Plexiform neurofibromas in NF1

Clinical aspects.

Neurofibromas are benign peripheral nerve sheath tumors characterized by unpredictable patterns of growth, variable cellular composition, and diverse appearances.1-5⇓⇓⇓⇓ Nearly all adults with NF1 will develop neurofibromas at some time during their lives. Classified as World Health Organization grade 1 tumors, such lesions may be present at birth or develop at any time during life. Clinically, neurofibromas may present as discrete tumors (dermal neurofibromas), diffuse tumors, plexiform neurofibromas, or tumors associated with spinal nerve sheaths (spinal neurofibromas). Neurofibromas are composed of neoplastic Schwann cells, perineural-like cells, and fibroblasts in a matrix of collagen fibers and mucosubstances.4 Growth of neurofibromas is initially along the course of nerve fibers. If the tumor arises from a relatively large nerve, it may be enclosed by the thickened epineurium and be confined to the nerve (a discrete lesion). Tumors arising from small nerves may spread diffusely into the dermis and soft tissue. Plexiform neurofibromas involve multiple nerves or fascicles and are expanded by tumor cells and collagen.

Solitary plexiform neurofibromas may occur in patients without other stigmata of NF1. In a recent review, 8 of 124 patients with plexiform neurofibromas had no other evidence of NF1.5 The pathogenesis of such lesions is unclear, but they may result from mosaicism of NF1 or a related gene. Although the majority of patients with localized forms of NF1 have no affected relatives with the disease, there are infrequent reports of patients with localized NF1 having children with findings diagnostic of NF1. Although genetic testing for some of the mutations of the NF1 gene exists, there is no evidence that such testing will be helpful in diagnosing NF1 in patients with isolated plexiform neurofibromas.

Discrete dermal neurofibromas tend to arise from a sensory nerve but may present as an exophytic or subcutaneous tumor. Dermal neurofibromas may cause discomfort or itching but are rarely associated with neurologic deficit. Their pattern of growth is often unpredictable, with periods of gradual sustained enlargement followed by apparent growth arrest. Hormonal influence may modulate their growth, as these tumors more frequently demonstrate increased growth during puberty and pregnancy. However, they do not transform into malignant tumors.6

Plexiform neurofibromas are slow-growing tumors, which may be present at birth or may become apparent later in life. Their incidence in patients with neurofibromatosis has not been well established, but they probably occur in anywhere between 25 and 50% of patients, with most series suggesting an incidence of 25 to 30%.2,3⇓ These tumors arise in various regions of the body, including the trunk, limbs, head, and neck. In all of these areas, they can cause dysfunction including cosmetic abnormalities, pain, and functional deficits. Plexiform neurofibromas can remain silent for many years and may be revealed only by imaging studies. Some tumors may grow to large sizes prior to clinical detection; in one study of 126 individuals 16 years of age or older with NF1, plexiform neurofibromas were found in the chest of 20% of patients and in the abdomen and pelvis of 44%.2 Spinal cord compression with associated neurologic dysfunction can occur, and masses that stretch along the peripheral nerve may cause nerve damage and resultant neurologic dysfunction. Plexiform neurofibromas can also present as congenital or early developmental lesions and result in severe cosmetic abnormalities or neurologic impairment; orbital lesions may compress the optic nerves, causing visual loss.

In contrast to discrete neurofibroma, plexiform neurofibromas often result in morbidity caused by continued tumor growth.6,7⇓ Diffuse or plexiform neurofibromas are typically associated with multiple nerves and can grow to large proportions, affecting an entire limb or body segment. These lesions often have a rich vascular network and may result in hemorrhage. In addition, the underlying bone may be stimulated to grow and result in limb length discrepancies or dysplasia; the latter is seen frequently in the orbital region (sphenoid wing dysplasia). Plexiform neurofibromas may also occasionally exhibit malignant transformation and mutate into malignant peripheral nerve sheath tumors.7-11⇓⇓⇓⇓ These spindle cell sarcomas tend to be poorly responsive to therapy, can metastasize, and are associated with a low 5-year survival rate.

