Preferentially Expressed Antigen in Melanoma (PRAME) Expression Utility in Distinguishing Desmoplastic and Spindle Cell Melanomas from Malignant Peripheral Nerve Sheath Tumors

Preferentially Expressed Antigen in Melanoma (PRAME) Expression Utility in Distinguishing Desmoplastic and Spindle Cell Melanomas from Malignant Peripheral Nerve Sheath Tumors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Preferentially Expressed Antigen in Melanoma (PRAME) Expression Utility in Distinguishing Desmoplastic and Spindle Cell Melanomas from Malignant Peripheral Nerve Sheath Tumors Katie Hall, Kathleen Torrko, Teresa Santiago, Michael Clay, Joshua Wisell This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1847991/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Spindle cell and desmoplastic melanoma (DM) are uncommon melanoma subtypes; both are composed of elongated “spindled cells” but differ from one another based on the extent of desmoplastic stroma separating individual tumor cells. These can be challenging lesions that often have absent or decreased staining for melanocyte specific markers and may resemble other tumors with spindle cell morphology and a similar immunohistochemical (IHC) staining profile, in particular malignant peripheral nerve sheath tumors (MPNSTs). We evaluated IHC expression of PRAME (Preferentially Expressed Antigen in Melanoma) in 41 DMs, 14 spindle cell melanomas, and 26 MPNSTs. PRAME expression was found in 68% of DM, 71% of spindle cell melanomas, and 84% of MPNSTs. However, when extent and intensity of expression are considered, significant differences emerge. The median H-score for all melanomas was 167.5 (range, 20 to 270) and 80 (range, 20 to 275) for all MPNSTs. Spindle cell melanoma, which often has the closest histologic appearance to MPNST, demonstrated the highest median H-score of 215 (range, 55 to 270). Furthermore, a strong and diffuse staining pattern was identified in 53% of all melanomas and 80% of spindle cell melanomas compared to strong, diffuse staining found in 29% of all MPNSTs and only 17% of MPNSTs with intact H3K27me3 expression. PRAME expression in malignant peripheral nerve sheath tumor MPNST PRAME expression in desmoplastic and spindle cell melanoma PRAME immunohistochemistry Figures Figure 1 Figure 2 Background Desmoplastic Melanomas Desmoplastic melanomas (DM) are an uncommon subtype of melanoma included in the WHO Classification of tumors and are generally defined by the presence of neoplastic spindle cells separated from one another by dense collagenous stroma. 1 – 5 Often, DMs present on chronically sun-damaged skin with a predilection for the head and neck region in older individuals with a median age of diagnosis being ten years later than conventional melanomas; however, cases have been reported in young patients as well as in a variety of anatomic locations. 2 , 3 , 6 Tumors usually present as an indurated, ill-defined plaque or nodule with overlying pigmentation in varying shades of tan and nearly half lacking pigmentation entirely. 2 , 4 , 6 These tumors can have a broad spectrum of histologic appearances ranging from traditional paucicellular scar-like morphology with only mild atypia to overtly malignant pleomorphic spindle cells. 3 – 5 DMs often show deep invasion in the reticular dermis or subcutis diffusely replacing the fat lobules. Characteristic diagnostic clues that may be present include prominent lymphocytic aggregates at the tumor edge and neural involvement. 1 – 3 , 6 While DMs have a well-known association with lentigo maligna melanoma, up to 30% of cases do not have an identifiable melanoma in situ (MIS) component. 4 Ancillary immunohistochemical stains are of limited value as virtually all DMs are distinctively negative for classic melanoma markers such as HMB45, Melan-A, and Tyrosinase. The majority label with S-100 protein and SOX10 in up to 96% and 92% of cases, respectively, according to one meta-review. 1 – 5 However, these stains may show more limited labeling when compared to conventional melanomas. Due to the heterogeneity in tumor morphology, classification criteria were set by Busam et al. in 2004 based on the percentage of invasive tumor cells displaying desmoplastic features (i.e., individual tumor cells separated from one another by fibrous stroma). 1 , 2 , 6 Pure DM (pDM) is defined by 90% or greater of the invasive component being desmoplastic, while mixed DM (mDM) is defined as having a desmoplastic component accounting for less than 90% but more than 10% of the invasive component. The non-desmoplastic portion of mDM is often composed of pleomorphic spindle or fusiform cells organized in fascicle-like structures or, less commonly, non-spindled epithelioid cells. When these criteria are strictly applied, the pure desmoplastic subtypes tend to behave more akin to sarcomas with a greater risk of local recurrence, increased hematogenous spread to the lung, and rare sentinel lymph node metastasis when compared to mDM and melanoma without any desmoplastic features. 1 Additionally, pDM is associated with longer disease-free survival than mixed DM and conventional melanomas of a similar thickness. 1 , 2 , 6 While not codified by the WHO Classification system, the commonly termed “spindle cell melanoma” is a morphologic variant of interest. They also demonstrate a predominantly spindle cell morphology but notably lack a desmoplastic component (i.e., cells individually surrounded by collagenous matrix). These have historically been considered to be on a histologic spectrum with DM; however, given the different clinical behavior between conventional melanoma and DM when strictly defined, there is potential value in distinguishing these two entities. 2 Melanomas with less than 10% of a desmoplastic component are not considered to be desmoplastic melanomas by some, but there is at least practical value in noting the presence of desmoplastic features in these lesions as it may let the pathologist evaluating a subsequent specimen know what she/he may find. The nosologic distinction between subtypes of melanoma notwithstanding, they pose a diagnostic challenge when the differential diagnosis includes other spindle cell lesions, particularly malignant peripheral nerve sheath tumor (MPNST). 2 , 7 Malignant Peripheral Nerve Sheath Tumor MPNST is an uncommon sarcoma composed of fascicles of spindle cells arranged in alternating hypo- and hypercellular areas with a tendency to show perivascular accentuation of tumor cells. The degree of cellular pleomorphism is often limited but may be striking, particularly in cases with heterologous differentiation. 8 , 9 These tumors frequently arise in a large peripheral nerve trunk or a pre-existing benign nerve sheath tumor and present as an enlarging mass or fusiform enlargement of a nerve, most commonly in the trunk or extremities, followed by the head and neck region. 7 , 8 While up to 50% of cases occur in the setting of Neurofibromatosis Type 1 (NF1), tumors may occur sporadically or in the setting of previous radiation. 7 Sporadic MPNST tends to occur between 20 to 60 years of age, with patients diagnosed with NF1 presenting at a younger age. 8 , 9 There is general agreement that the diagnosis of MPNST can be made when a lesion displays appropriate histomorphologic features and arises from a peripheral nerve or benign peripheral nerve sheath tumor. However, the diagnosis is quite challenging when these relationships are lacking or are unclear, particularly in sporadic cases. In these cases, it is broadly accepted that with the appropriate morphology, the diagnosis of a conventional MPNST can be made based on the identification of neural differentiation, ordinarily evidenced by focal or patchy S100-protein or SOX10 positivity, and or the loss of the methylation marker, H3K27me3. 7 , 8 While the loss of the H3K27me3 is considered to be highly specific in this context, up to 50% of MPNSTs demonstrate an intact staining pattern. The utility of the H3K27me3 marker is most significant in the post-radiation setting and in high-grade lesions. Such lesions will show loss of staining in 90% of cases. In contrast, low-grade lesions will show a loss pattern in only 30% of cases. 9 This diagnostic challenge is further complicated when considering epithelioid MPNST. This rare subtype is composed of plump, epithelioid cells with abundant eosinophilic cytoplasm that may be embedded in a hyalinized or myxoid extracellular matrix. It is traditionally not associated with NF1 and, uniquely, may be seen arising from conventional schwannoma. SOX10 and S100-protein IHC is likely to be strong and diffuse with a retained pattern of expression for H3K27me3. 