A case control study examining the population of epidermal and dermal pathology, inflammatory infiltrate and neural milieu of primary prurigo nodularis using S-100 and Toluidine blue stain and its therapeutic implications

doi:10.21203/rs.3.rs-2886265/v1

A cross-sectional observational study was in cases of untreated primary prurigo nodularis cases with histopathological assessment in 49 cases of prurigo from lesional and non lesional uninvolved skin with assessment of epidermal and dermal changes, composition of dermal infiltrate and S-100 & toluidine blue staining to assess the role of nerve and mast cells. The most common histological changes seen in lesional skin were hyperkeratosis( 98%), irregular hyperplasis (69.4%) hypergranulosis (69.4%), sub epidermal clefting( 6%), vertical collagen bundles (51.0%) & dermal fibrosis (48.9%). Chronic inflammatory infiltrate was seen in all cases (100%) and the most common inflammatory cells seen being found were lymphocytes (100%) followed by, eosinophils (18.4%), plasma cells (8.2%) patients neutrophils. (2.0%). There was a marked increase in the expression of S-100 (6.92 ± 3.40 Vs 3.94 ± 2.15, p < 0.001) and toludidine blue (4.99 ± 4.47 Vs 1.22 ± 1.28, p < 0.001) in the lesional skin as compared to the non lesional skin. Thus we can infer that the epidermal and dermal pathology is dependent on lymphocytes, mast cells and neural hyperplasia which perpetuate the pathology of PN by triggering the itch-inflammation cycle. Thus apart from immunosuppressive agents that target lymphocytes and their cytokines therapy targeted at mast cells and neural proliferation may be needed to treat prurigo nodularis.

Prurigo nodularis (PN), also referred to as Nodular Prurigo or Hyde's prurigo, is a chronic pruritic dermatosis which manifests as multiple papulonodular lesions occurring on the background of intense pruritus.¹ It is believed to be a reaction pattern consequent to chronic scratching with a heightened role of neuronal sensitization and consequential persistent itch-scratch-itch cycle.²

PN may be primary (idiopathic) or secondary. Secondary PN could be due to dermatological or systemic diseases, malignancies, psychogenic and neuropathic conditions.³ An atopic diathesis is implicated in almost half of PN patients, and superimposition with these diseases may alter the histopathological findings.⁴

The pathogenesis of PN is considered to be multifactorial, with numerous cells and cytokines working in conjunction to produce an immune response. It is believed that keratinocytes communicate with sensory nerve endings, especially pruriceptors, via synaptic-like contacts and the manifestations are due to these interactions and to interactions with other cells of their environment, such as mast cells, eosinophils, dendritic cells or T lymphocytes. Varied subsets of CD4 + cells, including TH1, TH2 and TH17, are thought to play significant role in the pathogenesis of PN which is evident by the abnormal cytokine levels (IL-4, IL-13 and IL-31).⁵ The prevalent hypothesis suggests that PN is primarily a Th2 mediated disease.^5,6 The itching leads to a focus on neuronal plasticity and Pautrier first reported hyperplasia of nerve fibres, referred to as “Pautrier’s neuroma”.^7,8 Subsequent studies revealed dermal neural hyperplasia with reduced intraepidermal nerve fibre density.⁹

The histopathology shows a constellation of features and is not characterized by a uniform pathology and this is due to differences in biopsy site time since lesion was last scratched and stage of a lesion, that is evolving, fully developed, or in healing stage.¹⁰ Also, the histological picture may often lag behind the clinical appearance.¹¹

The epidermis shows prominent orthohyperkeratosis, focal parakeratosis, focal or broad hypergranulosis and irregular acanthosis with hyperplasia which could be papillomatous, psoriasiform or pseudoepitheliomatous, with irregular elongation of rete ridges.¹⁰ Weigelt et al reported the "hairy palm sign", described as a hair follicle in the centre of each acanthotic downgrowth in non-volar skin with compact and thick stratum corneum.¹⁰

The dermis shows vascular hyperplasia, with increased small blood vessels in both papillary and reticular dermis along with angiogenesis.¹⁰ Collagen remodelling along with vertical orientation of collagen bundles is a prominent feature in papillary dermis, but also in reticular dermis in about one third of cases.¹¹ There is associated subepidermal fibrin deposition and stellate dermal fibroblasts that can lead to papillary fibrosis.¹¹ The inflammatory cell infiltrate in the dermis is usually perivascular and is lymphohistiocytic in almost all cases, with a minor role of mast cells, neutrophils and eosinophils.

