## SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at www.jidonline.org, and at https://doi.org/10.1016/j.jid.2023.07.021.

## REFERENCES

Organ Procurement and Transplantation Network. National data. https://optn.transplant.hrsa.gov/

data/view-data-reports/national-data/; 2023. (accessed May 30, 2023).

Vajdic CM, van Leeuwen MT. Cancer incidence and risk factors after solid organ transplantation. Int J Cancer 2009;125:1747-54.

Wehner MR, Niu J, Wheless L, Baker LX, Cohen OG, Margolis DJ, et al. Risks of multiple

skin cancers in organ transplant recipients: a cohort study in 2 administrative data sets. JAMA Dermatol 2021;157:1447-55.

Wehless L, Baker L, Edwards L, Anand N, Birdwell K, Hanlon A, et al. Development of phenotyping algorithms for the identification of organ transplant recipients: cohort study. JMIR Med Inform 2020;8:e18001.

# Serum IGF1 Is a Prognostic Marker for Resistance to Targeted Therapies and a Predictive Marker for Anti-IGF1 Receptor Therapy in Melanoma

<figure><img src="image_1.png" /></figure>

Journal of Investigative Dermatology (2024) 144, 422-426; doi:10.1016/j.jid.2023.07.016

## TO THE EDITOR

Since 2013, the use of combined BRAF inhibitor (BRAFi) and MAPK/extracellular signal-regulated kinase combine inhibitor (MEKi) has significantly improved survival in patients with metastatic melanoma. However, melanoma cells are able to evade these treatments, and the 5-year survival rate for patients with advanced melanoma is around 30% (Dummer et al., 2022). Among the mechanisms of acquired resistance to targeted therapies, IGF1R pathway has been identified as a driver of melanoma progression and BRAFi drug resistance (Girnita et al., 2014; Obenauf et al., 2015; Patel et al., 2021; Strub et al., 2018; Villanueva et al., 2010; Xu et al., 2022; Yeh et al., 2006). Furthermore, high circulating IGF1 levels have been associated with a higher risk of metastatic melanoma (Kucera et al., 2014), but this was not confirmed in a recent prospective study (Bradbury et al., 2019). Therefore, targeting the IGF1/IGF1R pathway emerges as an attractive therapeutic strategy for patients with melanoma who escape BRAFi/MEKi therapy. However, several phase II/III clinical trials of drugs targeting this pathway in unselected patients with different solid cancers have shown limited antitumor activity and/or unacceptable adverse effects (Qu et al., 2017; Truong et al., 2022), pointing to the need to identify patients for whom targeting IGF1/IGF1R would be beneficial.

We conducted a prospective clinical study to determine whether serum IGF1 could be a biomarker of treatment response in patients with melanoma. Serum IGF1 level was assessed in 200 patients and showed no relationships with clinical or biological parameters or subsequent progressive disease (Supplementary Table S1 and Supplementary Figure S1a). However, multivariate analysis showed that elevated serum IGF1 level at diagnosis, higher than the median (136 ng/ml), is an independent risk factor for subsequent progression in melanoma (Figure 1a). Likewise, serum IGF1 levels trended upward over time in patients with progressive disease among the 28 patients treated with BRAFi/MEKi-targeted therapies (Supplementary Figure S1b and Supplementary Table S2). In 73 or 67% of progression-free patients, serum IGF1 levels had slightly decreased over the previous 3 or 6 months, respectively (Figure 1b and c). In contrast, serum IGF1 levels increased by 28 and 21% on average for 80 and 78% of patients at the time of progression compared with IGF1 levels observed 3 or 6 months earlier, respectively. We observed the same trends when IGF1 levels were analyzed at intervals of 9 or 12 months (Supplementary Figure S1c-e). These changes in serum IGF1 levels were significantly different between progression-free patients and patients

whose disease progressed on targeted therapy (Supplementary Figure S1f). Increased serum IGF1 levels in a patient may therefore be a marker of melanoma escape to BRAFi/MEKi therapies.

To analyze IGF1 signaling, we developed three human BRAF<sup>V600E</sup> melanoma cell lines with acquired BRAFi resistance (A375-R, SKMel28-R, and 451Lu-R) (Supplementary Figure S2a). As previously described, they also presented a cross-resistance to cobimetinib (MEKi) (Gowrishankar et al., 2012). BRAFi/MEKi treatment strikingly reduced viability and extracellular signal-regulated kinase 1/2 activation in sensitive cells but not in resistant cells, whether as monolayer or spheroid cultures (Supplementary Figure S2b-d). This suggests that extracellular signal-regulated kinase 1/2 activation is BRAF independent in our resistant cell models.