8 While differentiating MPNST from melanoma with spindle cell morphology is not a common scenario one may encounter in the diagnosis of melanocytic lesions, in our tertiary referral practice, we have seen cases where this has been an issue, particularly in sporadic cases and sites where the ‘deep’ soft tissues can be sampled with superficial methods in the clinic, as in the head and neck. Furthermore, most DM arise in the head and neck region and often have more extensive neurotropism than DM at other sites. 2 , 4 , 5 Neurotropic patterns that have been described closely mimicking MPNSTs include “neural transformation,” in which the invasive component closely mimics the growth pattern of a nerve sheath tumor, as well as a rare “nerve-centered” pattern that is reserved when growth is confined entirely to the nerve or nerve sheath. 2 MPNSTs and the aforementioned melanoma subtypes can be nearly impossible to distinguish on morphology alone without an associated in situ lesion. Immunohistochemical studies have been considered to be of limited diagnostic use as there is considerable overlap in the expression patterns. Others have reported on these challenges surrounding this differential. 3 , 7 , 9 , 10 , 11 Prame PRAME (PReferentially expressed Antigen in MElanoma) is an antigen expressed in cutaneous and ocular melanomas. Lezcano et al . found that diffuse nuclear labeling with PRAME was present in up to 83% of the 155 primary melanomas tested. Of the primary melanomas, 20 were classified as desmoplastic melanomas, with 35% showing labeling for PRAME, and of the mixed desmoplastic melanomas, the non-desmoplastic portion preferentially expressed PRAME. 12 To our knowledge, Caldwell et al . have the only published study evaluating the diagnostic utility of PRAME immunohistochemistry (IHC) in MPNSTs compared to benign peripheral nerve sheath tumors with up to 65% of MPNSTs demonstrating PRAME labeling. 13 Due to the rarity of MPNSTs and DMs, the evaluation of large cohorts is problematic, and further investigation is warranted to characterize PRAME IHC labeling patterns fully. Our goal is to assess the diagnostic utility in the PRAME IHC marker in the context of distinguishing MPNSTs from histomorphologically similar melanomas, specifically spindle cell melanomas. Materials And Methods Case Selection After approval from the Colorado Multiple Institutional Review Board (IRB#, 21-2477), the University of Colorado Department of Pathology database was searched for patients from 1997 to 2021 with an established diagnosis of melanoma that at least in part contained a desmoplastic or spindle cell cytomorphology. The slides from the complete excision must have been available for review to be included in the study. Cases from primary mucosal sites were excluded. The cases were classified as spindle cell melanoma, pDMs, mDMs, and melanoma with desmoplastic features. Classification of pDM and mDM was done according to the aforementioned diagnostic criteria suggested by Busam et al. in 2004. 1 Tumors demonstrating a desmoplastic component accounting for less than 10% of the invasive tumor were included as melanomas with desmoplastic features. Tumors with spindle cell morphology without desmoplastic features were included as spindle cell melanomas. All cases were reviewed for diagnostic accuracy and categorization by two of the authors (KCH, JAW). Case selection for MPNSTs included specimens from patients seen at UCH and Children’s Hospital Colorado (CHCO) ages 8 to 64 years old with an established diagnosis of malignant peripheral nerve sheath tumor. Commercially available IHC studies including S-100 protein (Polyclonal; DAKO, predilute), SOX10 (SP267; Ventana, predilute), HMB45 (HMB45; DAKO, 1:50), Melan-A (DT101 + BC199; Abcam, 1:50), and Tri-Methyl-Histone H3 (Lys27) (H3K27me3) marker (Rabbit mAb C36B11; Cell Signaling, 1:200) were performed on all MPNST cases. Cases included as conventional MPNST included spindle cell sarcomas arranged in a fascicular growth pattern with geographic necrosis, conspicuous mitoses. While many cases arose from a nerve or a pre-existing nerve sheath tumor, this was not required. All cases with appropriate morphology, needed to demonstrate either patchy S100 or SOX10 staining and/or loss of H3K27me3 expression. Cases of epithelioid MPNST included sarcomas with epithelioid morphology arranged in a nodular growth pattern within a myxoid or hyalinized stroma, diffuse labeling of S100 and SOX10, and intact H3K27me3 expression. All any tumor demonstrating Melan-A or HMB-45 staining was not included. The H&E and IHC slides were reviewed by three of the authors (KCH, JAW, MRC) for diagnostic accuracy. PRAME Immunohistochemical Analysis Five-micrometer thick tissue secretions were cut from formalin-fixed and paraffin-embedded tissue blocks. A commercially available IHC antibody PRAME (EPR20330; Abcam, 1:500) was performed on all melanomas and MPNSTs on an automated Ventana Benchmark XT stainer platform. All cases were given a H-score based on the percentage of cells at each staining intensity level using the following formula: [1 x (% cells 1+) + 2 x (% cells 2+) + 3 x (% cells 3+)]. The intensity levels were determined based on the visibility of nuclear labeling. Conspicuous staining visible at 4x was scored as 3+. Inconspicuous staining at 4x or conspicuous staining at 10x was scored as 2+. Inconspicuous staining at 10x or conspicuous staining at 20x was scored as 1+. The in-situ component was also assigned an intensity score in melanomas with overlying MIS. Statistical Analyses Differences in proportions were analyzed using a Fisher's Exact test. Tests were two-sided with alpha = 0.05. Analyses were done using GraphPad Prism (ver 9.3). Results Melanomas A total of 58 melanomas were examined, including 45 primary, 12 recurrent lesions, and 1 satellite metastasis. The histologic breakdown was 41 DMs (17 pDMs and 24 mDMs), 14 spindle cell melanomas, and 3 melanomas with desmoplastic features. Patient ages ranged from 22 to 90 years of age (median, 64.5 y; mean, 65.9 y). Only tumors with meaningful staining (defined as staining in greater than 10% of tumor cells) were considered positive for our analyses. Of all melanoma cases examined, 40 (69%) tumors showed positive staining with a median H-score of 167.5 (range, 20 to 270). When considering different histologic subtypes, the subtypes with the most tumors showing PRAME expression were the mDMs (75%, n = 23) and spindle cell (71%, n = 10) melanoma groups, while pDMs (59%, n = 10) demonstrated the lowest number of positive tumors. Melanomas with spindle cell morphology demonstrated the highest median H-score at 215 (range 55 to 270), while pDMs had the lowest median H-score at 65 (range 20 to 250). When pDMs were excluded, 30 (73%, n = 41) of the remaining melanomas demonstrated PRAME labeling with a median H-score of 190 (range, 25 to 270). There was no difference in PRAME staining between primary or recurrent tumors. The single metastasis matched the highest H-score for PRAME recorded in the series (270). In the 24 cases with overlying MIS, 22 (92%) of the in-situ lesions demonstrated 3 + staining intensity. See Table 1 and Fig. 1 for a summary of PRAME labeling in melanomas. Table 1 Summary of PRAME Staining in Melanomas Tumor Group (n) Positive tumors, n (%) Median H Score (Range) All melanomas (58) 40 (69%) 167.5 (20 to 270) Histotype DMs (41) 28 (68%) 100 (20 to 270) pDMS (17) 10 (59%) 65 (20 to 250) mDM (24) 23 (75%) 177.5 (25 to 270) Spindle cell melanomas (14) 10 (71%) 215 (55 to 270) Melanomas with desmoplastic features (3) 2 (67%) 115 (40, 190) Melanomas excluding pDM (41) 30 (73%) 190 (25 to 270) Site Primary (45) 30 (67%) 130 (20 to 270) Recurrent (12) 9 (75%) 190 (50 to 250) Metastatic (1) 1 270 Of the 40 PRAME-positive melanomas, 21 (53%) demonstrated strong, diffuse staining (3 + intensity in greater than 50% of tumor cells), and spindle cell melanomas (80%, n = 10) were the most likely to show strong, diffuse labeling while pDMs (10%, n = 10) were the least likely to show this pattern. If pDMs are excluded, 20 (67%, n = 41) of the melanomas showed strong, diffuse labeling. Lastly, while not a measured outcome, we noted that PRAME expression showed greater intensity in the more cellular portions of tumors. MPNSTs Twenty-six MPNSTs were examined, including 16 primary, 5 recurrent, and 5 metastatic lesions. Patient ages ranged from 3 to 64 years (median, 24 y; mean 28.7 y). Fifteen tumors occurred in patients with an NF1 diagnosis, 9 occurred sporadically, and 2 occurred in sites that had been previously irradiated. Twenty-five tumors were of the conventional type, and one had epithelioid