The aim of our study was to perform a case control histopathological assessment of cases of primary PN with a focus on neural pathology and mast cells.

A cross-sectional observational study was conducted in the OPD of dermatology, venereology and leprosy and department of pathology in a tertiary hospital. To rule out the acquired causes of PN, biochemical tests and radiological tests were performed (complete blood cell count, fasting blood sugar, kidney and liver function tests, hepatitis B, C and HIV serology, ESR, stool for occult blood, Chest X-ray and ultrasound abdomen) . The inclusion criterion were clinically diagnosed and histologically confirmed cases of PN who are not on active treatment for prurigo nodularis for four weeks , with a age more than 18 years of either sex. (Table 1)

Histopathology was performed in 60 cases from a lesional and contralateral non-lesional non-involved skin, out of which 49 cases were histopathologically proven to be PN. Inflammatory cell density was semi-quantitatively measured as mild, moderate and dense.

Apart from haematoxylin and eosin, S-100 (Biocare Medical, 4C4.9, mouse monoclonal) was used to evaluate dermal nerve density and toluidine blue stain was used to visualise mast cells. Numbers of mast cells were counted in dermis at perivascular, periadnexal and interstitial locations in 10 fields with 40X magnification and an average per high power field was taken.

Qualitative variables were correlated using Chi-Square test/Fisher's exact test. To assess S-100, non-parametric tests were used to make statistical inference as data were notnormally distributed and paired Wilcoxon test was used to explore the difference between paired variables. A p value of < 0.05 was considered statistically significant.

A summary of the demographic profile is listed in Table 1. The lesional and non lesional samples were analysed in 4X, 10X and 40X magnification respectively (Fig 1 to 8). Out of the 49 samples evaluated, chronic inflammatory infiltrate was seen in all the cases (100%) and cell density was found to be mild in 17 (34.7%), moderate in 28 (57.1%) patients and dense in 4(8.2%) patients with the most common inflammatory cells seen being found were lymphocytes(100%), eosinophils in 9 (18.4%) patients, plasma cells 4 (8.2%) patients and (2.0%) neutrophils. (Table 2, Fig 3 to 8). Pseudoepitheliomatous hyperplasia was observed in 10 (20.4%) patients (Fig 1,3,5,8), and vertical collagen bundles were found to be present in 25 (51.0%) patients and absent in 24 (49.0%) patients. (Fig 1,3,4,7,8) Another previously undescribed histological finding that was observed was sub epidermal clefting seen in 3 PN samples (6.12%). (Table2, Fig 4,7)

S-100 IHC staining found a marked increase in the lesional skin (6.92 ± 3.40 Vs 3.94 ± 2.15, p<0.001) (Table 3, Fig 9,10). Also mast cells was pronounced in the lesional skin. (Table 4, Fig 11 to 13)

Our study aimed at comparing the histopathological epidermal and dermal changes including mast cells and neural hyperplasia in a large cohort of primary PM patients, and compare them with non-lesional skin samples and certain prominent findings included parakeratosis, dermal collagen remodelling with vertical orientation of collagen bundles, marked lymphocytic infiltrate, mast cells &neural hyperplasia.. Furthermore, we detected a new finding "epidermal clefting" in the PN skin. The features of PN in conjunction with our study are summarised in Table 5

A working hypothesis of PN is that specific Th2 cell response with targeted cytokines and neural hyperplasia lead to PN with an ancillary role of inflammatory cells. The induction of pruritus gives rise to neuronal inflammation and leads to accumulation of inflammatory cells in dermis and scratching leads to destruction of peripheral nerves in epidermis and consequent activation or retrograde signalling pathway which causes itch. It is thus pertinent to examine the key cellular and neural players in the disorder as that can entail a better knowledge of the disease and lead to the use of targeted therapies.¹⁸

