutations [30]. Moreover, epigenetic causes have also been found in the pathogenesis of the NF1-associated MPNST, such as mutations in SUZ12 or EED, which lead to inactivation of these two must components of polycomb repressive complex 2 (PRC2) [27].

Moreover, this kind of tumor is characterized by a high heterogeneity, meaning that different cells within the same tumor may respond differently to treatment. Thus, identifying a single target for therapy is therefore challenging, and, due to the rarity of MPNST, conducting large randomized trials is almost impossible. Since RAS proteins to be active should be associated with the plasmatic membrane, and this association is given by farnesylation, farnesyl transferase enzymes have been targeted in NF1-dependent MPNST cells, but in clinical trials, this strategy has not been successful [31]. An alternative to RAS inhibition is to target downstream of activated RAS. Sorafenib, a small-molecule RAF kinase and tyrosine kinase inhibitor has been shown to inhibit MAPK signaling and cell growth, inducing the cell-cycle arrest in G0/G1 phase in MPNST cell lines through B-Raf dependent inhibition [32]. However, sorafenib showed no significant response in a multicenter phase II trial, with overall survival of treated MPNST patients similar to controls [33].

MEK (MAP kinase) proteins are a family of kinases involved in the Ras-Raf-MEK-ERK (extracellular signal-related kinase) signaling pathway. This induces growth and survival in mammalian cells, activating alterations that drive cancer transformation. MEK proteins are kinases that phosphorylate in serine/threonine and tyrosine residue. They are classified into two isoforms, MEK1 and MEK2, ubiquitously expressed. Different mitogenic stimuli can bind to specific tyrosine kinase receptors (RTK), leading to its dimerization and autophosphorylation in tyrosine residues at the C-terminal domain. When activated, these activated receptors recruit and phosphorylate adaptor proteins such as Grb2 and SOS, which interact with Ras that function as molecular switcher GDP in GTP, causing its activation [34, 35]. Once activated Ras recruits and activates Raf kinases, which in turn interact and activate MEK1/2, which finally activates ERK1/2. Differently from the others, these latter have many cytosolic and nuclear substrates regulating different cellular processes, such as cell proliferation, survival, differentiation, motility, and angiogenesis [36]. While MEK 1/2 has not been considered an oncogene as long as activated MEK mutations are relatively rare in human tumors, MEK constitutive activation results in cellular transformation, and it is implicated in the progression of human tumors [37]. So, since 1995 new molecules   that   inhibit   MEK   activity   have progressed   into   clinical   trials (e.g., refametinib, selumetinib, trametinib, cobimetinib). MEK inhibitors are commonly classified as molecules competing directly for the ATP–binding site (ATPcompetitive), and the ATP non- competitive inhibitors bind to a unique allosteric site adjacent to the ATP site, ensuring their high specificity [38]. Despite about thirteen MEK inhibitors being tested and continuous screening or molecular modifications being made to improve the inhibitory activity, only trametinib (GSK1120212), a selective inhibitor of MEK 1/2, has been proven effective in a phase III clinical study.