Spinal neurofibromas are often difficult to classify as either discrete or diffuse lesions.11 Although they can involve multiple nerve roots, they may also appear as discrete compact lesions. The incidence of spinal neurofibromas in patients with NF1 has not been well delineated by prospective studies done in patients of different ages, but such lesions are thought to be quite common, are often multiple along the spine, and can cause motor or sensory deficits as they grow. Most spinal neurofibromas are located within the vertebral foramina and cause problems by compression of the nerve roots. In some patients, they are found essentially along all vertebral regions, and it can be difficult to determine which lesion is symptomatic. A familial form of NF1, manifest by café-au-lait spots and multiple spinal neurofibromas symmetrically affecting multiple spinal nerve roots, but with little other stigmata or NF1, has been described.12

Biologic aspects.

There are several impediments that have limited progress in designing optimal therapies for NF1-associated neurofibromas, including a more complete understanding of 1) the contribution of each cell type in a neurofibroma to its genesis and continued growth, 2) the specific consequences of absent NF1 gene function on cell growth control, and 3) the role of additional genetic and biologic factors that influence neurofibroma formation and growth. Histologically, dermal and plexiform neurofibromas are composed of “neoplastic” Schwann cells accompanied by a varying number of other participating cellular and noncellular components. Embedded in a rich mucosubstance collagen matrix are both non-neoplastic Schwann cells that retain one functional NF1 allele (NF1+/−), “neoplastic” Schwann cells lacking NF1 gene expression, as well as NF1+/− fibroblasts, perineurial cells, and mast cells. Although it is presumed that the NF1-deficient Schwann cell is the “neoplastic” component of this tumor, NF1+/− fibroblasts and mast cells might also contribute to tumorigenesis.

A number of recent studies have demonstrated that the “neoplastic” Schwann cells lack NF1 gene expression by multiple methods including loss of heterozygosity, RNA and protein expression, and fluorescent in situ hybridization (FISH).13-19⇓⇓⇓⇓⇓⇓ The NF1 gene codes for a large cytoplasmic protein, termed neurofibromin, which functions in part as a tumor suppressor (negative growth regulator) by inactivating the RAS signaling molecule.20,21⇓ In many cells, activation of RAS results from the binding of specific mitogenic growth factors (e.g., epidermal growth factor, fibroblast growth factor, etc.) to their cognate receptors (epidermal and fibroblast growth factor receptors). This association results in recruitment of specific activators of RAS to the cell membrane and the initiation of a cascade of signaling events (such as RAF and MAPK activation) that culminate in increased cell proliferation (figure 1). Neurofibromin functions to inactivate RAS and prevents RAS mitogenic signaling, resulting in reduced cell proliferation. Loss of neurofibromin as a consequence of inactivation of both copies of the NF1 gene in the “neoplastic” Schwann cells is associated with elevated levels of activated RAS.22 In another “benign” tumor type common in NF1, the optic nerve glioma, loss of the NF1 gene product, neurofibromin, is likewise associated with increased RAS pathway activation.23

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Figure 1. Growth regulatory pathways important for neurofibromatosis type 1 (NF1)-associated neurofibroma formation and progression. The binding of a mitogenic growth factor, such as epidermal growth factor (EGF) or fibroblast growth factor (FGF), to its specific receptor (EGF-R and FGF-R) results in receptor activation (denoted by asterisk). This activation recruits guanine nucleotide exchange factors (GEF) to the cell membrane, where they can accelerate the conversion of inactive GDP-bound RAS to its active GTP-bound form. Activation of RAS is associated with an additional post-translational modification mediated by farnesyltransferase proteins (FTP) that facilitates the translocation of RAS to the cell membrane, where it can initiate the cascade of activating interactions involving its specific downstream effectors (e.g., RAF and MAPK) that culminate in increased cell proliferation. Neurofibromin functions as a negative growth regulator (tumor suppressor) by accelerating the conversion of active GTP-bound RAS to its inactive GDP-bound form.

In contrast, neurofibroma-derived fibroblasts do not demonstrated elevated RAS activity.23 However, in vitro studies suggest that neurofibroma-associated human fibroblasts or mouse NF1+/− fibroblasts may contribute to the pathogenesis of this tumor.23 In this regard, mouse fibroblasts with one functioning NF1 allele (NF1+/−), analogous to the fibroblasts in the human neurofibromas, display abnormal wound healing and continued fibroblast proliferation in vivo.24 These results argue that the non-neoplastic fibroblast is not “normal” and may contribute to the development of the neurofibroma by responding aberrantly to proliferative signals imparted by “neoplastic” Schwann cells.