The inflammatory cell infiltrate in the dermis was perivascular in location with prominent cells being lymphocytes, mast cells and eosinophils. In our study, we found lymphocytic infiltrate in all the cases. Lymphocytic cells, especially subsets of CD4+ T helper cells are thought to play significant role in the pathogenesis of PN, as evidenced by their presence in all the lesional samples in our study. Different cytokines induce different T helper cells (Th cells), which in turn contribute to the inflammatory cascade. Th2 and Th17 cytokines are depicted to have a profound role.⁵In a previous study, lesional and non-lesional PN skin were compared with the use of STAT 1,3, and 6 IHC, as surrogate markers for Th1, Th2/Th17/Th22 and Th2 effector response and a marked expression of STAT 3 and 6 was noted.⁶ This suggests that signature cytokines seem to be IL-4, IL-6, IL-31, IL-17& IL 22, which mediate the epidermal and dermal pathology of PN.¹⁹

Other inflammatory cells observed include eosinophils and neutrophils. Eosinophils release eosinophilic granule proteins, which have cytotoxic activities and result in exacerbation of inflammation and damaging nervous tissue,^20,21Eosinophils also release NGF, thereby contributing to the neurohyperplasia.²²

Mast cells are found to be increased in number in PN lesional skin as depicted in our study using toluidine blue stain.[Fig]. Morphologically, they become dendritic with an enlarged cell body size with reduction in cytoplasmic granules, suggestive of granular release into surrounding tissue.^23,24As they lie in close proximity to nerves , they express increased levels of NGFr, and are one of the major sources of NGFin addition to many itch mediators like histamine, prostaglandins and tryptase.^24,25Additionally, they induce fibroblast proliferation and synthesis of collagen, the orientation of which is modified in PN.²⁷ A recent paper used MRGPRX2 (protein and mRNA) using immunohistochemistry, and found a pronounced expression on mast cells in PN which shows that this cell is important in PN.²⁷

An important aspect of the pathophysiology of PN is the neural proliferation seen in PN which was described as the Pautrier's neuroma. While some authors have concurred with this hypothesis there are contradictory findings in otherstudies.^{28, 29}In our study, we used S-100, a special neural stain to detect the proliferative changes of the nerve and detect neural hyperplasia with a significant difference between lesional and non-lesional, which validates the role of neural hyperplasia in PN.³⁰

This was also noted by Harris et al( 25 patients) and Johansson et al ( 7 patients), ^{31, 32}while Doyle et al and Rowland Payne et al did not find the presence of neuromas and observed neural hyperplasia in only two out of 46 patients respectively.^{33, 34}Weigelt et al had opined that neuroma formation is an uncommon finding in PN and not a significant criterion for disease diagnosis, however, a special stain was not used in their study.¹⁰ Ours is the largest study that used S-100 and neural hyperplasia is a prominent finding and can explain the role of neurotrophins which via NGF receptors (NGFr)and nerve growth factor (NGF), released by keratinocytes, as well as inflammatory cells such as the mast cells, lymphocytes, and eosinophils can lead to neuritic growth and arborisation, with concomitant up-regulation of neuropeptides, like Substance P (SP) in PN. Neuropeptides, such as SP and CGRP, also lead to persistence of neural hyperplasia.³⁵Dermalhyperinnervationof sensory SP positive nerves may contribute to the intense itching sensation independent of pruritus, as well as modulate cellular functional responses by activating and moderating differentiation of immune cells like lymphocytes and macrophages.³⁶ Similarly, CGRP contributes to inflammatory cell infiltration and vasodilatation, forming a link between skin inflammation and nervous system by induction of neurogenic vasodilatation and neurogenic inflammation.³⁷

Our findings reiterate the role of nerve proliferation and also focus on mast cells in the pathogenesis of PN, in addition to lymphocytes. The neural hyperplasia and the concomitant epidermal proliferation can be explained by neurotrophins and the systemic and cutaneous Th2 immune polarization with pronounced STAT 6 expression.^6,18These findings may help us to use directed therapeutic agents that work on the neural element like nortryptiline, pregabalin and thalidomide.^18,38,39AsIL-4, IL-13 and IL 31 are the prominent cytokines which mediate their action via specific JAK receptors (IL-4=JAK1, JAK3; IL-13=JAK1 & TYK2 & IL-31=JAK1) thus JAK inhibitors would be potentially useful in PN , as they can inhibit multiple cytokines, unlike the specific cytokine directed therapeutic agents (nemolizumab, dupilumab, tralokinumab, lebrikizumab and vixarelimab).⁴⁰

A drawback in our study is that we did not include the atopic sub type of PN and did not perform cytokine signatures in our biopsy specimens. A larger cohort of patients with the use of pathogen directed therapeutic agents with post therapy tissue analysis would have been the ideal study.