morphology. Fifteen tumors demonstrated H3K27me3 loss with intact staining in the remaining 11. Of all 26 cases, 21 (81%) demonstrated meaningful PRAME staining (greater than 10% of tumor cells staining) with a median H-score of 80 (range, 20 to 275). Of note, we saw one primary tumor with conventional morphology that showed diffuse 1 + cytoplasmic staining without convincing nuclear staining; this case was considered negative for our analysis. Tumors that arose in the NF1 setting were more likely to show PRAME labeling when compared to those in the sporadic setting (93% vs. 56%, p value = 0.047 by Fischer Exact Test); however, the median H-score for tumors associated with NF1 was lower for than those in the sporadic setting, 80 (range, 30 to 275) and 160 (range, 60 to 240), respectively. Recurrent tumors demonstrated a higher median H-score at 210 (range, 100 to 220) when compared to primary and metastatic lesions. MPNSTs with H3K27me3 loss all had positive PRAME staining (100%, n = 15), while only 6 (55%, n = 11) tumors with intact H3K27me3 staining had PRAME positivity. The one tumor with epithelioid morphology did not demonstrate PRAME (H score = 0). See Table 2 and Fig. 2 for a summary of PRAME staining in the MPNSTs. Table 2 Summary of PRAME Staining in MPNSTs Tumor Group (n) Positive staining, n (%) Median H Score (Range) All MPNSTs (26) 21 (81%) 80 (20 to 275) Histotype Conventional (25) 21 (84%) 80 (20 to 275) Epithelioid (1) 0 N/A Setting NF1 (15) 14 (93%) 80 (30 to 275) Sporadic (9) 5 (56%) 160 (60 to 240) Radiation (2) 2 (100%) 40 (20, 60) Site Primary 13 (81%) 60 (20 to 275) Recurrent 3 (60%) 210 (100 to 200) Metastatic 5 (100%) 60 (43 to 265) H3k27me Expression H3K27me3 loss (15) 15 (100%) 60 (30 to 270) H3K27me3 intact (11) 6 (55%) 130 (20 to 275) Of the 21 positive MPNSTs, only 6 (29%) demonstrated strong, diffuse staining (3 + intensity in greater than 50% of tumor cells), with the aforementioned groups showing similar percentages. Although this is not a measured outcome, we did note that a subset of MPNSTs demonstrated perivascular accentuation with PRAME staining. Such cases had more significant intensity staining localized near large vessels and less staining in otherwise viable-appearing tumor cells further away (Fig. 2 ). Discussion If any degree of expression is accepted, we found no difference in the relative number of tumors positive for PRAME labeling between melanomas and MPNSTs; however, differences are appreciable if PRAME expression's intensity and extent are considered. If these groups are compared based on the degree of strong, diffuse staining, melanomas were more likely to show this staining pattern compared to MPNSTs. Additionally, our group chose to compare melanomas with spindle cell morphology and all melanomas that excluded the pDMs to MPNSTs as we felt that these were the two groups that were the most morphologically similar to MPNSTs. We found that when pDMs were excluded, the remaining set of melanomas was more likely to show strong and diffuse expression compared to MPNST (67% vs. 29%; p value = 0.01, Fisher’s exact test). This was also identified in melanomas with spindle cell morphology when compared to MPNSTs (80% vs. 29%; p value = 0.02, Fisher’s exact test). See Table 3 . Furthermore, we felt that the most diagnostically problematic MPNSTs were those with intact H3K27me3 staining, and only one of those demonstrated strong and diffuse PRAME expression. Table 3 Comparison of Tumor Types with the Strong, Diffuse Staining Tumor Group (n = # of positive tumors) Strong, Diffuse staining, n (%) p value° compared to all MPNSTs p value° compared to MPNSTs with intact H3K27me3* All MPNSTs (21) 6 (29%) MPNSTs, intact H3k27me3 (6) 1 (17%) All melanomas (40) 21 (53%) 0.10 0.19 Melanomas excluding pDM (30) 20 (67%) 0.01 0.06 Spindle cell melanomas (10) 8 (80%) 0.02 0.04 ° p value by Fisher's exact test Overall, the staining characteristics seen in MPNSTs are in keeping with that of a recent study by Cadwell et al. 2021 who revealed that 66% (n = 82) of MPNSTs had positive PRAME staining, and only 15 tumors demonstrated strong, diffuse (> 50% of tumor nuclei) staining. This study also noted perivascular accentuation of staining within tumor cells with a similar conclusion that these findings are likely secondary to the rapid growth and tissue ischemia. 13 While this pattern was not seen in the melanomas, all melanomas tested were smaller cutaneous lesions that were unlikely to demonstrate the same degree of ischemia; it is unknown if this pattern could be seen in larger metastatic melanomas exhibiting rapid growth. Other studies investigating the utility of PRAME IHC for lesions in the differential for melanoma noted some degree of staining in other lesions tested, most notably clear cell sarcomas. However, PRAME was often limited in these lesions and diffuse, intense staining favored the diagnosis of melanoma. 12 , 14 , 15 Furthermore, we recognize that tumors with intermediate staining in either intensity (1–2+) or quantity (50–75% of tumor cells) may not be reproducible between laboratory IHC platforms or pathologist interpretation. This has been recognized by others. 14 Although to our knowledge an analysis of equipment, antigen retrieval methods, and titration of antibodies across PRAME IHC studies has not been published, we recognize that these and other factors such as the age of paraffin blocks and tissue fixation methods may contribute to this variability and should be considered during evaluation. A significant limitation in our study was difficulty determining the percentage of cells staining in desmoplastic melanomas, specifically in cases with pure desmoplastic morphology. This was a challenge as these tumors are occult by nature, with tumor cells being dispersed throughout the dermis and often obscured by accompanying lymphoid aggregates. Additionally, this study only included small numbers of some of the aforementioned MPNST groups, including H3K27me3-intact and those associated with prior radiation, in particular. Additional studies with larger numbers would be beneficial to fully characterize staining patterns in these tumors. In conclusion, correlation with patient history and clinical and radiographic findings remains paramount in distinguishing these lesions; however, in difficult cases, PRAME IHC can provide discriminating power in favoring the diagnosis of melanoma when labeling of tumor cells is present in a strong and diffuse pattern. Moreover, the value of PRAME may be accentuated when the differential diagnosis centers on specific subsets of these tumors, namely those melanomas with limited desmoplastic features (strictly defined) and MPNSTs with H3K27me3 expression. Declarations ACKNOWLEDGEMENTS Sponsorship for this project was provided by the University of Colorado Department of Pathology and the St. Jude Children’s Research Hospital Department of Pathology. CONFLICT OF INTEREST There are no sources of support, including pharmaceutical or industry support, that require acknowledgement. There was no funding received for this work from any of the following organizations: National Institutes of Health (NIH); Wellcome Trust; Howard Hughes Medical Institute (HHMI); or other(s). ETHICS APPROVAL AND CONSENT TO PARTICIPATE Approval for this study was waived after review from the Colorado Multiple Institutional Review Board (IRB#, 21-2477). This study was performed in accordance with the Declaration of Helsinki. AUTHOR CONTRIBUTIONS K.H., J.W., and M.C. performed study concept and design, development methodology, acquisition and interpretation of data, writing, review and revision of the paper; K.T. provided statistical analysis; T.S. provided technical and material support. All authors read and approved the final paper. FUNDING Funding for this project was provided by the University of Colorado Department of Pathology and the St. Jude Children’s Research Hospital Department of Pathology. DATA AVAILABILITY STATEMENT The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Busam KJ, Mujumdar U, Hummer AJ, Nobrega J, Hawkins WG, Coit DG, Brady MS. Cutaneous desmoplastic melanoma: reappraisal of morphologic heterogeneity and prognostic factors. Am J Surg Pathol. 28 , 1518–1525(2004) Busam KJ. Cutaneous desmoplastic melanoma. Adv Anat Pathol. 12 , 92–102(2005) Busam KJ. Desmoplastic Melanoma. Surg Pathol Clin. 2 , 511–520 (2009) Chen LL, Jaimes N, Barker CA, Busam KJ, Marghoob AA. Desmoplastic melanoma: a review. J Am Acad Dermatol. 68 , 825–833(2013) Weissinger SE, Keil P, Silvers DN, Klaus BM, Moller P, Horst BA, Lennerz JK. A diagnostic algorithm to distinguish desmoplastic from spindle cell melanoma. 