Less is known about the contribution of the mast cell to the development of the neurofibroma. Although some patients describe itching in regions that later develop cutaneous neurofibromas, no direct role for the mast cell has been demonstrated. Mouse NF1+/− mast cells exhibit abnormal growth properties and dysregulated RAS signaling.25 Further studies will be required to determine whether the mast cell plays a direct role in neurofibroma tumor formation or whether it represents an “innocent bystander” cell trapped within the tumor.

Although loss of NF1 function in Schwann cells is associated with neurofibroma formation, there is abundant evidence that additional cooperating events may be important for neurofibroma genesis and growth. These changes include increased expression of growth factors and growth factor receptors, including epidermal and platelet-derived growth factor receptors26,27⇓ and vascular endothelial growth factor,28 which may promote new blood vessel formation. In addition, neurofibroma-derived Schwann cells can invade chick allantoic membranes and survive as explants in rat sciatic nerve.29,30⇓ The angioinvasive properties of neurofibroma-derived Schwann cells may result from increased production of proteins that break down the extracellular matrix, such as matrix metalloproteinases (MMP), whose expression is elevated in neurofibromas.31 These results suggest that other biologic properties in addition to cell growth are altered in human neurofibromas (figure 2).

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Figure 2. Additional factors may also contribute to tumor formation or progression. In this model of neurofibroma pathogenesis, the increased mitogenic signaling that results from inactivation of the NF1 tumor suppressor and increased RAS activation is sufficient to initiate neurofibroma formation. Neurofibroma progression may result from additional genetic or biologic changes that increase the expression of vascular growth factors (GF; e.g., vascular endothelial growth factor [VEGF]) or matrix metalloproteinase enzymes (MMP) to promote angiogenesis and tumor infiltration. Last, malignant transformation requires further genetic changes that inactivate key cell cycle regulators, like the p16, p53, or p27-Kip1 tumor suppressors. Loss of these cell cycle regulators results in increased cell proliferation and the accumulation of additional genetic changes important for malignant transformation. Green denotes pathways and molecules that promote increased cell proliferation; red denotes molecules that reduce cell growth. GF-R = growth factor receptor.

As benign plexiform neurofibromas can transform into malignant peripheral nerve sheath tumors (MPNST), studies have focused on identifying cooperating genetic events that might be associated with malignant transformation (see figure 2). Functional inactivation of several key cell cycle regulators, including p53, p27-Kip1, and p16, have been identified in MPNST compared with their benign neurofibroma counterparts.32-38⇓⇓⇓⇓⇓⇓ Loss of the function or expression of these cell cycle regulators results in increased cell proliferation and might permit the accumulation of additional genetic mutations important for malignant transformation. In support of the notion that alterations in cell cycle growth regulators are critical for MPNST formation, two groups have demonstrated that mice with targeted mutations in the NF1 and p53 genes develop MPNST when both the NF1 and p53 genes are inactivated.39,40⇓

Treatment.

Historically, the treatment of patients with plexiform neurofibromas in NF1 has been empiric. Patients have been followed clinically and radiographically, with intervention reserved for those who are symptomatic or those with clear-cut tumor enlargement. The majority of patients requiring intervention have been treated by surgery; however, because of the infiltrative nature of the tumors, outcome after surgery is often suboptimal, with a high incidence of tumor regrowth. In one series of 168 tumors operated on in patients collected from a large multidisciplinary institutional NF1 clinic, 74 cases were found to progress after surgery.41 Factors associated with progression after surgery included younger age at the time of diagnosis, subtotal tumor resection, and a nonextremity location. As can be expected, extent of resection and tumor location are often inter-related, as tumors of the face may be difficult to excise because of concerns over postoperative cosmesis and tumors of the mediastinum and spine may insinuate with vital structures and thus be less amenable to total resection. In this series, patients younger than 10 years were more likely to have tumor progression than older patients after surgery; the reason(s) for such an association remains unclear but may be partially related to an increase in growth of the tumor at the time of puberty.