Funding sources: Nil

Conflict of interest disclosures – Nil, Ethics approval: Obtained

Authorship

Diksha Agrawal- Conception and design, acquisition of data, analysis and interpretation of data, drafting the manuscript.
Kabir Sardana- Conception and design, acquisition of data, analysis and interpretation of data, drafting the manuscript.
Sinu Rose Mathachan- Design,Revision of critically important intellectual content, analysis and interpretation of data, , drafting the manuscript.
Minakshi Bhardwaj- Conception and design, analysis and interpretation of data, drafting the manuscript.
Arvind Ahuja- Analysis and interpretation of data, drafting the manuscript.
Swasti Jain- Analysis and interpretation of data, drafting the manuscript.
Sanjeet Panesar- Data analysis, statistics

Acknowledgment: The patients in this manuscript have given written informed consent for publication of their case details.

Hyde JN, Montgomery FHA. Practical treatise on diseases of the skin for the use of students and practitioners. Lea &Febiger, Philadelphia, 1909
Pereira MP, Steinke S, Zeidler C, Forner C, Riepe C. European academy of dermatology and venereology European prurigo project: expert consensus on the definition, classification and terminology of chronic prurigo. J EurAcadDermatolVenereol. 2018 Jul; 32(7):1059-1065
Zeidler C, Tsianker A, Pereira M, Stander H, Yosipovitch G et al. Chronic Prurigo of Nodular Type: A Review. ActaDermVenereol 2018; 98: 173-9.
Iking A, Grundmann S, Chatzigeorgakidis E, Phan NQ, Klein D, Ständer S. Prurigo as a symptom of atopic and non‐atopic diseases: Aetiological survey in a consecutive cohort of 108 patients. J EurAcadDermatolVenereol 2013; 27: 550–7
Fukushi S, Yamasaki K, Aiba S. Nuclear localization of activated STAT6 and STAT3 in epidermis of prurigo nodularis. Br J Dermatol. 2011 Nov;165(5):990-6.
Agrawal D, Sardana K, Mathachan SR, Bhardwaj M, Ahuja A, Jain S. A prospective study examining the expression of STAT 1, 3, 6 in prurigo nodularis lesions with its immunopathogenic and therapeutic implications. J Cosmet Dermatol. 2022 Sep;21(9):4009-4015
Zeidler C, S. Stander. The pathogenesis of Prurigonodularis – ‗Super-Itch‘ in exploration. Eur J Pain 20 (2016) 37--40
Pautrier LM. [Le neurome de la lichenification circonscrite nodulaire chronique (lichen rubber obtusus corne prurigo nodularis).Ann Dermatol Syph 1934;7:897.
Bobko S, Zeidler C, Osada N, Riepe C, Pfleiderer B, Pogatzki-Zahn E, et al. Intraepidermal Nerve Fibre Density is Decreased in Lesional and InterlesionalPrurigoNodularis and Reconstitutes on Healing of Lesions. ActaDermVenereol. 2016 Mar; 96(3):404-6.
Weigelt N, Metze D, Ständer S. Prurigonodularis: systematic analysis of 58 histological criteria in 136 patients. J CutanPathol. 2010; 37(5): 578–58.
Johnston RB. Tumors of the epidermis. Patterson JW editor. Weedon ‘s skin pathology. 4th ed. Elsevier Heath Sciences; 2016
Rothmann S. A case for diagnosis; lichen obtusus? prurigonodularis? Arch DermSyphilol 1948; 57(3 Pt. 2): 477.
McKenzie AW, Stubbing DG, Elvy BL. Prurigonodularis and gluten enteropathy. Br J Dermatol 1976; 95: 89.
Mattila JO, Vornanen M, Vaara J, Katila ML. Mycobacteria in prurigonodularis: the cause or a consequence? J Am AcadDermatol 1996; 34: 224
Miyauchi H, Uehara M. Follicular occurrence of prurigonodularis. J CutanPathol 1988; 15: 208
Doyle JA, Conolly SM, Hunziker N, Winkelmann RK. Prurigonodularis: a reappraisal of the clinical and histologic features. J CutanPathol 1979; 6: 392
Jacob CI, Patten SF. Strongyloides stercoralis infection presenting as generalized prurigonodularis and lichen simplex chronicus. J Am AcadDermatol 1999; 41: 357.
Agrawal D, Sardana K, Mathachan SR, Bhardwaj M, Ahuja A, Jain S. A Prospective Study Examining the Effect of Selected Topical and Systemic Drugs on Pruritus Grading System Score and STAT 6 Expression in Patients of Prurigo Nodularis. Indian J Dermatol. 2021 Nov-Dec;66(6):638-644.
Seif F, Khoshmirsafa M, AazamiH, et al. The role of JAK-STAT signaling pathway and its regulators in the fate of T helper cells. Cell Commun Signal. 2017; 15:23.
Perez GL, Peters MS, Reda AM, Butterfield JH, Peterson EA, Leiferman KM. Mast cells, neutrophils, and eosinophils in prurigonodularis. Arch Dermatol. 1993 Jul 1;129(7):861-5.
Sunohara N, Furukawa S, Nishio T, Mukoyama M, Satoyoshi E. Neurotoxicity of human eosinophils towards peripheral nerves. J. Neurol. Sci. 1989; 92: 1–7.
Matsumura S, Terao M, Murota H, Katayama I. Th2 cytokines enhance TrkA expression, upregulate proliferation, and downregulate differentiation of keratinocytes. J Dermatol Sci. 2015 Jun 1;78(3):215-23.