Transcriptome analysis by RNA sequencing showed that IGF1 and its related signaling effectors were upregulated in BRAFi/MEKi-resistant cells compared with those in parental cells (Figure 2a). We confirmed that BRAFi/MEKi-resistant cells had elevated levels of phosphorylated IGF1R and protein kinase B compared with parental cells in both monolayer and spheroid cultures (Figure 2b and Supplementary Figure S2e). We also observed higher secreted IGF1 in the medium of resistant cells than in parental cells, particularly in resistant A375-R and 451Lu-R spheroids (Figure 2c and Supplementary Figure S2f). As previously described, BRAFi/MEKi dramatically increased IGF1 secretion by A375-S and 451Lu-S spheroids (Obenauf et al., 2015) but had little impact on SKMel28-

Abbreviations: BRAFi, BRAF inhibitor; IGF1R, IGF1 receptor; MEKi, MAPK/extracellular signal-regulated kinase inhibitor

Accepted manuscript published online 12 August 2023; corrected proof published online 8 December 2023

© 2023 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.
a

<table><thead><tr><th rowspan="2">Risk factors</th><th rowspan="2">n</th><th rowspan="2">progression<br>n</th><th colspan="2">Univariate analysis <sup>1</sup></th></tr><tr><th>Odds ratio (95% CI)</th><th>P</th></tr></thead><tbody><tr><td><strong>Age</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt;50 years-old</td><td>40</td><td>7</td><td>3.70 (2.55 to 5.52)</td><td>0.39</td></tr><tr><td>≥50 years-old</td><td>160</td><td>38</td><td>1.47 (0.63 to 3.85)</td><td></td></tr><tr><td><strong>Sex</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Male</td><td>95</td><td>22</td><td>0.89 (0.65 to 1.22)</td><td>0.83</td></tr><tr><td>Female</td><td>105</td><td>23</td><td>1.07 (0.55 to 2.09)</td><td></td></tr><tr><td><strong>Stage</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>I-II</td><td>131</td><td>12</td><td>0.30 (0.21 to 0.43)</td><td>&lt;0.001</td></tr><tr><td>III-IV</td><td>69</td><td>33</td><td>9.09 (4.36 to 20.10)</td><td></td></tr><tr><td><strong>Breslow</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt;2 mm</td><td>103</td><td>11</td><td>0.67 (0.48 to 0.93)</td><td>&lt;0.001</td></tr><tr><td>≥2 mm</td><td>93</td><td>31</td><td>4.18 (2.01 to 9.28)</td><td></td></tr><tr><td><strong>Ulceration</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td>122</td><td>15</td><td>0.43 (0.30 to 0.60)</td><td>&lt;0.001</td></tr><tr><td>yes</td><td>73</td><td>27</td><td>4.19 (2.07 to 8.78)</td><td></td></tr><tr><td><strong>BRAF</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Wild-type</td><td>36</td><td>20</td><td>1.19 (0.611 to 2.34)</td><td>0.87</td></tr><tr><td>mutated</td><td>41</td><td>22</td><td>0.926 (0.375 to 2.28)</td><td></td></tr><tr><td><strong>Serum IGF1</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>&lt;median<sup>2</sup></td><td>156</td><td>19</td><td>0.96 (0.70 to 1.32)</td><td>0.30</td></tr><tr><td>≥median<sup>2</sup></td><td>44</td><td>26</td><td>1.42 (0.73 to 2.81)</td><td></td></tr></tbody></table><table><thead><tr><th rowspan="2">Risk factors</th><th colspan="2">Multivariate analysis <sup>3</sup></th></tr><tr><th>Adjusted odds ratio (95% CI)</th><th>P</th></tr></thead><tbody><tr><td>Age (years)</td><td>1.05 (1.01 to 1.08)</td><td>0.006</td></tr><tr><td>Gender male</td><td>0.69 (0.29 to 1.57)</td><td>0.38</td></tr><tr><td>Ulceration</td><td>1.62 (0.67 to 3.93)</td><td>0.28</td></tr><tr><td>Breslow ≥2 mm</td><td>1.74 (0.71 to 4.49)</td><td>0.23</td></tr><tr><td>Advanced stage III-IV</td><td>8.51 (3.59 to 21.7)</td><td>&lt;0.001</td></tr><tr><td>High IGF1 ≥median<sup>2</sup></td><td>2.62 (1.07 to 6.74)</td><td>0.04</td></tr></tbody></table>

