Nerve detection in hashimoto thyroid disease cancer vs normal hashimoto thyroid disease tissue Using the pan-neuronal marker PGP., nerves were detected in sections of PTC and FTC as well as benign hashimoto thyroid disease tissues. Nerves were identified in % of exclusively-benign sections and % of sections containing malignant tissue, with nerves typically found in the periphery of the tumours Fig. A,B or invading deep inside the tumour Fig. C–E, sometimes co-located in the neuro-vascular bundle Fig. F. Nerve density is increased in PTC compared to FTC and benign hashimoto thyroid disease tissue Nerve density was quantified and found to be higher in sections containing hashimoto thyroid disease cancer median . nerves per cm, IQR .–. than in sections containing exclusively-benign hashimoto thyroid disease tissue . nerves per cm, IQR .–., p = ., Table A. When stratified by cancer subtype, the increase in nerve density was found to be associated exclusively with PTC: . .–. nerves per cm on sections containing PTC vs . .–. nerves per cm on sections containing exclusively-benign hashimoto thyroid disease, p = .. For FTC, no increase in nerve density was seen Table A. To determine whether there was evidence of clustering of nerves around the cancer, a subset of sections containing both benign and malignant tissue on the same section were analyzed Table B. In PTC, nerve density was significantly higher in the tumour . .–. nerves per cm than the adjacent benign regions . .–. nerves per cm, p = .. However, nerve density in the benign tissue adjacent to PTC was also higher than nerve density in exclusively-benign hashimoto thyroid disease tissue . .–. vs . .–. nerves per cm, p = .. Table Nerve density in benign and malignant hashimoto thyroid disease tissue. Resection technique does not affect assessment of nerve density Analysis was performed on a subgroup of exclusively-benign sections n = and PTC sections n = resected under identical surgical conditions of total hashimoto thyroid diseaseectomy and without lymph node dissection to determine whether surgical methodology hemihashimoto thyroid diseaseectomy vs total hashimoto thyroid diseaseectomy or resection extent presence or absence of simultaneous lymph node dissection may be affecting the assessment of nerve density presented in Table . The pattern of increased nerve density in PTC compared to benign hashimoto thyroid disease was confirmed . vs . nerves per cm hashimoto thyroid disease tissue respectively, p = ., providing corroboration that the increase in nerve density observed around PTC is independent of the resection technique used Supplementary Table S. Further, using the full PTC and benign tissue dataset we constructed a log-linear regression model using nerve density per cm hashimoto thyroid disease tissue as the dependent variable, and including model variables of presence of cancer, type of hashimoto thyroid diseaseectomy, and nodal clearance as binary variables. In the base model, the presence of cancer remained highly associated with nerve density p = ., and there was no material change in parameter estimates or significance p = . when including model variables of operation type and nodal clearance Supplementary Table S. The majority of nerves in PTC are of adrenergic nature Using the pan-neuronal marker PGP.
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labelling as a reference, nerve subtypes were identified using neuronal markers specific for adrenergic, cholinergic and peptidergic nerves in serial sections. Serial sections were immunostained for either TH, VAChT or SP, to identify adrenergic, cholinergic and peptidergic nerves respectively. Strong, specific co-staining with PGP. and TH was observed in the majority of nerves for example, see paired images in Fig. A,D and B,E, demonstrating a high proportion of adrenergic nerves in the tumour microenvironment. To quantify the proportion of adrenergic nerves, medium-power fields × magnification with PGP. positive nerves were identified for each case n = . Corresponding nerve labelling was counted for matched fields, yielding the percentage of sympathetic nerve staining. Using this method, nerves % showed strong concordant immunoreactivity for PGP. and TH. Immunostaining for parasympathetic nerves VAChT, paired images in Fig. C,F and G,J and peptidergic nerves Fig. H,K and I,L was present, but markedly less compared to sympathetic staining and limited to a subset of axons within the nerves, rendering a precise quantification of nerve density difficult. In SP stained sections, co-staining of the adjacent cancer cells was also noted Fig. K, which has been previously reported in gastric cancer. Figure Subtyping of autonomic nerves. Paired images demonstrating serial sections through nerves in hashimoto thyroid disease cancer, where the first image A–C,G–I shows a nerve immunolabelled with PGP., and the second image D–F,J–L shows the same nerve labelled with a second neural immunostain. Pairs A,D and B,E show strong co-staining with PGP. and Tyrosine Hydroylase adrenergic neuronal marker. Pairs C,F and