Liang Y, Jacobi JA, Marcusson M, Haak-Frendscho M, Johansson O. Dendritic mast cells in prurigonodularis skin. Eur J Dermatol. 1999; 9: 297–9.
Lee MR, Schumack S. Prurigonodularis: A review. Australas J Dermatol 2005; 46: 211-20.
Schmelz M, Schmidt R, Weidner C, Hilliges M, Torebjörk HE, Handwerker HO. Chemical response pattern of different classes of C-nociceptors to pruritogens and algogens. J. Neurophysiol. 2003; 89: 2441–8.
Groneberg DA, Serowka F, Peckenschneider N, Artuc M, Grützkau A, et al. Gene expression and regulation of nerve growth factor in atopic dermatitis mast cells and the human mast cell line-1. J Neuroimmunol. 2005; 161: 87- 92
Ogasawara H, Noguchi M. Therapeutic Potential of MRGPRX2 Inhibitors on Mast Cells. Cells. 2021 Oct 27;10(11):2906
Cowan MA. Neurohistological changes in prurigonodularis. Arch Dermatol 1964; 89:754–8
Lindley RP, Payne CM. Neural hyperplasia is not a diagnostic prerequisite in nodular prurigo. A controlled morphometric microscopic study of 26 biopsy specimens. J CutanPathol. 1989 Feb;16(1):14-8.
Sedaghat F, Notopoulos A. S100 protein family and its application in clinical practice. Hippokratia. 2008 Oct;12(4):198.
Harris B, Harris K, Penneys NS. Demonstration by S-100 protein staining of increased numbers of nerves in the papillary dermis of patients with prurigonodularis. J Am AcadDermatol 1992; 26:56-8
Johansson O, Liang Y, Heilborn JD, Marcusson JA. Langerhans cells in prurigonodularis investigated by HLA-DR and S-100 immunofluorescence double staining. J Dermatol Sci. 1998; 17(1) :24-32.
Doyle JA, Connolly SM, Hunziker N, Winkelmann RK. Prurigonodularis: a reappraisal of the clinical and histologic features. J CutanPathol 1979; 6: 392–403.
Rowland Payne CM, Wilkinson JD, McKee PH, Jurecka W, Black MM. Nodular prurigo- a clinicopathological study of 46 patients. Br J Dermatol. 1985; 113(4):431– 9
Verge VM, Richardson PM, Wiesenfeld-Hallin Z, et al. Differential influence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide suppression in adult sensory neurons. J Neurosci 1995; 15:2081–96
Haas S, Capellino S, Phan NQ, et al. Low density of sympathetic nerve fibers relative to substance P-positive nerve fibers in lesional skin of chronic pruritus and prurigo nodularis. J Dermatol Sci. 2010;58(3):193–197
Ansel JC, Armstrong CA, Song IS, Quinlan KL, Olerud JE et al. Interactions of the skin and nervous system. J. Invest. Dermatol. 1997; 2: 23–6
Briatico G, Scharf C, Di Brizzi EV, Pellerone S, Buononato D, Squadrilli F, Babino G, Argenziano G, Giorgio CM. Amitriptyline and azathioprine: an effective therapeutic approach in prurigo nodularis resistant to conventional therapies. J Dtsch Dermatol Ges. 2023 Mar;21(3):291-293
. Labib A, Ju T, Vander Does A, Yosipovitch G. Immunotargets and Therapy for Prurigo Nodularis. Immunotargets Ther. 2022 Apr 26;11:11-21
Müller S, Bieber T, Ständer S. Therapeutic potential of biologics in prurigo nodularis. Expert Opin Biol Ther. 2022 Jan;22(1):47-58.
Inui K, Ugajin T, Namiki T, Yokozeki H. Chronic prurigo: A retrospective study of 168 cases. J Dermatol. 2020; 47(3):283–289.
Tanaka M, Aiba S, Matsumura N, Aoyama H, Tagami H. Prurigo nodularis consists of two distinct forms: early-onset atopic and late-onset non-atopic. Dermatology 1995; 190: 269–76.
Johansson O, Liang Y, Marcusson JA, Reimert CM. Eosinophil cationic protein-and eosinophil-derived neurotoxin/eosinophil protein X-immunoreactive eosinophils in prurigo nodularis. Arch Dermatol Res. 2000 Aug 1;292(8):371-8.
Liang Y, Marcusson JA, Jacobi HH, Haak-Frendscho M, Johansson O. Histamine-containing mast cells and their relationship to NGFr-immunoreactive nerves in prurigonodularis: a reappraisal. J. Cutan. Pathol 1998; 25: 189–98
Schmelz M, Schmidt R, Weidner C, Hilliges M, Torebjörk HE, Handwerker HO. Chemical response pattern of different classes of C-nociceptors to pruritogens and algogens. J. Neurophysiol. 2003; 89: 2441–8.
Hashimoto T, Satoh T, Yokozeki H. Protective role of STAT6 in basophil-dependent prurigo-like allergic skin inflammation. J Immunol. 2015 May 15;194(10):4631-40.
Redrup AC, Howard BP, MacGlashan DW Jr, Kagey-Sobotka A, Lichtenstein LM, Schroeder JT. Differential regulation of IL-4 and IL-13 secretion by human basophils: their relationship to histamine release in mixed leukocyte cultures. J Immunol. 1998 Feb 15;160(4):1957-64.
Hashimoto T, Rosen JD, Sanders KM, Yosipovitch G. Possible roles of basophils in chronic itch. Exp Dermatol. 2019; 28(12): 1373-9.