Because of concerns about malignant transformation, radiation therapy has not been widely used for plexiform neurofibromas. Over the last three decades, nonsurgical management has focused on the use of a variety of different drugs (see the table). However, despite clinical interest and need, there has been a paucity of clinical trials for patients with plexiform neurofibromas in NF1. The earliest therapeutic trials were aimed at targets thought to be integral in the progression of plexiform neurofibromas, such as mast cell function and angiogenesis. More recent approaches have targeted the “neoplastic” Schwann cell or the role of fibroblasts in tumor progression.

The first studies were performed with the antihistamine agent ketotifen fumarate.42-44⇓⇓ These innovative studies, focused predominantly on superficial, primarily dermal, neurofibromas, used clinical criteria for eligibility. All patients with symptoms of itching and/or pain were eligible for the study. Treatment was aimed at improving symptomatology, especially relieving itching due to the neurofibroma. The first trial, using a double-blind crossover design, treated 20 patients (mean age 30.5 years); not all patients had clear-cut NF1, as patients with isolated neurofibromas could be entered if they had pruritus or dysesthetic pain. The second study was an open-label trial of 25 patients (mean age 27.1 years) with symptomatic neurofibromas. Eligible patients were to have progressive or continually symptomatic lesions causing discomfort or disability; radiographic progression was not a necessity for patient entry.

Because of the variability of entry criteria and the subjective endpoints, based primarily on patient self-reporting, evaluation of the efficacy of ketotifen fumarate is difficult. Patients entered in the ketotifen fumarate studies were evaluated for both change in clinical symptomatology, using a disability score, and change in size of the lesion, either by direct or by radiographic measurement. The investigators of the study concluded that ketotifen fumarate resulted in symptomatic relief of pruritus or dysesthetic pain or both in a subset of patients and improvement in the disability score.42-44⇓⇓ However, radiographic response and clinical shrinkage of tumor were not well documented. These trials were performed primarily in the pre-MRI era, and only a subset of patients was evaluated by CT. In addition, owing partially to availability of the drug, the study treated predominantly teenagers and adults.

The largest prospective treatment trial for plexiform neurofibromas performed to date was a randomized noncomparative phase II trial that treated 57 evaluable patients with either cis-retinoic acid or interferon-α. Retinoic acid was used for its maturation effects and α-interferon for its nonspecific anti-inflammatory and anti-angiogenic properties. This study entered both children and adults. The median age of patients was 11 years (mean age 5.4 years), skewing the population to a relatively young patient group. This trial was designed to enter only patients with progressive plexiform neurofibromas. Patients were to have unequivocal radiographic progression or evidence of tumor growth by direct measurement within 1 month prior to entry, but some patients were entered on the basis of increasing clinical symptomatology. There was no stratification based on tumor location or age. Although the majority of patients had plexiform neurofibromas of the head and neck (n = 35), others had growths of the chest and spine (n = 9) or viscera (n = 8). Patients on study were to have direct measurements of tumor size and repeat neuroimaging evaluations, by either CT or MRI, every 3 months while on treatment. Standard oncologic phase II response criteria were used to evaluate for efficacy: That is, a complete response was considered total resolution of all lesions; a partial response, a >50% decrease in the sum of the greatest perpendicular diameters of measurable lesions; a minor response, a 25 to 50% decrease in the sum of the greatest perpendicular diameters of measurable lesions; and stable disease, a ≤25% decrease in measurable lesion size.

At 18 months of follow-up, 86% of patients treated with retinoic acid and 96% of patients treated with interferon were considered to be at least stable (P. Phillips, Children’s Hospital of Philadelphia, personal communication, 2002). Three patients treated with retinoic acid and two treated with interferon had a 10 to 20% reduction in surface measurement; however, no patient had a demonstrable radiographic response. Symptomatic/physiologic improvement was noted in 14% (eight patients) of the population, with five of the symptomatic improvements occurring in those who had received interferon. Symptomatic improvement included relief of pain, resolution of bradycardia in a patient with a vagal nerve tumor, and, in one patient, a resolution of orthopnea.