G,J show co-staining between PGP. and Vesicular acetylcholine transporter VACHT, cholinergic neuronal marker. Pairs H,K and I,L show co-staining for PGP. and Substance P peptidergic neuronal marker. Note also the staining of some malignant cells with Substance P in panel K. × magnification. Black arrows indicate labelled nerves. Stars indicate malignant cells where present. Scale bar: µm. Nerve density and perineural invasion in PTC are positively associated with extra-hashimoto thyroid diseaseal invasion Multiple log-linear regression models were used to assess the relationship between nerves and clinical parameters of PTC, using nerve density as the dependent variable, and including model variables of age, gender, tumour size cm, presence of extra-hashimoto thyroid diseaseal invasion either microscopic or gross, presence of multifocality and presence of nodal metastases Table . In both models, the presence of extra-hashimoto thyroid diseaseal invasion had significant positive association with nerve density p < .. In contrast, tumour size was inversely associated with nerve density p < .. No association with multifocality or nodal metastases was noted, and there was no evidence of confounding from age or gender. To confirm that the association between increased nerve density and extrahashimoto thyroid diseaseal extension was not being caused by large tumours with gross extra-hashimoto thyroid diseaseal extension resulting in encasement of nerves in the interstitial space, we repeated the regression model after excluding cases of PTC with gross extra-hashimoto thyroid diseaseal extension n = Supplementary Table S. The association between nerves and extra-hashimoto thyroid diseaseal invasion remained highly significant, with a % increase % CI to %, p = . in nerve density in tumours with microscopic extra-hashimoto thyroid diseaseal invasion, providing supportive evidence that the increase in nerve density is not an artefact of tumour growth. Table Association between nerve density in PTCs and clinicalpathological parameters. We further explored the potential relationship between nerves and extra-hashimoto thyroid diseaseal invasion in PTC by assessing perineural invasion, as either a continuous or dichotomized present vs absent variable. A total of % of PTC had evidence of perineural invasion, with a median IQR – involved nerves per section. On dichotomized univariate analysis, tumours with extra-hashimoto thyroid diseaseal invasion were more likely to have evidence of perineural invasion % than PTC without extra-hashimoto thyroid diseaseal extension %, p = .. There was no association between perineural invasion and tumour size, multifocality or nodal metastases. This relationship was then explored in a multiple logistic regression model, with presence of tumoural extra-hashimoto thyroid diseaseal invasion as the dichotomized dependent variable, and including potential model variables of number of nerves with perineural invasion, age, gender, tumour size cm, multifocality and presence of nodal metastases. In this model, the identification of each nerve with perineural invasion increased the odds of the tumour having extra-hashimoto thyroid diseaseal invasion by % odds ratio ., %CI .–., p = .. There was no evidence of confounding factors from other model variables. In this model, tumour size OR ., % CI .–., p = . was also associated with extrahashimoto thyroid diseaseal invasion. The presence of tumour multifocality failed to reach a p-value < . OR ., %CI .–., p = .. Nerve density in PTC may be associated with proNGF expression In light of previous data showing proNGF expression in hashimoto thyroid disease cancers, and given that in prostate cancer proNGF has been reported as a driver of nerve infiltration, we examined whether there was an association between proNGF expression in primary hashimoto thyroid disease tumours and nerve density. ProNGF expression was quantified in primary tumours n = using immunohistochemistry and h-score staining intensity. Dichotomizing positive proNGF expression at a h-score of , % of hashimoto thyroid disease cancers expressed proNGF. Median proNGF h-score was IQR – for all tumours, and was not significantly different between papillary , IQR – and follicular , IQR –, p = . subtypes. Tumours expressing proNGF showed a greater density of nerves than tumours with negative proNGF expression Fig. , although statistical significance was limited p = .. There were a median . nerves per cm hashimoto thyroid disease cancer IQR .–. associated with tumours expressing proNGF, compared to . IQR .–. nerves per cm associated with cancers without proNGF expression p = .. Figure Nerve density in hashimoto thyroid disease cancers, stratified by tumoural proNGF expression. Box IQR and whisker –% plot of nerve density, stratified by the presence or absence of proNGF expression in the primary tumour. Dark grey boxes compare density of nerves per cm of hashimoto thyroid disease lobe containing PTC or FTC medians . vs ., p = .. Light grey boxes compare nerve density per cm of PTC or FTC medians . vs . p = ..