Table 1: Demographic profile of PN patients

Patient characteristics	Number of patients (Percentage)
Age (Mean ± SD)	37.49 ± 16.04 years
Gender
Male	33 (67.3%)
Female	16 (32.7%)
Duration of Disease (Mean ± SD)	2.69 ± 3.43 years
Number of lesions
<10	3 (6.1%)
10-100	29 (59.2%)
>100	17 (34.7%)
Lesion Morphology
Excoriated	46 (93.9%)
Intact	3 (6.1%)
Type of lesion
Papule	48 (98.0%)
Nodule	47 (95.9%)
Plaque	21 (42.9%)
Lesion Distribution
Upper Limb	41 (83.7%)
Lower Limb	48 (98.0%)
Trunk	35 (71.4%)
Buttocks	21 (42.9%)

Table 2: Characteristic histological findings in prurigo nodularis (comparison to Weigelt et al)

Histological criteria	Lesional skin (n=49)	Non-lesional skin (n=49)	Weigelt et al¹⁰ (n=136)	Using chi square test (between lesional skin of index cases versus Weigelt et al) p-value
EPIDERMAL FINDINGS
Hyperkeratosis	48(98%)
Orthohyperkeratosis	47 (95.9%)	21 (42.9%)	120 (88.2%)	0.191
Parakeratosis	20 (40.8%)	0 (0.0%)	6 (4.4%)	<0.001(s)

2. Epidermal Hyperplasia	48 (98%)	0 (0.0%)	125 (91.9%)	0.141
Pseudoepitheliomatous hyperplasia	10 (20.4%)	0 (0.0%)	39 (28.7%)	0.262
Psoriasiform hyperplasia	4 (8.2%)	0 (0.0%)	4 (2.9%)	0.123
Irregular hyperplasia	34 (69.4%)	3 (6.1%)	79 (58.1%)	0.164