MEK inhibitors: trametinib

Trametinib (GSK1120212, JTP-74057) is an allosteric inhibitor of MEK kinase. It falls into the second-generation non-competitive category, with potent activity against purified MEK 1/2 kinases [39]. In vitro preclinical studies demonstrate inhibition of tumor cell growth by cell-cycle arrest, through the de-phosphorylation of ERK 1/2. A trametinib phase I clinical trial on 206 patients with advanced solid tumors shows finite toxicities and a global response rate of 10% with 33% on B-Raf mutant melanoma [40]. These results bring researchers to carry out several phase II/III clinical trials of trametinib alone or in combination with other agents, especially on V600E/K B-Raf mutations in advanced melanoma, but also on advanced non-small cell lung cancer (NSCLC) which showed the same mutations. Results of these trials point out Trametinib extends the progression-free survival (PFS) of patients to 4.8 months, giving an 81% overall survival in patients with B-Raf mutated metastatic melanoma. Furthermore, combinations of Dabrafenib and Trametinib led to improved progression-free survival compared to single treatment [41]. In a randomized phase II clinical trial on NSCLC, Trametinib demonstrated similar efficacy to docetaxel in KRAS-mutant patients with moderate activity as a monotherapy [42]. Hence, the authors conclude that trametinibbased combination regimens may show improved efficacy. Only more recently, trametinib was used for the treatment of MPNSTs too. In vitro, results demonstrated the reduction of the MAPK pathway thanks to significant inhibition of p-ERK and downstream cyclin D1 levels [43]. Unfortunately, in vivo, tumor growth inhibition was transient. It resulted in resistance and reactivation of target pathways. In contrast, the combination therapy with other targets of interest in MPNST pathogenesis (mTOR, MNK, BRD, MET) demonstrated tumor regression with synergistic responses [44, 45]. A meta-analysis of 8 studies evaluates the effectiveness and safety of trametinib in NF1-related nerve tumors indicating a satisfactory stabilization but an inefficiency to shrink tumors in NF1-related nerve tumors [46]. Even though the safety profile of trametinib is satisfactory, there is no evidence that single-agent MEKi is effective in treating MPNST. Indeed, in NF-1 biallelic loss, it is capable alone to sustain a complete response to the metastatic recurrence in an adolescent patient in only 3 months of treatment. After 15 months after the second relapse, there was no recurrence of the primary chest lesion since the second surgery or at any other site [47].

Multitarget TK inhibitors: pazopanib

A significant role in the development of numerous human malignancies is carried out by tyrosine kinases Receptors (RTKs), such as vascular endothelial growth factor receptors (VEGFRs), fibroblastic growth factor receptors (FGFRs), and plateletderived growth factor receptors (PDGFRs). In the last decade, many tyrosine-kinase inhibitors (TKIs) targeting osteosarcoma oncogenic pathways have been explored [48, 49]. Several studies have suggested that pazopanib may be effective against MPNST. Pazopanib is a tyrosine kinase inhibitor that blocks the vascular endothelial growth factor receptors (VEGFR-1/2/3), plateletderived growth factor receptors (PDGFR-α/-β), and c-kit. In a phase II study on patients with relapsed or refractory advanced Soft Tissue Sarcoma (STS), Pazopanib showed interesting activity prolonging progression-free and overall survivals and was well tolerated in patients with relapsed, advanced STS [50]. A phase III clinical study was done randomly on 372 patients assigned to receive pazopanib or placebo, with no subsequent cross-over. Results indicated that the survival progression-free was a 3-fold increase in PFS over placebo. Overall survival grew in 2 months with pazopanib [51]. Since MPNST manifests in subjects who have lost the activity of NF1 or are deleted, and this lack leads to Ras activation, which in turn increases cells’ dependence on the VEGF-VEGFR pathway, pazopanib may be effective against MPNST. Yoshihiro Nishida et al. examining the efficacy of pazopanib in a phase II clinical trial on 12 patients with advanced MPNST, demonstrated the effect of pazopanib against tumors with higher grades of malignancy, and thereby prolong life [52].

In light of this evidence, it is clear that monotherapy is not the right choice because the clinical activity of TKIs was relatively short due to primary or acquired resistance related to the activation of RTK downstream pathways [53], such as RAS/RAF/ MAPK and/or PI3K-Akt-mTOR signaling pathways. Strategies involving combination therapies are being explored as potential approaches to treat MPNST. A vertical blockade combining upstream with the RTK inhibitor Pazopanib and downstream with the MEK inhibitor Trametinib should greatly suppress MAPK signaling. The antitumor activity was studied both in vitro and in vivo, demonstrating a synergistic antitumor activity through the strengthening of apoptosis and decreased ERK1/2 and Akt signaling activation in osteosarcoma, thyroid cancer, and advanced cholangiocarcinoma [54, 55]. Moreover, a phase Ib/II clinical study on the safety and efficacy of target therapy with Pazopanib and Trametinib was performed both in advanced solid tumors and differentiated thyroid cancers and in 25 patients with advanced soft tissue sarcoma. Although, no patient performed a complete response, a stable disease was obtained in the 48% of cases. Indeed, results demonstrated a progression-free survival median of 2.27 months (95% CI). The 4-month PFS rate was gained in 21.1% of cases with a median overall survival of 9.0 months [24, 56].