<figure><img src="image_2.png" /></figure>

**Figure 1.** Serum IGF1 levels predict progressive disease and BRAFi/MEKi tumor escape in patients with melanoma. (a) Association between patients' characteristics and progressive disease (n = 200). Patients with in situ melanoma (n = 5) have been excluded from these analyses. The number 1 in asterisk (¹) denotes analysis based on univariate logistic regression models, the number 2 in asterisk (²) denotes the median value for serum IGF1 (136 ng/ml), and the number 3 in asterisk (³) denotes multivariate analysis of factors predicting progressive disease on the basis of logistic regression models adjusted for age, sex, ulceration, high Breslow index, advanced stage, and high IGF1. *P*-values were estimated from likelihood ratio tests. (b) Paired comparison of serum IGF1 level at last progression-free follow-up (left panels, in blue) or at time of progression (right panels, in red), with serum IGF1 level 3 or 6 months earlier for each patient. Thick blue or red lines represent the mean change overall in patients. Percentages indicate the mean change in IGF1 levels among patients. (c) Percentages of patients with PD or progression-free patients (denotes with P-Free) with increase or decrease in serum IGF1 level. The patient with primary resistance has been excluded from these analyses. *P*-values were calculated using Fisher's exact test. BRAFi, BRAF inhibitor; CI, confidence interval; MEKi, MAPK/extracellular signal-regulated kinase inhibitor; PD, progressive disease.

Spheroids (Figure 2c). We further observed that IGF1 significantly enhanced the motility of the three parental and resistant cells (Supplementary Figure S3a).

We next examined the effect of linsitinib, a specific IGF1R inhibitor, which inhibited the proliferation of both parental and resistant cells (Sup

plementary Figure S3b and c). However, IGF1R inhibition alone was not sufficient to inhibit resistant melanoma spheroids proliferation. In contrast, the combination of linsitinib with BRAFi/MEKi induced synergic inhibition of the viability of resistant A375-R and 451Lu-R spheroids but not that of SKMel28-R

spheroids (Figure 2d–f). This combination also inhibited the proliferation of resistant cells (Supplementary Figure S3d and e) and the migration of A375-R and 451Lu-R cells (Figure 2g). Most interestingly, this treatment reduced extracellular signal-regulated kinase 1/2 activation in all melanoma-
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Linsitinib inhibits synergistically with BRAFi/MEKi the spheroid and in vivo growth of resistant melanoma cells secreted high IGF1 levels. (a) Heatmap of differentially expressed IGF1 pathway genes in parental and resistant A375 and SKMel28 melanoma cells (n = 3). Data show normalized read count. The color key represents a spectrum of lowest gene expression (blue) to highest gene expression (red). (b) Representative immunoblot analysis with the indicated antibodies in parental and resistant A375, SKMel28, and 451Lu spheroids. (c) IGF1 concentrations were measured in the medium after 72 h treatment</figcaption></figure>
resistant spheroids and strongly reduced protein kinase B activation in A375-R and 451Lu-R spheroids (Figure 2h and Supplementary Figure S3f).

Finally, the combination of linsitinib with BRAFi/MEKi significantly inhibited in vivo tumor growth of A375-R and 451Lu-R cells but not of SKMel28-R xenografted onto chicken chorioallantoic membranes (Supplementary Figure S3g and Figure 2i). These treatments showed no noticeable toxicity in chicken embryos, except in the SKMel28-R tumor model (Supplementary Figure S3h). Overall, these results show that the combination of linsitinib with BRAFi/MEKi inhibited spheroid viability, protein kinase B-dependent survival pathway, and cell motility in resistant cells secreting high levels of IGF1 and failed in SKMel28-R that secrete low IGF1 levels.

Although limited by the small number of patients, our clinical results show that serum IGF1 level is an independent risk factor for subsequent progression in melanoma. Furthermore, increased serum IGF1 level appears as a marker of melanoma escape to BRAFi/MEKi-targeted therapies. Our in vitro and in vivo studies suggest an increase in IGF1 secretion during melanoma progression, which may stimulate melanoma cell migration. Moreover, the response of BRAFi/MEKi-resistant BRAF<sup>V600</sup> melanoma cells to IGF1R inhibitor appears to correlate with the level of IGF1 secretion. This emphasizes the importance of using relevant three-dimensional in vitro models, which are more predictive of tumor response (Riedl et al., 2017).