A phase I study utilizing thalidomide, again targeting antiangiogenesis as a means to control neurofibroma growth, has been recently completed for patients with progressive plexiform neurofibromas.45 As this was a phase I study, patients were required to have symptomatic lesions not amenable to treatment by conventional measures but did not require evidence of radiographic progression prior to study entry. The predominant aim of the study was to determine the toxicity of the agent used and the maximal tolerated dose of the agent safely deliverable; clinical and radiographic efficacy were secondary endpoints. An intrapatient dose escalation of the thalidomide was performed, and 20 patients, between the ages of 6 and 41 years (mean age 16.5 years) at the time of treatment, were entered on study. Eight of the 20 patients abandoned the study for a variety of reasons, including 2 being lost to follow-up, 2 having a MPNST on biopsy, and 2 for acute dose-limiting toxicity including neuropathy and hives. Interestingly, although efficacy was not a primary endpoint, four patients were noted to have a decrease in size of their lesions (by either radiographic or direct surface measure) and five had symptomatic improvement. Building on the results of these two previous studies, which used interferon and thalidomide, and the concept that antiangiogenic compounds may be synergistic, there is interest in combining the agents to achieve greater efficacy.

The second phase I study used an oral farnesyl protein transferase inhibitor in 17 patients with plexiform neurofibromas (B. Widemann, National Cancer Institute, personal communication, 2002). Farnesyl protein transferase inhibitors block the post-translational isoprenylation of RAS and other farnesylated proteins.46 The RAS proteins are integral to mitogenic cell signaling pathways, and proper farnesylation is essential for the function of RAS proteins. As described previously, neurofibromin, the product of the NF1 gene, contains a domain capable of inactivating RAS by accelerating RAS-GTP hydrolysis (see figure 1). Decreased levels of neurofibromin have been associated with constitutively activated RAS-GTP status; thus, inhibition of RAS farnesylation may inhibit growth of tumors in patients with NF1. This was the first study to directly aim at the molecular genetic underpinnings of aberrant Schwann cells. The study used the criterion of a partial response as a measure of efficacy, and no patients on the study had a radiographic partial response. A phase II study utilizing a randomized placebo-controlled crossover design with the same agent is ongoing.

Another promising agent, 5-methyl-1-phenyl-2-(1H)-pyridone (Pirfenidone, Marnac, Dallas, TX), is already in clinical trials in adults with progressive plexiform and spinal neurofibromas. Pirfenidone is a broad-spectrum antifibrotic drug that modulates actions of cytokines such as platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, intracellular adhesion molecules, and transforming growth factor-β1.47,48⇓ Inhibition of these cytokines decreases proliferation and collagen matrix synthesis in human fibroblasts. The antifibrotic effects of pirfenidone have been documented in vitro and in animal experiments in vivo and have been used in human fibrosing conditions.49 The histopathology of neurofibromas is characterized by slender spindle cells with abundant extracellular matrix of dense, wavy collagen fibers and mucoid material. The pirfenidone treatment approach is designed to affect the potential growth-promoting effects of fibroblasts in plexiform neurofibromas, which may have an important role in the pathogenesis of these tumors. Recent work from laboratories at the Children’s National Medical Center has demonstrated significant overexpression of fibroblast growth factor and platelet-derived growth factor in five plexiform neurofibromas by gene expression profiling, increasing the rationale for the use of pirfenidone.

Because of concerns over both short-term and long-term toxicities, including mutagenesis, conventional chemotherapy has not been widely employed in patients with progressive plexiform neurofibromas. Based on promising results in patients with desmoid tumors, the combination of vincristine and methotrexate is under study for plexiform neurofibromas in patients with NF1.50

A major issue in all of these studies comprises the entry criteria used. Although the one prospective randomized phase II study attempted to enter only those patients with progressive tumors, a subset of patients, even on that study, were entered because of clinical progression without documented radiographic progression. This variability in entry criteria makes evaluation of the efficacy of agents across studies difficult, especially given the erratic natural history of plexiform neurofibromas.

At the current time, a study evaluating the natural history of plexiform neurofibromas is ongoing (B.R. Korf, unpublished results). This study was designed to enter 300 patients with plexiform neurofibromas stratified by both age (patients younger or older than 18 years) and location (patients were stratified as regards having tumors of the head or neck, trunk and extremities externally visible, or trunk and extremities not externally visible). An interesting and somewhat unexpected finding of this study is that the majority of patients entered on study, to date, are younger than 18 years at the time of entry. This natural history study is utilizing a variety of criteria to determine if the tumor progresses, including volumetric radiographic measurements. A major goal of the study is to determine the incidence of progression of plexiform neurofibromas over a finite period of time. Data concerning this important issue are scant, and without more information, evaluating future phase II studies will be difficult. An updated listing of institutional review-approved studies in the United States supported by the National Neurofibromatosis Foundation is available at their web site (www.nf.org).