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Jour G, Andeen NK, Al-Rohil R, Aung PP, Mehrotra M, Duose D, et al. Novel enriched pathways in superficial malignant peripheral nerve sheath tumours and spindle/desmoplastic melanomas. J Pathol. 244 ( 1 ), 97–106 (2018). Owosho AA, Estilo CL, Huryn JM, Chi P, Antonescu CR. A Clinicopathologic Study of Head and Neck Malignant Peripheral Nerve Sheath Tumors. Head Neck Pathol. 12 ( 2 ), 151–159 (2018). Lezcano C, Jungbluth AA, Nehal KS, Hollmann TJ, Busam KJ. PRAME Expression in Melanocytic Tumors. Am J Surg Pathol. 42 , 1456–1465 (2018). Cadwell CR, Yuksek GE, Hirbe AC, Srihari D, LeBoit P, Dahiya S, et al. Preferentially Expressed Antigen in Melanoma (PRAME) Expression in Malignant, but Not Benign, Peripheral Nerve Sheath Tumors. J Neuropathol Exp Neurol. 80 , 384–386 (2021). Lezcano C, Jungbluth, AA, Busam, KJ. PRAME Immunohistochemistry as an Ancillary Test for the Assessment of Melanocytic Lesions. Surgical pathology clinics. 14 ( 2 ), 165–175 (2021). Raghavan SS, Wang JY, Toland A, Bangs CD, Rieger KE, Novoa RA, et al. Diffuse PRAME expression is highly specific for malignant melanoma in the distinction from clear cell sarcoma. J. Cutan Pathol. 47 , 1226–1228(2020). Additional Declarations There is NO conflict of interest to disclose. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1847991","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":121641932,"identity":"8765df87-999a-4c57-b9cd-28f8893f3481","order_by":0,"name":"Katie Hall","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDklEQVRIie3NMUvDQBTA8RceJMu1WSNS/AoXAnWp5qskBDq5uHVwOBAylbqmiN8hIDhfeOAkdHXIUgKZHFIEySDWS6JbD+rmcH8OHjzudwdgMv3DuAToz9hBOay8YVhCQ3yBA7HRjn5Jf1tLAsBudITx48jU2RSygfJMkd07uylD9355vm1hNsnlYTJjCRQZ1H6Ko8dT9lzHWfniCw/mgZZAAsSALEWekNkUce/KFxwo1pGpWwF9AoUpshrZF4U9iWCvJYGnfgH1piI2jlKy8o5IkFriZxUvlpySFO3g5GFFcfY6v14LngRrDeGbuGraBV3cObfb5u2DQjdL8l27uJysNOQHHrExmUwm0x/6BnoWYCXCQq8tAAAAAElFTkSuQmCC","orcid":"","institution":"University of Colorado","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Katie","middleName":"","lastName":"Hall","suffix":""},{"id":121641933,"identity":"f78b6f90-ee37-4458-a11a-c6b6f56c4ae6","order_by":1,"name":"Kathleen Torrko","email":"","orcid":"","institution":"University of Colorado","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kathleen","middleName":"","lastName":"Torrko","suffix":""},{"id":121641934,"identity":"32e50b81-5ae0-44df-948e-c97e480a8401","order_by":2,"name":"Teresa Santiago","email":"","orcid":"","institution":"St. Jude Children's Research Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Teresa","middleName":"","lastName":"Santiago","suffix":""},{"id":121641935,"identity":"c96e480c-6bea-4117-bc0c-fd2b5f36f957","order_by":3,"name":"Michael Clay","email":"","orcid":"https://orcid.org/0000-0003-1659-8927","institution":"University of Colorado Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Clay","suffix":""},{"id":121641936,"identity":"e2f8354b-c4af-4a31-ae45-7ed3aaab00a6","order_by":4,"name":"Joshua Wisell","email":"","orcid":"","institution":"University of Colorado","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"Wisell","suffix":""}],"badges":[],"createdAt":"2022-07-11 20:56:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1847991/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1847991/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":24059839,"identity":"15c4485e-bf57-40b1-9a97-e23e377e6c1b","added_by":"auto","created_at":"2022-07-19 19:50:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4059923,"visible":true,"origin":"","legend":"\u003cp\u003e1A (H\u0026amp;E, 20x) and 1B (PRAME, 20x).\u0026nbsp;A pure desmoplastic melanoma with neurotropic features associated with minimal nesting (upper, central portion), these nests demonstrate brighter PRAME staining the remainder of the tumor cells separated by collagen fibers.\u0026nbsp;This tumor received an H-score of 60.\u0026nbsp;1C (H\u0026amp;E, 20x) and 1D (PRAME, 20x). A mixed desmoplastic melanoma demonstrates individually separated tumor cells (upper, left) juxtaposed to areas of tumor cells that create fascicle-like structures (lower, right).\u0026nbsp;Again, PRAME more strongly stains the more cellular portions of the tumor.\u0026nbsp;This tumor received an H-score of 160.\u0026nbsp;1E (H\u0026amp;E, 20x) and 1F (PRAME, 20x).\u0026nbsp;A spindle cell melanoma shows tumor cells growing in short fascicle-like structures.\u0026nbsp;The PRAME strongly stains the majority of the tumor cells.\u0026nbsp;This tumor received an H-score of 220.\u003c/p\u003e","description":"","filename":"Figure1CKMY.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1847991/v1/8375965288dd0cc2b7736e18.jpg"},{"id":24059840,"identity":"a035c3cb-e956-466a-a151-ce318df638ab","added_by":"auto","created_at":"2022-07-19 19:50:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4986124,"visible":true,"origin":"","legend":"\u003cp\u003e2A (H\u0026amp;E, 20x) and 2B (H\u0026amp;E, 20x).\u0026nbsp;A conventional MPNST with spindle cells arranged in fascicle-like structures with minimal pleomorphism.\u0026nbsp;PRAME stains groups of tumor cells with a weak to moderate intensity. This tumor received an H-score of 60.\u0026nbsp;2C (H\u0026amp;E, 20x) and 2D (H\u0026amp;E, 20x).\u0026nbsp;A conventional MPNST composed of spindle cells with perivascular condensation of tumor cells.\u0026nbsp;PRAME shows moderate staining of plump tumor cells adjacent to vessels with little or no staining in areas away from vessels.\u0026nbsp;This tumor received an H-score of 80. 2E (H\u0026amp;E, 20x) and 2F (PRAME, 20x).\u0026nbsp;An epithelioid MPNSTs with a lobular growth pattern composed of plump epithelioid cells.\u0026nbsp;This tumor received an H-score of 0.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2CKMY.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1847991/v1/3545d365e1fbea49e74afeda.jpg"},{"id":25439562,"identity":"177ff6a0-0e32-43a4-9200-8c7b12f43a3d","added_by":"auto","created_at":"2022-08-20 01:00:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1369138,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1847991/v1/cac2fa7d-dd47-4d77-9e30-c56a2924c883.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Preferentially Expressed Antigen in Melanoma (PRAME) Expression Utility in Distinguishing Desmoplastic and Spindle Cell Melanomas from Malignant Peripheral Nerve Sheath Tumors","fulltext":[{"header":"Background","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eDesmoplastic Melanomas\u003c/h2\u003e \u003cp\u003eDesmoplastic melanomas (DM) are an uncommon subtype of melanoma included in the WHO Classification of tumors and are generally defined by the presence of neoplastic spindle cells separated from one another by dense collagenous stroma.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Often, DMs present on chronically sun-damaged skin with a predilection for the head and neck region in older individuals with a median age of diagnosis being ten years later than conventional melanomas; however, cases have been reported in young patients as well as in a variety of anatomic locations.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Tumors usually present as an indurated, ill-defined plaque or nodule with overlying pigmentation in varying shades of tan and nearly half lacking pigmentation entirely.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThese tumors can have a broad spectrum of histologic appearances ranging from traditional paucicellular scar-like morphology with only mild atypia to overtly malignant pleomorphic spindle cells.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e DMs often show deep invasion in the reticular dermis or subcutis diffusely replacing the fat lobules. Characteristic diagnostic clues that may be present include prominent lymphocytic aggregates at the tumor edge and neural involvement.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e While DMs have a well-known association with lentigo maligna melanoma, up to 30% of cases do not have an identifiable melanoma in situ (MIS) component.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Ancillary immunohistochemical stains are of limited value as virtually all DMs are distinctively negative for classic melanoma markers such as HMB45, Melan-A, and Tyrosinase. The majority label with S-100 protein and SOX10 in up to 96% and 92% of cases, respectively, according to one meta-review.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e However, these stains may show more limited labeling when compared to conventional melanomas.\u003c/p\u003e \u003cp\u003eDue to the heterogeneity in tumor morphology, classification criteria were set by \u003cem\u003eBusam et al.