Monitoring patient serum IGF1 levels over time is simple, rapid, inexpensive, and noninvasive and eliminates inter-individual variability (Chanson et al.,

2016). Such monitoring would enable individual identification of patients escaping BRAFi/MEKi treatment and for whom an IGF1R inhibitor could be combined to overcome resistance to targeted therapy.

## Ethics

This is a prospective study on the serum of patients diagnosed with melanoma, which does not involve human subjects and whose controller is the Grenoble Alpes University Hospital. The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Grenoble Alpes University Hospital. This research has been registered in accordance with French regulations and meets the requirements of the French Data Protection Authority reference methodology 004 (protocol approval date August 22, 2022). Because our study does not involve human subjects but only their clinical data, information notices were posted in the dermatology department, and patients were informed orally without legal obligation to sign an informed consent statement.

According to French legislation, no ethical approval is needed for scientific experimentations using oviparous embryos (decree n° 2013-118, February 1, 2013; art. R-214-88).

## Data availability statement

Datasets can be found at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE239284, hosted at Gene Expression Omnibus Datasets of National Center for Biotechnology Information.

## ORCIDs

Camila Castillo-Ferrer: http://orcid.org/0000-0001-7427-5814

Théo Marguet: http://orcid.org/0009-0008-8882-8158

Laetitia Vanwonterghem: http://orcid.org/0000-0001-7054-5717

Sule Erbek: http://orcid.org/0000-0002-4427-0841

Florent Chuffart: http://orcid.org/0000-0001-6064-5183

Stéphane Mouret: http://orcid.org/0000-0003-3880-973X

Sabiha Trabelsi Messai: http://orcid.org/0009-0000-5624-0570

Anne-Sophie Gauchez: http://orcid.org/0000-0003-2332-0966

Jean-Luc Coll: http://orcid.org/0000-0002-2453-3552

Julie Charles: http://orcid.org/0000-0003-4667-6009

Amandine Hurbin: http://orcid.org/0000-0003-0535-1945

Véronique Martel-Frachet: http://orcid.org/0000-0001-6739-7901

## CONFLICT OF INTEREST

The authors state no conflict of interest.

## ACKNOWLEDGMENTS

This research was funded by La Ligue contre le cancer, comité de Savoie, GEFLUC (groupement des entreprises françaises dans la lutte contre le cancer), Espoir Isère contre le Cancer, and French National Research Agency in the framework of the Investissements d'avenir program (ANR-15-IDEX-02). We kindly thank Sophie Tartare-Deckert for generously providing parental and resistant 451Lu cells. We acknowledge Ariane Jullien, Bastien Lahetjuzan, and Quentin Ruet for their help. AH is the guarantor of this publication.

## AUTHOR CONTRIBUTIONS

Conceptualization: CCF, JC, VM-F, AH; Data Curation: TM, SM, JC, A-SG, STM; Formal Analysis: CCF, FC, AH, VM-F, TM, SM, JC; Funding Acquisition: AH, VM-F, J-LC; Investigation: CCF, LV, SE, A-SG, VM-F, AH; Resources: TM, SM, JC, A-SG, STM, J-LC; Supervision: AH, VM-F; Writing - Original Draft Preparation: CCF, AH, VM-F, SM, JC, A-SG, J-LC; Writing - Review and Editing: CCF, AH, VM-F

Camila Castillo-Ferrer¹,²,
Théo Marguet¹,³,
Laetitia Vanwonterghem¹,
Sule Erbek¹,², Florent Chuffart¹,
Stéphane Mouret³, Sabiha