Clinical trial design.

As noted, given the unpredictable natural history of plexiform neurofibromas and their infiltrative nature, clinical trial design becomes a major issue in evaluating approaches for patients with plexiform neurofibromas as well as comparing results across studies. Although such lesions do grow, it may be difficult to apply standard oncologic criteria to assess the efficacy of an agent. In most pediatric and adult oncologic trials evaluating new means of therapy for patients (phase II trials), efficacy is assessed by the ability of the agent either to increase survival or progression-free survival or to result in shrinkage of the tumor.

To date, no study in patients with NF1 has demonstrated objective (radiographic) responses in the ≥50% range. This may be because of the indolent nature of the plexiform neurofibromas and the unlikeliness of any treatment to cause such a dramatic response in the tumor growth. Alternatively, it may be caused by the lack of efficacy of agents used. Another possible reason for the inability to document tumor response is the difficulty in measuring the size of plexiform neurofibromas and determining a change in size over time. Plexiform neurofibromas are notoriously large and irregular in shape, and standard two-dimensional imaging by either CT or more recently by MRI is quite difficult (figures 3 and 4⇓).

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Figure 3. A 20-year-old man with lumbosacral plexiform neurofibromas. Inversion recovery coronal T2-weighted MRI of the lumbar–sacral junction reveals extensive fusiform enlargement of most of the visualized nerve roots. There is good contrast definition between the neurofibromas and the surrounding soft tissues; however, contrast between the neurofibromas and the subarachnoid fluid is poor. Central foci of low T2 signal within each nerve root (“target” sign) are a typical finding in plexiform neurofibroma.

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Figure 4. Extensive facial plexiform neurofibroma in an 8-year-old boy. Inversion recovery axial (A) and coronal fat-suppressed T2-weighted (B) MRI reveal extensive involvement of the subcutaneous and deep soft tissues of nearly the entire right side of the face. Accurate demarcation of the plexiform neurofibroma from surrounding fatty tissue, lymph nodes, and salivary glands cannot be made because of their similar contrast characteristics.

In the aforementioned ongoing natural history study of patients with NF1, the reproducibility of volumetric MR data analysis is being analyzed. If such analysis is found to be reproducible and easily performable, it may be of major utility. Other imaging techniques have not been widely used in the evaluation of patients with NFI and plexiform neurofibromas. MR neurography is a high-resolution means to evaluate peripheral nerves and may, in the future, be of utility in evaluation of response for patients on treatment trials for plexiform neurofibromas.51 However, at this point, the technique is relatively cumbersome and is useful predominantly for small lesions. The size of plexiform neurofibromas may make this technique, in its current state, impossible to apply to clinical studies. PET has been evaluated in intracranial lesions in patients with NF1 (the majority thought to be low-grade astrocytomas), but the utility of this technique has not been evaluated in patients with plexiform neurofibromas. PET may be helpful in identifying malignant change in plexiform neurofibromas.52

In attempts to overcome the issue of the erratic nature of plexiform neurofibromas, other trial designs have been investigated for patients with progressive lesions. One approach is to use the endpoint of progression-free survival, instead of objective response, as a measure for efficacy. Although this is a rational endpoint, it does not negate the problem of the erratic natural history of the disease. The use of a randomized, crossover, double-blinded, placebo-controlled study design does overcome some of these issues, as patients serve as their own controls for endpoints such as time to progression. Because there is no other standard effective treatment for plexiform neurofibromas and these tumors may remain quiescent, even after rapid growth for a period of time, the use of a placebo arm could be postulated. Such study designs are still predicated on the concept that the population to be studied is uniform and that demonstration of progression denotes that the tumor will continue to progress over a finite period of time. This assumption has never been formally demonstrated and may, indeed, not be true for plexiform neurofibromas in patients with NF1.