Case Report

An 18-year-old man presented with a massive intracranial hemorrhage and underwent surgical removal of the hematoma. The first diagnostic question was initially arteriovenous malformation, a widespread feature in NF-1 patients since 18F-fluorodeoxyglucose Positron Emission Tomography (18FDG-PET) was negative [57]. However, the final pathological diagnosis revealed eMPNST arising from peripheral nerves localized in the right parietal lobe, with a very high proliferative index (Ki67>70%). The histological analysis also revealed a high heterogeneity of the tumor tissue with Vimentin positivity, partial positivity to CD10 and CD56, and isolated positivity to S-100, HMB-45, and Melan A. After diagnosis, the patient was referred to the Department of Oncology due to the multiple recurrences and invasive nature of the tumor. Adjuvant radiotherapy was administered with a dose of 50 Gray (Volumetric Modulated Arch Therapy using high energy 15 MV photons for 30 seconds) in 30 fractions over 6 weeks (1 per day for 5 days a week). Subsequently, ICE chemotherapy was initiated according to the protocol published by Lafay-Cousin et al. in 2016 [24], consisting of ifosfamide 3g/m2 intravenously (IV) over 3 hours on day 1 and day 2; etoposide 150 mg/m2 IV over 1 hour on day 1 and day 2, and carboplatin 500 mg/m2 IV over 2 hours on day 3. Chemotherapy cycles were given every 21-28 days based on the blood cell count. Before treatment cell count was: 6.63 10³ cells/ µl of white blood cells (WBC); 5.32x10⁶ cells/µl of red blood cells (RBC); 231x10³ cells/µl of platelets (PLT). After the first cycle, the blood cell count was: 1.26x10³ cells/μl of WBC; 4.63x10⁶ cells/µl RBC, and 112x10³ cells/μl PLT. Granulocyte colony-stimulating factor (PegfilGraStim 8mg) was administered to stimulate the proliferation of white blood cells. ICE therapy was interrupted after four cycles, due to a very low blood cell count (0.69x10³ cells/μl WBC; 4.02 10⁶ cells/µl RBC; 108x10³ cells/µl PLT), necessitating discontinuation of the treatment. At the end of all treatments, a brain NMR for potential recurrence was performed, and the results revealed a recurrence of the disease (Figure 2 A and C). Meanwhile, genomic sequencing was performed on primary cells derived from tumor tissue. It resulted in two point mutations in the NF1 gene: deletion of glutamic acid in position 740 (E740*) in cysteine- and serine-rich domain (CSRD), leading to a truncate neurofibromin, and lysin deletion in position 2257 (K2257*) in C-terminal domain (CTD domain), that produces a variant missing of C-terminal domain. As a consequence of these mutations, the amplification of the following genes was observed: CCND2; KRAS; FGF23; FGF6; and KDM5A. Based on genetic results, we decided to treat the patient with a combined targeted therapy including pazopanib 400 mg in association with trametinib 2 mg, in a single administration per day as an alternative systemic treatment. The treatment was highly tolerated, and blood cell count was normal (4.45x10³ cells/µl WBC; 4.40x10⁶ cells/μl RBC; 354x10³ cells/µl PLT). The only side effect was hair whitening. Serial disease response assessments by NMR imaging showed complete resolution of the lesion with a complete metabolic response 24 months after the start of the surgery (Figure 2).

Figure 2: Brain MRI with gadolinium: A e C) Sagittal and axial section of early follow-up MRI (6 months after surgery) revealed evidence of recurrence disease; B and D) Sagittal and axial section of 16 months later the combinatory therapy with pazopanib and trametinib.