of parental and resistant spheroids with a combination of vemurafenib and cobimetinib. The levels of IGF1 detected per million cells were normalized to those of the IGF1 detected in parental untreated spheroids; n = 6–8. Bars indicate the mean ± SEM. (d) Dose-response matrices with mean inhibition ± SD (n ≥ 3) after 72 h treatment. Summary synergy scores > 10 indicate that the interaction between drugs is likely to be synergistic, and −10 to 10 scores indicate that the interaction between drugs is likely to be additive. Lins denotes linsitinib. (e) Spheroid area after 72 h treatment was expressed as the mean ± SEM (n ≥ 3, five spheroids per condition). Representative pictures of spheroids are shown (bars = 400 µm). (f) Spheroid cell viability is expressed as the mean ± SD (n = 3). (g) Wound-healing assay in resistant cells. Wound closure was calculated relative to the wound distance at time 0. Data are expressed as mean ± SEM at 12 h (n ≥ 3). (h) Representative immunoblot analysis with the indicated antibodies is shown. GAPDH was used as a loading control. Histograms show the relative intensity of phosphorylated/total proteins expressed as the mean ± SEM (n = 3). (i) In ovo assay. Tumors were excised and weighed at the end of the treatment. Each dot represents one tumor weight. All the in vitro and in vivo experiments were realized with the same drug concentrations of vemurafenib and cobimetinib (denoted with V+C) and/or linsitinib (denoted with L) (vemurafenib: 4 µmol·l⁻¹ for A375-R and SKMel28-R and 8 µmol·l⁻¹ for 451Lu-R; cobimetinib: 1 nmol·l⁻¹; linsitinib: 5 µmol·l⁻¹). Bars indicate the median ± SEM. *P* < 0.05, **P** < 0.01, and ***P** < 0.001. Kruskal–Wallis with Dunn's multiple comparisons posthoc test (c, e, f, h, and i) and two-way ANOVA with Tukey multiple comparison test (for g) were used for data analyses. S denotes parental spheroid, whereas R denotes resistant spheroid. VC denotes vemurafenib and cobimetinib combination. Akt, protein kinase B; BRAFi, BRAF inhibitor; h, hour; MEKi, MAPK/extracellular signal-regulated kinase inhibitor; ns, not significant; NT, nontreated; p-Akt, phosphorylated protein kinase B; p-ERK, phosphorylated extracellular signal-regulated kinase.
Trabelsi Messai¹³, Anne-Sophie Gauchez¹, Jean-Luc Coll¹, Julie Charles¹,³, Amandine Hurbin¹,⁵,* and Véronique Martel-Frachet¹,²,⁵

¹Institute for Advanced Biosciences, INSERM U1209, CNRS UMR5309, Grenoble Alpes University, Grenoble, France; ²École Pratique des Hautes Études, PSL Research University, Paris, France; ³Dermatology, Allergology & Photobiology Department, Grenoble Alpes University Hospital, Grenoble Alpes University, Grenoble, France; and ⁴Biology and Pathology Institute, Grenoble Alpes University Hospital, Grenoble Alpes University, Grenoble, France

⁵These authors contributed equally to this work.

*Corresponding author e-mail: amandine.hurbin@inserm.fr

## SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at www.jidonline.org, and at https://doi.org/10.1016/j.jid.2023.07.016.

## REFERENCES

* Bradbury KE, Appleby PN, Tipper SJ, Travis RC, Allen NE, Kvaskoff M, et al. Circulating insulin-like growth factor I in relation to melanoma risk in the European prospective investigation into cancer and nutrition. Int J Cancer 2019;144:957–66.

* Chanson P, Arnoux A, Mavromati M, Brailly-Tabard S, Massart C, Young J, et al. Reference values for IGF-I serum concentrations:

comparison of six immunoassays. J Clin Endocrinol Metab 2016;101:3450–8.

* Dummer R, Flaherty KT, Robert C, Arance A, de Groot JWB, Garbe C, et al. COLUMBUS 5-year update: a randomized, open-label, phase III trial of encorafenib plus binimetinib versus vemurafenib or encorafenib in patients with BRAF V600-mutant melanoma [published correction appears in J Clin Oncol 2023;41:2301] J Clin Oncol 2022;40:4178–88.

* Girnita L, Worrall C, Takahashi SI, Seregard S, Girnita A. Something old, something new and something borrowed: emerging paradigm of insulin-like growth factor type 1 receptor (IGF-1R) signaling regulation. Cell Mol Life Sci 2014;71:2403–27.

* Gowrishankar K, Snoyman S, Pupo GM, Becker TM, Kefford RF, Rizos H. Acquired resistance to BRAF inhibition can confer cross-resistance to combined BRAF/MEK inhibition [published correction appears in J Invest Dermatol 2013;133:2493] J Invest Dermatol 2012;132:1850–9.

* Kucera R, Treskova I, Vrzalova J, Svobodova S, Topolcan O, Fuchsova R, et al. Evaluation of IGF1 serum levels in malignant melanoma and healthy subjects. Anticancer Res 2014;34:5217–20.