3. Hypergranulosis	34 (69.4%)	2 (4.1%)	71 (52.2%)	0.037(s)
DERMAL FINDINGS
1. Vertically oriented collagen bundles	25 (51.0%)	0 (0.0%)	87 (64.0%)	0.112
2. Dermal fibrosis	24 (48.9%)	2 (4.1%)	97 (71.3%) (papillary dermis)	0.005(s)
3. Dermal melanophages	12 (24.5%)	1 (2.0%)	13 (9.6%)	0.008(s)
4. Inflammatory Infiltrate	49 (100.0%)	28 (57.1%)	-
- Mild	17 (34.7%)	26 (53.1%)	-	-
- Moderate	28 (57.1%)	2 (4.1%)	-	-
- Dense	4 (8.2%)	0 (0.0%)	-	-
5. Inflammatory cells
- Lymphocytes	49 (100.0%)	27 (55.1%)	128 (94.1%)	Test can not be applied
- Eosinophils	9 (18.4%)	1 (2.0%)	64 (47.1%)	<0.001(s)
- Plasma Cells	4 (8.2%)	0 (0.0%)	2 (1.5%)	0.086
- Neutrophils	1(2.0%)	0 (0.0%)	45 (33.1%)	<0.001(s)
- Mast cells (n=32)	32 (100%)	30 (93.75%)	0 (0.0%)*	Test can’t be applied

* No special stain was used to visualise mast cells in Weigelt et al study

Table 3: Assessment of lesional and non-lesional skin for S-100 (n = 49)

Variable	S-100			P Value
Variable	Mean ± SD	Median (IQR)	Range	P Value
Lesional skin	6.92 ± 3.40	7.00 (5.00)	2.00 – 14.00	<0.001*
Non- Lesional skin	3.94 ± 2.15	4.00 (3.00)	0.00 – 10.00	<0.001*
Absolute Difference	2.98 ± 3.17	2.00 (3.00)	11.00 – 4.00
Percent Difference	35.1% ± 43.8%	44.4% (36.5)	100% - 133%

*Significant finding using Wilcoxon rank test

Table 4: Assessment of lesional and non-lesional skin for Toluidine blue stain. (n = 32)

Variable	Toluidine blue stained mast cells			P Value
Variable	Mean ± SD	Median (IQR)	Range	P Value
Lesional skin	4.99 ± 4.47	4.2 (3.52)	0.1- 24.0	<0.001*
Non- Lesional skin	1.22 ± 1.28	0.85 (1.18)	0.0 - 5.6	<0.001*
Absolute Difference	3.77 ± 3.74	2.9 (3.6)	0-18.4

*Significant finding using Wilcoxon rank test

Table 5: Summary of histological and neural findings in prurigo nodularis in conjunction with the present study

	Findings
Epidermis	Thick compact orthohyperkeratosis (hairy palm sign)¹⁰ Focal parakeratosis Epidermal hyperplasia Irregular Pseudoepitheliomatous Psoriasiform Hypergranulosis Elongation and broadening of the interpapillary pegs / rete ridges^12,13 Epidermal downgrowth¹⁴ Epidermal clefting Necrotic keratinocytes (solitary or in groups)¹⁰ Spongiosis¹⁵ Rarely: multinucleated keratinocytes, increased mitotic figures ^10,16 Superficial erosion (depending on biopsy area)¹⁰
Dermis	Dermal fibrosis (papillary/ reticular)¹¹ Vertically oriented collagen bundles Increased number of fibroblasts Increased number of capillaries, more dilatation.¹² Occasionally subepidermal accumulation of fibrin Dermal melanophages Dermal edema¹⁷ Inflammatory Infiltrate (predominantly perivascular> interstitial)¹¹ Lymphocytes Eosinophils Plasma cells Neutrophils Mast cells (special stain needed) Occasionally extravasal erythrocytes
Neural hyperplasia	Best detected when compared with non-lesional skin. Can be considered as a significant criterion.
Mast cells	Significantly increased in comparison to non-lesional skin. Can be considered as a significant criterion. Toluidine blue special stain used.

No competing interests reported.

A case control study examining the population of epidermal and dermal pathology, inflammatory infiltrate and neural milieu of primary prurigo nodularis using S-100 and Toluidine blue stain and its therapeutic implications

Status:

Version 1

Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

Declarations

References

Tables

Additional Declarations

Status:

Version 1