Discussion

Little data is available on prognostic factors and effective therapy for this tumor, but in both adult and pediatric cases, maximal safe resection is crucial. Reports on pediatric intracranial sarcomas, and especially on MPNST, are limited to single institutional case reports, and in all cases, a complete tumor resection promotes the chances of patient survival. Furthermore, the majority of long-term survivors have received multimodal treatment to achieve local tumor control with radiotherapy and chemotherapy [24-26]. Here we describe the comprehensive and multidisciplinary approach required to treat a complex and aggressive intracerebral MPNST in an 18-year-old NF-1 patient. The poor prognosis, high rates of recurrence and metastasis, and genetic heterogeneity of MPNSTs make them particularly difficult to treat with monotherapy. MPNSTs often harbor mutations and alterations in key signaling pathways that induce intrinsic or acquired resistance to singleagent therapies. Thus, understanding the molecular basis of MPNST can lead to targeted therapies and personalized medicine approaches. In this contest, a vertical blockade combining upstream the RTKs inhibitor Pazopanib, and downstream with the MEK inhibitor Trametinib should greatly suppress MAPK signaling. The antitumor activity was studied both in vitro and in vivo, demonstrating a synergistic antitumor activity through the strengthening of apoptosis and decreased ERK1/2 and Akt signaling activation in osteosarcoma models [54]. Moreover, a phase Ib/II clinical study on the safety and efficacy of target therapy with Pazopanib and Trametinib was performed in patients with advanced sarcoma. Although, no patient performed a complete response, a stable disease was obtained in the 48% of cases. Indeed, results demonstrated a progression-free survival median of 2.27 months (95% CI). The 4-month PFS rate was gained in 21.1% of cases with a median overall survival of 9.0 months [56]. For the right management of this kind of malignancy, the network among neurosurgeons, pathologists, molecular biologists, and oncologists is crucial for significantly increasing overall survival. In this kind of network, the neurosurgeon is responsible for the surgical removal of the tumor, which is often the primary treatment for MPNST. Critical is the tumor complete resection for reducing the risk of recurrence, although the balance between the tumor removal and the preservation of nerve function is needed. The close work of pathologists with oncologists is important for diagnosing MPNST and determining its characteristics, which are essential for guiding treatment decisions and prognosis. Pathologists identify specific markers that can influence treatment options. They provide critical diagnostic information to oncologists for therapy decisions. Moreover, the interaction with molecular biologists is essential to reach, through advanced diagnostic techniques, the genetic and molecular characteristics of MPNSTs, including mutations, gene expression profiles, and molecular pathways involved in tumor growth and progression. This can help us to predict response and/or resistance to specific treatments. All these professional figures work together to develop and apply personalized targeted therapies. Combined target therapy represents a promising approach for treating MPNSTs. In particular, combining MEK and RTK inhibitors is promising for treating MPNSTs by targeting multiple critical pathways involved in tumor growth, survival, and progression. This approach could enhance anti-tumor efficacy, overcome resistance mechanisms, and improve patient outcomes. In this scenario, oncologists manage the overall treatment plan, including chemotherapy, radiation therapy, and novel therapeutic approaches. They monitor the patient’s response to treatment and adjust protocols as needed, integrating the surgical outcomes from neurosurgeons, pathologists’ diagnostic insights, and biologists’ molecular findings. Moreover, this network fosters research into new treatments and understanding of MPNST, potentially leading to innovative therapies, improved patient outcomes, and ultimately improved patient prognosis and quality of life.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Credit authorship contribution statement

Conceptualization: AF and MTG; Writing original: draft AF and MTG; Investigation: GT, AdB, FM, AC, SB; Methodology: GT, AdB, AA, VM, AC, FM; Formal analysis: AA, APezone; Data curation: AC, APezone, AP, MM, PdM; Writing review and editing: AP, AP, MG, MM, PdP and MTG; Supervision: AP, MG, PdM and MTG; Funding acquisition: MG and MTG.

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