\u003c/em\u003e in 2004 based on the percentage of invasive tumor cells displaying desmoplastic features (i.e., individual tumor cells separated from one another by fibrous stroma).\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Pure DM (pDM) is defined by 90% or greater of the invasive component being desmoplastic, while mixed DM (mDM) is defined as having a desmoplastic component accounting for less than 90% but more than 10% of the invasive component. The non-desmoplastic portion of mDM is often composed of pleomorphic spindle or fusiform cells organized in fascicle-like structures or, less commonly, non-spindled epithelioid cells. When these criteria are strictly applied, the pure desmoplastic subtypes tend to behave more akin to sarcomas with a greater risk of local recurrence, increased hematogenous spread to the lung, and rare sentinel lymph node metastasis when compared to mDM and melanoma without any desmoplastic features.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Additionally, pDM is associated with longer disease-free survival than mixed DM and conventional melanomas of a similar thickness.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWhile not codified by the WHO Classification system, the commonly termed \u0026ldquo;spindle cell melanoma\u0026rdquo; is a morphologic variant of interest. They also demonstrate a predominantly spindle cell morphology but notably lack a desmoplastic component (i.e., cells individually surrounded by collagenous matrix). These have historically been considered to be on a histologic spectrum with DM; however, given the different clinical behavior between conventional melanoma and DM when strictly defined, there is potential value in distinguishing these two entities.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Melanomas with less than 10% of a desmoplastic component are not considered to be desmoplastic melanomas by some, but there is at least practical value in noting the presence of desmoplastic features in these lesions as it may let the pathologist evaluating a subsequent specimen know what she/he may find. The nosologic distinction between subtypes of melanoma notwithstanding, they pose a diagnostic challenge when the differential diagnosis includes other spindle cell lesions, particularly malignant peripheral nerve sheath tumor (MPNST).\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMalignant Peripheral Nerve Sheath Tumor\u003c/h2\u003e \u003cp\u003eMPNST is an uncommon sarcoma composed of fascicles of spindle cells arranged in alternating hypo- and hypercellular areas with a tendency to show perivascular accentuation of tumor cells. The degree of cellular pleomorphism is often limited but may be striking, particularly in cases with heterologous differentiation.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e These tumors frequently arise in a large peripheral nerve trunk or a pre-existing benign nerve sheath tumor and present as an enlarging mass or fusiform enlargement of a nerve, most commonly in the trunk or extremities, followed by the head and neck region.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e While up to 50% of cases occur in the setting of Neurofibromatosis Type 1 (NF1), tumors may occur sporadically or in the setting of previous radiation.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Sporadic MPNST tends to occur between 20 to 60 years of age, with patients diagnosed with NF1 presenting at a younger age.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThere is general agreement that the diagnosis of MPNST can be made when a lesion displays appropriate histomorphologic features and arises from a peripheral nerve or benign peripheral nerve sheath tumor. However, the diagnosis is quite challenging when these relationships are lacking or are unclear, particularly in sporadic cases. In these cases, it is broadly accepted that with the appropriate morphology, the diagnosis of a conventional MPNST can be made based on the identification of neural differentiation, ordinarily evidenced by focal or patchy S100-protein or SOX10 positivity, and or the loss of the methylation marker, H3K27me3.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e While the loss of the H3K27me3 is considered to be highly specific in this context, up to 50% of MPNSTs demonstrate an intact staining pattern. The utility of the H3K27me3 marker is most significant in the post-radiation setting and in high-grade lesions. Such lesions will show loss of staining in 90% of cases. In contrast, low-grade lesions will show a loss pattern in only 30% of cases.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis diagnostic challenge is further complicated when considering epithelioid MPNST. This rare subtype is composed of plump, epithelioid cells with abundant eosinophilic cytoplasm that may be embedded in a hyalinized or myxoid extracellular matrix. It is traditionally not associated with NF1 and, uniquely, may be seen arising from conventional schwannoma. SOX10 and S100-protein IHC is likely to be strong and diffuse with a retained pattern of expression for H3K27me3.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWhile differentiating MPNST from melanoma with spindle cell morphology is not a common scenario one may encounter in the diagnosis of melanocytic lesions, in our tertiary referral practice, we have seen cases where this has been an issue, particularly in sporadic cases and sites where the \u0026lsquo;deep\u0026rsquo; soft tissues can be sampled with superficial methods in the clinic, as in the head and neck. Furthermore, most DM arise in the head and neck region and often have more extensive neurotropism than DM at other sites.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Neurotropic patterns that have been described closely mimicking MPNSTs include \u0026ldquo;neural transformation,\u0026rdquo; in which the invasive component closely mimics the growth pattern of a nerve sheath tumor, as well as a rare \u0026ldquo;nerve-centered\u0026rdquo; pattern that is reserved when growth is confined entirely to the nerve or nerve sheath.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e MPNSTs and the aforementioned melanoma subtypes can be nearly impossible to distinguish on morphology alone without an associated in situ lesion. Immunohistochemical studies have been considered to be of limited diagnostic use as there is considerable overlap in the expression patterns. Others have reported on these challenges surrounding this differential.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003ePrame\u003c/h2\u003e\u003cp\u003ePRAME (PReferentially expressed Antigen in MElanoma) is an antigen expressed in cutaneous and ocular melanomas. \u003cem\u003eLezcano et al\u003c/em\u003e. found that diffuse nuclear labeling with PRAME was present in up to 83% of the 155 primary melanomas tested. Of the primary melanomas, 20 were classified as desmoplastic melanomas, with 35% showing labeling for PRAME, and of the mixed desmoplastic melanomas, the non-desmoplastic portion preferentially expressed PRAME.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e To our knowledge, \u003cem\u003eCaldwell et al\u003c/em\u003e. have the only published study evaluating the diagnostic utility of PRAME immunohistochemistry (IHC) in MPNSTs compared to benign peripheral nerve sheath tumors with up to 65% of MPNSTs demonstrating PRAME labeling.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Due to the rarity of MPNSTs and DMs, the evaluation of large cohorts is problematic, and further investigation is warranted to characterize PRAME IHC labeling patterns fully. Our goal is to assess the diagnostic utility in the PRAME IHC marker in the context of distinguishing MPNSTs from histomorphologically similar melanomas, specifically spindle cell melanomas.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCase Selection\u003c/h2\u003e \u003cp\u003eAfter approval from the Colorado Multiple Institutional Review Board (IRB#, 21-2477), the University of Colorado Department of Pathology database was searched for patients from 1997 to 2021 with an established diagnosis of melanoma that at least in part contained a desmoplastic or spindle cell cytomorphology. The slides from the complete excision must have been available for review to be included in the study. Cases from primary mucosal sites were excluded.\u003c/p\u003e \u003cp\u003eThe cases were classified as spindle cell melanoma, pDMs, mDMs, and melanoma with desmoplastic features. Classification of pDM and mDM was done according to the aforementioned diagnostic criteria suggested by \u003cem\u003eBusam et al.