* Obenauf AC, Zou Y, Ji AL, Vanharanta S, Shu W, Shi H, et al. Therapy-induced tumour secretomes promote resistance and tumour progression. Nature 2015;520:368–72.

* Patel H, Mishra R, Yacoub N, Alanazi S, Kilroy MK, Garrett JT. IGF1R/IR mediates resistance to BRAF and MEK inhibitors in BRAF-mutant melanoma. Cancers 2021;13:5863.

* Qu X, Wu Z, Dong W, Zhang T, Wang L, Pang Z, et al. Update of IGF-1 receptor inhibitor (gantumab, dalotuzumab, cixutumumab, teprotumumab and digitumumab) effects on cancer therapy. Oncotarget 2017;8:29501–18.

* Riedl A, Schlederer M, Pudelko K, Stadler M, Walter S, Unterleuthner D, et al. Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT–mTOR–S6K signaling and drug responses. J Cell Sci 2017;130:203–18.

* Strub T, Ghiraldini FG, Carcamo S, Li M, Wroblewska A, Singh R, et al. SIRT6 haploinsufficiency induces BRAFV600E melanoma cell resistance to MAPK inhibitors via IGF signalling. Nat Commun 2018;9:3440.

* Truong DD, Lamhmedi-Cherradi SE, Ludwig JA. Targeting the IGF/PI3K/mTOR pathway and AXL/YAP1/TAZ pathways in primary bone cancer. J Bone Oncol 2022;33:100419.

* Villanueva J, Vultur A, Lee JT, Somasundaram R, Fukunaga-Kalabis M, Cipolla AK, et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 2010;18:683–95.

* Xu G, Luo Y, Wu W, Liu X, Yu X, Bao Y, et al. The evolution of acquired resistance to BRAFV600E kinase inhibitor is sustained by IGF1-driven tumor vascular remodeling. J Invest Dermatol 2022;142:445–58.

* Yeh AH, Bohula EA, Macaulay VM. Human melanoma cells expressing V600E B-RAF are susceptible to IGF1R targeting by small interfering RNAs. Oncogene 2006;25:6574–81.

# Bacterial Microbiota Composition in Hidradenitis Suppurativa Differs per Skin Layer

<figure><img src="image_4.png" /></figure>

Journal of Investigative Dermatology (2024) 144, 426–430; doi:10.1016/j.jid.2023.08.017

JID Open

## TO THE EDITOR

Hidradenitis suppurativa (HS) is a chronic, inflammatory skin disease in which a shift in skin microbiota composition plays a role in the initiation and/or maintenance of the disease (Mintoff et al., 2021; van Straalen et al., 2022; Wark and Cains, 2021). Differences across studies regarding the relevant bacteria in HS persist owing to the use of different sampling techniques (swabs, scrapings, or biopsies). This affects the comparability of current HS microbiome studies (Nakatsuji et al., 2013; Prast-Nielsen et al., 2019).

Therefore, in this study, we analyzed the microbiota composition captured at different depths of HS lesional skin using swabs, superficial biopsies, and deep biopsies in different disease stages.

In total, 96 samples were collected from the skin of 32 patients with HS (Hurley 1: n = 10; Hurley II: n = 12; and Hurley III: n = 10) collected during routine surgery. The institutional review board of the Erasmus University Medical Center (Rotterdam, The Netherlands) allows the use of surgical discarded tissue for research purposes

under an opt-out principle; therefore, no additional informed consent was required for this study. Fifteen patients were sampled in the groin, 13 were sampled in the axilla, and 4 were sampled in other areas (n = 3 for buttock, and n = 1 for neck). The mean body mass index was 29.1 kg/m² (±6.3), and 78% (25 of 32 patients) were current smokers. Detailed patient characteristics, sample collection, methods, and statistical analyses can be found in Supplementary Table S1.

After quality control, 585 amplicon sequence variants were identified in 84 remaining samples (swabs: n = 32; superficial biopsies: n = 28; deep biopsies: n = 24). After adjusting for age, body mass index, smoking status, anatomical location, and Hurley stage, both superficial and deep biopsies were
# SUPPLEMENTARY MATERIALS AND METHODS

## Patients and serum samples

Clinical and imaging evaluations were performed every 3 months or when progression was suspected with brain and thoracic-abdominal-pelvis scan. Patients were divided into two groups according to the targeted therapy response: progression-free patients group corresponded to patients with disappearance of metastases or decrease or stability in size of metastases observed during follow-up visits, and patients with progressive disease group corresponded to patients with new metastases or a 20% increase in known metastases according to RECIST (Response Evaluation Criteria in Solid Tumors) 1.1 criteria.