Because plexiform neurofibromas often are indolent and may cause increasing dysfunction, even without clear-cut evidence of radiographic progression, other methods have been explored to determine the efficacy of an agent. In the early trials with ketotifen, a symptomatic score was developed primarily to determine the effect of the drug on pruritus and, to a lesser extent, pain.53,54⇓ Although a variety of neurologic rating scales have been used for patients with neurologic impairments, none has been reproducibly applied to patients with NF1, much less for patients with NF1 and plexiform neurofibromas. If such scales are to be used to capture the diverse manifestations of compromise caused by the plexiform neurofibromas, the scale must measure not only the neurologic impairment but also the impact of the plexiform neurofibromas on other issues such as pain, ambulation, discomfort, general well-being, and functional independence. There has been recent interest in utilizing quality-of-life scales for this type of assessment because of their ease in reporting and the breadth of information captured. A variety of different quality-of-life scales are under evaluation or have been suggested as possible outcome measures for patients on studies. The Health-Related Quality-of-Life Scale is a multi-attribute scale that evaluates the health status of attributes such as sensation, mobility, emotion, cognition, self-care, and fertility.53 It also has measures of vision, hearing, speech, ambulation, dexterity, emotion, and cognition. The Health-Related Quality-of-Life Scale can be used in patients ranging from 6 years of age through adulthood. For children between 6 and 12 years, a parental form of the scale is available, whereas for patients older than 12 years, a self-reporting form is used. A similar, somewhat less detailed scale is the Rand 36-Item Health Survey.54 This scale also evaluates physical functioning, mental health, and cognition. Still another potential scale is the NIH Impact of Pediatric Illness Scale (F. Balis, National Cancer Institute, personal communication, 2002). This questionnaire has been developed to assess the effects of chronic illness and treatment on the everyday behavior of children and evaluate adaptive behavior, emotional functioning, physical status, and CNS symptoms. The assessment usually takes <30 minutes but is limited by its lack of application to older patients. None of the aforementioned scales has been validated in patients with plexiform neurofibromas in NF1.

Future directions.

Despite the difficulties in evaluating the efficacy of agents, a variety of studies utilizing biologically based approaches are either underway or soon to begin for patients with plexiform neurofibromas associated with NF1. The ability to design more rational therapies for NF1-associated neurofibromas and, for that matter, other manifestations of NF1 is heavily predicated on an improved understanding of the molecular and cellular biology of the cells involved in neurofibroma formation and growth. Some of these insights possibly derived from further experiments are more easily addressed utilizing mouse models and derivative cells and will likely provide critical insights into human tumors. There is still, however, a basic lack of fundamental information regarding neurofibromas in patients with NF1. Further studies will require access to tumor specimens and necessitate the development of multicenter-coordinated collection systems to provide these tissues to scientists. Key questions that still must be addressed include 1) What genetic or protein expression changes are associated with neurofibroma formation? 2) What cells participate in neurofibroma formation? and 3) What biologic markers correlate with tumor growth or arrest in response to therapy? Although paraffin-embedded specimens are of utility and can be used for FISH and immunohistochemistry-based analysis, their utility for RNA and protein activity studies is limited. Fresh snap-frozen tissue or similarly obtained tissue will be required for large-scale gene expression profiling to identify transcripts associated with tumor formation and progression. Gene expression profiling experiments afford the opportunity to identify potential transcripts that might serve as surrogate markers for tumor growth and response to therapy. In addition, fresh tissue is required to develop xenographs or primary culture models for in vitro preclinical models of human neurofibromas suitable for rapid screening of potential rational therapies.

The advances in the laboratory must be coupled with greater uniformity in the improved design of the clinical studies needed. Definition of what constitutes progressive disease has to be agreed upon to standardize criteria for patient entry. More refined means of assessment, be they volumetric analysis, other forms of neuroimaging, or quality-of-life scales, have to be validated before they can be used as determinants of the efficacy of agents. Future clinical trials, given the number of patients available for treatment, will necessitate the development of multi-institutional or national trial structures. A better understanding of the natural history of plexiform neurofibromas is required to determine the optimum time for initiation of treatment and the types of treatment most likely to succeed. However, independent of all these hurdles, there is a clear clinical need to expeditiously develop well-designed, statistically sound studies for children and adults with NF1 and plexiform neurofibromas. Potential therapies are already available, and the number is likely to increase as more is learned about the pathogenesis of NF1.