\u003c/em\u003e in 2004.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Tumors demonstrating a desmoplastic component accounting for less than 10% of the invasive tumor were included as melanomas with desmoplastic features. Tumors with spindle cell morphology without desmoplastic features were included as spindle cell melanomas. All cases were reviewed for diagnostic accuracy and categorization by two of the authors (KCH, JAW).\u003c/p\u003e \u003cp\u003eCase selection for MPNSTs included specimens from patients seen at UCH and Children\u0026rsquo;s Hospital Colorado (CHCO) ages 8 to 64 years old with an established diagnosis of malignant peripheral nerve sheath tumor. Commercially available IHC studies including S-100 protein (Polyclonal; DAKO, predilute), SOX10 (SP267; Ventana, predilute), HMB45 (HMB45; DAKO, 1:50), Melan-A (DT101\u0026thinsp;+\u0026thinsp;BC199; Abcam, 1:50), and Tri-Methyl-Histone H3 (Lys27) (H3K27me3) marker (Rabbit mAb C36B11; Cell Signaling, 1:200) were performed on all MPNST cases. Cases included as conventional MPNST included spindle cell sarcomas arranged in a fascicular growth pattern with geographic necrosis, conspicuous mitoses. While many cases arose from a nerve or a pre-existing nerve sheath tumor, this was not required. All cases with appropriate morphology, needed to demonstrate either patchy S100 or SOX10 staining and/or loss of H3K27me3 expression. Cases of epithelioid MPNST included sarcomas with epithelioid morphology arranged in a nodular growth pattern within a myxoid or hyalinized stroma, diffuse labeling of S100 and SOX10, and intact H3K27me3 expression. All any tumor demonstrating Melan-A or HMB-45 staining was not included. The H\u0026amp;E and IHC slides were reviewed by three of the authors (KCH, JAW, MRC) for diagnostic accuracy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePRAME Immunohistochemical Analysis\u003c/h2\u003e \u003cp\u003eFive-micrometer thick tissue secretions were cut from formalin-fixed and paraffin-embedded tissue blocks. A commercially available IHC antibody PRAME (EPR20330; Abcam, 1:500) was performed on all melanomas and MPNSTs on an automated Ventana Benchmark XT stainer platform. All cases were given a H-score based on the percentage of cells at each staining intensity level using the following formula: [1 x (% cells 1+)\u0026thinsp;+\u0026thinsp;2 x (% cells 2+)\u0026thinsp;+\u0026thinsp;3 x (% cells 3+)]. The intensity levels were determined based on the visibility of nuclear labeling. Conspicuous staining visible at 4x was scored as 3+. Inconspicuous staining at 4x or conspicuous staining at 10x was scored as 2+. Inconspicuous staining at 10x or conspicuous staining at 20x was scored as 1+. The in-situ component was also assigned an intensity score in melanomas with overlying MIS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eDifferences in proportions were analyzed using a Fisher's Exact test. Tests were two-sided with alpha\u0026thinsp;=\u0026thinsp;0.05. Analyses were done using GraphPad Prism (ver 9.3).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMelanomas\u003c/h2\u003e \u003cp\u003eA total of 58 melanomas were examined, including 45 primary, 12 recurrent lesions, and 1 satellite metastasis. The histologic breakdown was 41 DMs (17 pDMs and 24 mDMs), 14 spindle cell melanomas, and 3 melanomas with desmoplastic features. Patient ages ranged from 22 to 90 years of age (median, 64.5 y; mean, 65.9 y). Only tumors with meaningful staining (defined as staining in greater than 10% of tumor cells) were considered positive for our analyses.\u003c/p\u003e \u003cp\u003eOf all melanoma cases examined, 40 (69%) tumors showed positive staining with a median H-score of 167.5 (range, 20 to 270). When considering different histologic subtypes, the subtypes with the most tumors showing PRAME expression were the mDMs (75%, n\u0026thinsp;=\u0026thinsp;23) and spindle cell (71%, n\u0026thinsp;=\u0026thinsp;10) melanoma groups, while pDMs (59%, n\u0026thinsp;=\u0026thinsp;10) demonstrated the lowest number of positive tumors. Melanomas with spindle cell morphology demonstrated the highest median H-score at 215 (range 55 to 270), while pDMs had the lowest median H-score at 65 (range 20 to 250). When pDMs were excluded, 30 (73%, n\u0026thinsp;=\u0026thinsp;41) of the remaining melanomas demonstrated PRAME labeling with a median H-score of 190 (range, 25 to 270). There was no difference in PRAME staining between primary or recurrent tumors. The single metastasis matched the highest H-score for PRAME recorded in the series (270). In the 24 cases with overlying MIS, 22 (92%) of the in-situ lesions demonstrated 3\u0026thinsp;+\u0026thinsp;staining intensity. See Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for a summary of PRAME labeling in melanomas.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of PRAME Staining in Melanomas\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor Group (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive tumors, n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedian H Score (Range)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAll melanomas (58)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40 (69%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e167.5 (20 to 270)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHistotype\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMs (41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28 (68%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100 (20 to 270)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epDMS (17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (59%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65 (20 to 250)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emDM (24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23 (75%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e177.5 (25 to 270)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpindle cell melanomas (14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (71%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e215 (55 to 270)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMelanomas with desmoplastic features (3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e115 (40, 190)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMelanomas excluding pDM (41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30 (73%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e190 (25 to 270)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSite\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary (45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30 (67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130 (20 to 270)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecurrent (12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (75%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e190 (50 to 250)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetastatic (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e270\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOf the 40 PRAME-positive melanomas, 21 (53%) demonstrated strong, diffuse staining (3\u0026thinsp;+\u0026thinsp;intensity in greater than 50% of tumor cells), and spindle cell melanomas (80%, n\u0026thinsp;=\u0026thinsp;10) were the most likely to show strong, diffuse labeling while pDMs (10%, n\u0026thinsp;=\u0026thinsp;10) were the least likely to show this pattern. If pDMs are excluded, 20 (67%, n\u0026thinsp;=\u0026thinsp;41) of the melanomas showed strong, diffuse labeling. Lastly, while not a measured outcome, we noted that PRAME expression showed greater intensity in the more cellular portions of tumors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMPNSTs\u003c/h2\u003e \u003cp\u003eTwenty-six MPNSTs were examined, including 16 primary, 5 recurrent, and 5 metastatic lesions. Patient ages ranged from 3 to 64 years (median, 24 y; mean 28.7 y). Fifteen tumors occurred in patients with an NF1 diagnosis, 9 occurred sporadically, and 2 occurred in sites that had been previously irradiated. Twenty-five tumors were of the conventional type, and one had epithelioid morphology. Fifteen tumors demonstrated H3K27me3 loss with intact staining in the remaining 11.\u003c/p\u003e \u003cp\u003eOf all 26 cases, 21 (81%) demonstrated meaningful PRAME staining (greater than 10% of tumor cells staining) with a median H-score of 80 (range, 20 to 275). Of note, we saw one primary tumor with conventional morphology that showed diffuse 1\u0026thinsp;+\u0026thinsp;cytoplasmic staining without convincing nuclear staining; this case was considered negative for our analysis.