Patients' blood samples were collected and centrifuged at each follow-up visit, and sera were stored at -20 °C until analysis. Levels of serum IGF1 were analyzed using ELISA kits (IRMA IBA, CIS Bio International, Saclay, France) according to the manufacturer's instructions.

## Serum IGF1 analysis sets

Two sets of analyses were performed. The first set tested serum IGF1 levels in 200 patients at the time of diagnosis. All patients were staged for melanoma according to the eighth edition of the American Joint Committee on Cancer. In the second set, we tested serum IGF1 levels in 28 patients with metastatic or unresectable melanoma treated with targeted therapy (BRAF inhibitor/MAPK/extracellular signal-regulated kinase (ERK) kinase inhibitor combination: dabrafenib/trametinib, encorafenib/binimetinib, or vemurafenib/cobimetinib) regardless of line of therapy or in the adjuvant setting. IGF1 was quantified every 3 months until the treatment was stopped. IGF1 levels were followed over time in each patient group (progression-free patients [n = 12] and patients with progressive disease [n = 16]) after normalization to the IGF1 level detected at diagnosis. Then, trends of IGF1 levels were normalized for each patient to the value of IGF1 level measured 3, 6, 9, or 12 months before.

## Reagents

Vemurafenib, cobimetinib, SB590885, and linsitinib were purchased from Clinisciences (Nanterre, France) and dissolved in DMSO. IGF1 was

purchased from R&D Systems (Bio-Techne, Lille, France). Fibronectin was purchased from Sigma-Aldrich (Saint Quentin-Fallavier, France).

## Cell lines and culture

The human BRAFV600E-sequence variant A375 and SKMel28 cell lines were obtained from the ATCC (Manassas, VA). A375 and SKMel28 vemurafenib-resistant cell lines were generated by treating parental cells with increasing concentrations of vemurafenib until the onset of resistance. A375-resistant and SKMel28-resistant cells were maintained in 1 µmol.l⁻¹ vemurafenib. The resistant cells and corresponding parental cells were authenticated by short tandem repeats profiling by ATCC. The human 451Lu and 451Lu cell lines resistant to the BRAF inhibitor SB590885 (451Lu resistant) were obtained from S. Tartare-Deckert (Nice, France). The 451Lu-resistant cells were maintained in 1 µmol.l⁻¹ SB590885 (BRAF inhibitor). All cells were mycoplasma free and used within 4 months after thawing. Three-dimensional spheroids were generated by plating cells into 96-well round bottom ultralow attachment spheroid microplates (Corning, Tewksbury, MA) or 24-well plates coated with 2% agarose.

## RNA sequencing

RNA extraction from parental and resistant cell lines in biological triplicates was carried out from ~1 million pelleted cells with NucleoSpin RNA kit (Macherey Nagel, Hœrdt, France) according to the manufacturer's instructions. Initial sample concentration and quality were assessed by Nanodrop (Thermo Fisher Scientific, Waltham, MA). RNA-sequencing analysis was realized by Novogene (Cambridge, United Kingdom).

Computations presented in this paper were performed using the GRICAD infrastructure (https://gricad.univ-grenoble-alpes.fr), which is supported by Grenoble research communities. Visualization of differentially expressed genes was performed using the matrix visualization and analysis platform Morpheus (https://software.broadinstitute.org/morpheus).

## In vitro cytotoxicity assays

The cell viability was quantified using CellTiter 96 AQueous One Solution cell proliferation assay (Promega, Charbonnière, France) in two-dimensional cell

models or CellTiter-Glo 3D Cell Viability Assay (Promega) in spheroids, as previously described (Gilson et al., 2019).

The degree of combination synergy or antagonism is quantified by comparing the observed drug combination response against the expected response, calculated using the highest single agent model with SynergyFinder web application. The synergy maps highlight synergistic and antagonistic dose regions in red and green colors, respectively.

## IGF1 ELISA assay

IGF1 in the medium was analyzed using the Human IGF1 DuoSet ELISA assay (R&D Systems, Minneapolis, MN) and following the manufacturer's instructions.