\u003c/p\u003e \u003cp\u003eTumors that arose in the NF1 setting were more likely to show PRAME labeling when compared to those in the sporadic setting (93% vs. 56%, p value\u0026thinsp;=\u0026thinsp;0.047 by Fischer Exact Test); however, the median H-score for tumors associated with NF1 was lower for than those in the sporadic setting, 80 (range, 30 to 275) and 160 (range, 60 to 240), respectively. Recurrent tumors demonstrated a higher median H-score at 210 (range, 100 to 220) when compared to primary and metastatic lesions. MPNSTs with H3K27me3 loss all had positive PRAME staining (100%, n\u0026thinsp;=\u0026thinsp;15), while only 6 (55%, n\u0026thinsp;=\u0026thinsp;11) tumors with intact H3K27me3 staining had PRAME positivity. The one tumor with epithelioid morphology did not demonstrate PRAME (H score\u0026thinsp;=\u0026thinsp;0). See Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for a summary of PRAME staining in the MPNSTs.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of PRAME Staining in MPNSTs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor Group (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive staining, n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedian H Score (Range)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAll MPNSTs (26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21 (81%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80 (20 to 275)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHistotype\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConventional (25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21 (84%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80 (20 to 275)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpithelioid (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSetting\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF1 (15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (93%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80 (30 to 275)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSporadic (9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (56%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e160 (60 to 240)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadiation (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40 (20, 60)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSite\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (81%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60 (20 to 275)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecurrent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (60%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e210 (100 to 200)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetastatic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60 (43 to 265)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eH3k27me Expression\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH3K27me3 loss (15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60 (30 to 270)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH3K27me3 intact (11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (55%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130 (20 to 275)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOf the 21 positive MPNSTs, only 6 (29%) demonstrated strong, diffuse staining (3\u0026thinsp;+\u0026thinsp;intensity in greater than 50% of tumor cells), with the aforementioned groups showing similar percentages. Although this is not a measured outcome, we did note that a subset of MPNSTs demonstrated perivascular accentuation with PRAME staining. Such cases had more significant intensity staining localized near large vessels and less staining in otherwise viable-appearing tumor cells further away (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIf any degree of expression is accepted, we found no difference in the relative number of tumors positive for PRAME labeling between melanomas and MPNSTs; however, differences are appreciable if PRAME expression's intensity and extent are considered. If these groups are compared based on the degree of strong, diffuse staining, melanomas were more likely to show this staining pattern compared to MPNSTs. Additionally, our group chose to compare melanomas with spindle cell morphology and all melanomas that excluded the pDMs to MPNSTs as we felt that these were the two groups that were the most morphologically similar to MPNSTs. We found that when pDMs were excluded, the remaining set of melanomas was more likely to show strong and diffuse expression compared to MPNST (67% vs. 29%; p value\u0026thinsp;=\u0026thinsp;0.01, Fisher\u0026rsquo;s exact test). This was also identified in melanomas with spindle cell morphology when compared to MPNSTs (80% vs. 29%; p value\u0026thinsp;=\u0026thinsp;0.02, Fisher\u0026rsquo;s exact test). See Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Furthermore, we felt that the most diagnostically problematic MPNSTs were those with intact H3K27me3 staining, and only one of those demonstrated strong and diffuse PRAME expression.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of Tumor Types with the Strong, Diffuse Staining\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor Group\u003c/p\u003e \u003cp\u003e(n = # of positive tumors)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStrong, Diffuse staining, n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep value\u0026deg; compared to all MPNSTs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value\u0026deg; compared to MPNSTs with intact H3K27me3*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAll MPNSTs (21)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (29%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMPNSTs, intact H3k27me3 (6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (17%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAll melanomas (40)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21 (53%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMelanomas excluding pDM (30)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 (67%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSpindle cell melanomas (10)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 (80%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u0026deg; p value by Fisher's exact test\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOverall, the staining characteristics seen in MPNSTs are in keeping with that of a recent study by \u003cem\u003eCadwell\u003c/em\u003e et al. 2021 who revealed that 66% (n\u0026thinsp;=\u0026thinsp;82) of MPNSTs had positive PRAME staining, and only 15 tumors demonstrated strong, diffuse (\u0026gt;\u0026thinsp;50% of tumor nuclei) staining. This study also noted perivascular accentuation of staining within tumor cells with a similar conclusion that these findings are likely secondary to the rapid growth and tissue ischemia.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e While this pattern was not seen in the melanomas, all melanomas tested were smaller cutaneous lesions that were unlikely to demonstrate the same degree of ischemia; it is unknown if this pattern could be seen in larger metastatic melanomas exhibiting rapid growth.\u003c/p\u003e \u003cp\u003eOther studies investigating the utility of PRAME IHC for lesions in the differential for melanoma noted some degree of staining in other lesions tested, most notably clear cell sarcomas. However, PRAME was often limited in these lesions and diffuse, intense staining favored the diagnosis of melanoma.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Furthermore, we recognize that tumors with intermediate staining in either intensity (1\u0026ndash;2+) or quantity (50\u0026ndash;75% of tumor cells) may not be reproducible between laboratory IHC platforms or pathologist interpretation. This has been recognized by others.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Although to our knowledge an analysis of equipment, antigen retrieval methods, and titration of antibodies across PRAME IHC studies has not been published, we recognize that these and other factors such as the age of paraffin blocks and tissue fixation methods may contribute to this variability and should be considered during evaluation.\u003c/p\u003e \u003cp\u003eA significant limitation in our study was difficulty determining the percentage of cells staining in desmoplastic melanomas, specifically in cases with pure desmoplastic morphology. This was a challenge as these tumors are occult by nature, with tumor cells being dispersed throughout the dermis and often obscured by accompanying lymphoid aggregates. Additionally, this study only included small numbers of some of the aforementioned MPNST groups, including H3K27me3-intact and those associated with prior radiation, in particular. Additional studies with larger numbers would be beneficial to fully characterize staining patterns in these tumors. In conclusion, correlation with patient history and clinical and radiographic findings remains paramount in distinguishing these lesions; however, in difficult cases, PRAME IHC can provide discriminating power in favoring the diagnosis of melanoma when labeling of tumor cells is present in a strong and diffuse pattern. Moreover, the value of PRAME may be accentuated when the differential diagnosis centers on specific subsets of these tumors, namely those melanomas with limited desmoplastic features (strictly defined) and MPNSTs with H3K27me3 expression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSponsorship for this project was provided by the University of Colorado Department of Pathology and the St. Jude Children\u0026rsquo;s Research Hospital Department of Pathology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no sources of support, including pharmaceutical or industry support, that require acknowledgement. There was no funding received for this work from any of the following organizations: National Institutes of Health (NIH); Wellcome Trust; Howard Hughes Medical Institute (HHMI); or other(s).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproval for this study was waived after review from the Colorado Multiple Institutional Review Board (IRB#, 21-2477). \u0026nbsp;This study was performed in accordance with the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.H., J.W., and M.C. performed study concept and design, development methodology, acquisition and interpretation of data, writing, review and revision of the paper; K.T. provided statistical analysis; T.S. provided technical and material support. All authors read and approved the final paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for this project was provided by the University of Colorado Department of Pathology and the St. Jude Children\u0026rsquo;s Research Hospital Department of Pathology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBusam KJ, Mujumdar U, Hummer AJ, Nobrega J, Hawkins WG, Coit DG, Brady MS. Cutaneous desmoplastic melanoma: reappraisal of morphologic heterogeneity and prognostic factors. Am J Surg Pathol. \u003cb\u003e28\u003c/b\u003e, 1518\u0026ndash;1525(2004)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBusam KJ. Cutaneous desmoplastic melanoma. Adv Anat Pathol. \u003cb\u003e12\u003c/b\u003e, 92\u0026ndash;102(2005)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBusam KJ. Desmoplastic Melanoma. Surg Pathol Clin. \u003cb\u003e2\u003c/b\u003e, 511\u0026ndash;520 (2009)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen LL, Jaimes N, Barker CA, Busam KJ, Marghoob AA. Desmoplastic melanoma: a review. J Am Acad Dermatol. \u003cb\u003e68\u003c/b\u003e, 825\u0026ndash;833(2013)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeissinger SE, Keil P, Silvers DN, Klaus BM, Moller P, Horst BA, Lennerz JK. A diagnostic algorithm to distinguish desmoplastic from spindle cell melanoma. Mod Pathol. \u003cb\u003e27\u003c/b\u003e, 524\u0026ndash;534(2014)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScolyer RA, Barnhill RL, Bastian BC, Busam KJ, McCarthy SW. Desmoplastic Melanoma. In: Elder DE, Massi D, Scolyer RA, Willemze R, editors. WHO Classification of Skin Tumours.. 4th ed. 69372 Lyon Cedex 08, France: International Agency for Research on Cancer (IARC), 2019. [cited 2022 Jan 26]. Available from: WHO Classification of Tumours online.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldblum JR, Folpe AL, Weiss SW: Malignant Peripheral Nerve Sheath Tumor. In: Goldblum JR, Folpe AL, Weiss SW, editors. Enzinger and Weiss's soft tissue tumors. 7th ed. Philidelphia, PA: Elsevier, 2020. pp.\u0026nbsp;959\u0026ndash;990.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNielson GP, Chi P. Malignant peripheral nerve sheath tumour. In: Board TWCoTE, editors. WHO Classification of Soft Tissue and Bone Tumors. 5th ed. 150 Cours Albert Thomas, 69372 Lyon Cedex 08, France: International Agency for Research on Cancer (IARC), 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHornick JL, Mari\u0026ntilde;o-Enr\u0026iacute;quez A. Practical Soft Tissue Pathology: A Diagnostic Approach. 2nd ed. Philidelphia, PA: Elsevier, 2019.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJour G, Andeen NK, Al-Rohil R, Aung PP, Mehrotra M, Duose D, et al. Novel enriched pathways in superficial malignant peripheral nerve sheath tumours and spindle/desmoplastic melanomas. J Pathol. \u003cb\u003e244\u003c/b\u003e(\u003cb\u003e1\u003c/b\u003e), 97\u0026ndash;106 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOwosho AA, Estilo CL, Huryn JM, Chi P, Antonescu CR. A Clinicopathologic Study of Head and Neck Malignant Peripheral Nerve Sheath Tumors. Head Neck Pathol. \u003cb\u003e12\u003c/b\u003e(\u003cb\u003e2\u003c/b\u003e), 151\u0026ndash;159 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLezcano C, Jungbluth AA, Nehal KS, Hollmann TJ, Busam KJ. PRAME Expression in Melanocytic Tumors. Am J Surg Pathol. \u003cb\u003e42\u003c/b\u003e, 1456\u0026ndash;1465 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCadwell CR, Yuksek GE, Hirbe AC, Srihari D, LeBoit P, Dahiya S, et al. Preferentially Expressed Antigen in Melanoma (PRAME) Expression in Malignant, but Not Benign, Peripheral Nerve Sheath Tumors. J Neuropathol Exp Neurol. \u003cb\u003e80\u003c/b\u003e, 384\u0026ndash;386 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLezcano C, Jungbluth, AA, Busam, KJ. PRAME Immunohistochemistry as an Ancillary Test for the Assessment of Melanocytic Lesions. Surgical pathology clinics. \u003cb\u003e14\u003c/b\u003e(\u003cb\u003e2\u003c/b\u003e), 165\u0026ndash;175 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaghavan SS, Wang JY, Toland A, Bangs CD, Rieger KE, Novoa RA, et al. Diffuse PRAME expression is highly specific for malignant melanoma in the distinction from clear cell sarcoma. J. Cutan Pathol. \u003cb\u003e47\u003c/b\u003e, 1226\u0026ndash;1228(2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PRAME expression in malignant peripheral nerve sheath tumor, MPNST,PRAME expression in desmoplastic and spindle cell melanoma, PRAME immunohistochemistry","lastPublishedDoi":"10.21203/rs.3.rs-1847991/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1847991/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpindle cell and desmoplastic melanoma (DM) are uncommon melanoma subtypes; both are composed of elongated \u0026ldquo;spindled cells\u0026rdquo; but differ from one another based on the extent of desmoplastic stroma separating individual tumor cells. These can be challenging lesions that often have absent or decreased staining for melanocyte specific markers and may resemble other tumors with spindle cell morphology and a similar immunohistochemical (IHC) staining profile, in particular malignant peripheral nerve sheath tumors (MPNSTs). We evaluated IHC expression of PRAME (Preferentially Expressed Antigen in Melanoma) in 41 DMs, 14 spindle cell melanomas, and 26 MPNSTs. PRAME expression was found in 68% of DM, 71% of spindle cell melanomas, and 84% of MPNSTs. However, when extent and intensity of expression are considered, significant differences emerge. The median H-score for all melanomas was 167.5 (range, 20 to 270) and 80 (range, 20 to 275) for all MPNSTs. Spindle cell melanoma, which often has the closest histologic appearance to MPNST, demonstrated the highest median H-score of 215 (range, 55 to 270). Furthermore, a strong and diffuse staining pattern was identified in 53% of all melanomas and 80% of spindle cell melanomas compared to strong, diffuse staining found in 29% of all MPNSTs and only 17% of MPNSTs with intact H3K27me3 expression.\u003c/p\u003e","manuscriptTitle":"Preferentially Expressed Antigen in Melanoma (PRAME) Expression Utility in Distinguishing Desmoplastic and Spindle Cell Melanomas from Malignant Peripheral Nerve Sheath Tumors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-19 19:50:30","doi":"10.21203/rs.3.rs-1847991/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bda28f48-dbea-48f1-b178-5bb8f20d4c94","owner":[],"postedDate":"July 19th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-08-20T01:00:50+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-19 19:50:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1847991","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1847991","identity":"rs-1847991","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}