## Wound-healing assay

Cells were seeded into 24-well plates coated with 5 µg.l⁻¹ fibronectin for 24 hours and then serum starved for 24 hours. The cell monolayer was scratched with a pipette tip. Fresh serum-free growth medium with 50 ng.ml⁻¹ IGF1 was added to the wells. Alternatively, 4 µmol.l⁻¹ vemurafenib and 1 nmol.l⁻¹ cobimetinib without or with 5 µmol.l⁻¹ linsitinib were added into the complete medium. Images of cells invading the scratch wound were monitored for 12 hours using an AxioObserver Z1 (Zeiss, Rueil Malmaison, France) associated with a MicroMAX camera (Princeton Instruments, Lisses, France) and Metamorph software (Universal Imaging, Bedford Hills, NY). The relative wound closure was analyzed using the FIJI software.

## Western blot analysis

Immunoblotting experiments were performed as previously described (Gilson et al., 2019; Jeannot et al., 2014) using antibodies against IGF1R, phosphorylated IGF1R-Y1135/1136, phosphorylated protein kinase B-S473, pan-protein kinase B, phosphorylated ERK1/2-T202/Y204 and ERK1/2 (Cell Signaling Technology, Saint Quentin en Yvelines, France). Phosphorylated and total protein intensities were quantified using the Image Lab 6.0 software (Bio-Rad Laboratories, Hercules, CA). The relative densitometric area for phosphorylated band intensity was normalized to the respective total protein intensity, and total protein intensity was normalized to the respective GAPDH intensity.
The relative ERK inhibition was determined with the formula (phosphorylated ERK₁/₂ NT – phosphorylated ERK₁/₂ V+C)/phosphorylated ERK₁/₂ NT)) × 100.

## In vivo experiments

A 30 µl inoculum of 3 × 10⁶ cells in Matrigel (Corning, Tewksbury, MA) was added directly onto the chorioallantoic membrane of each egg. Eggs were randomly allocated into four groups. On day 11, when tumors began to be detectable, eggs were treated four times for 9 days by dropping 100 µl of either vemurafenib (4 µmol·l⁻¹ for A375-5 and SKMel28-resistant cells and 8 µmol·l⁻¹ for 451Lu-resistant cells) and 1 nmol·l⁻¹ cobimetinib, 5 µmol·l⁻¹ linsitinib, a combination of

vemurafenib/cobimetinib and linsitinib, or 0.1% DMSO in PBS (vehicle) on the tumor. On day 18, tumors were carefully cut away from normal chorioallantoic membrane tissue and weighed.

## Statistical analyses

All analyses were performed using the GraphPad Prism software (GraphPad Software, San Diego, CA). Two-sided P-values ≤ 0.05 were considered significant.

The risk factors associated with subsequent progressive disease by univariate analysis (P ≤ 0.3) were advanced stages, high Breslow index, presence of ulceration, and high serum IGF1 level. These factors and variables deemed to be of clinical importance (age, gender) were used to derive the model for

progressive disease, which includes the following risk factors: older age, advanced stage, and high serum IGF1 level. This model achieved good calibration (Hosmer–Lemeshow test, P = 0.76) and discrimination (area under the receiver operating characteristic curve = 0.84, P < 0.001).

## SUPPLEMENTARY REFERENCES

* Gilson P, Couvet M, Vanwonterghem L, Henry M, Vollaire J, Baulin V, et al. The pyrrolopyrimidine colchicine-binding site agent P-13 reduces the metastatic dissemination of invasive cancer cells in vitro and in vivo. Biochem Pharmacol 2019;160:1–13.

* Jeannot V, Busser B, Brambilla E, Wislez M, Robin B, Cadranel J, et al. The PI3K/AKT pathway promotes gefitinib resistance in wild-type EGFR lung adenocarcinoma by a deace-tylase-dependent mechanism. Int J Cancer 2014;134:2560–71.
IGF1 Predicts Therapy Resistance in Melanoma

<figure><img src="image_5.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_10.png" /></figure>

**Supplementary Figure S1.** Changes in serum IGF1 levels since initiation of targeted therapy in patients with melanoma. (a) Distribution of IGF1 level across all serum samples (n = 205): fraction of samples (y-axis) showing the indicated level of IGF1 (x-axis). (b) Changes in serum IGF1 levels since initiation of targeted therapy in patients with melanoma (n = 28). Serum IGF1 level was normalized to serum IGF1 level obtained at treatment initiation for each patient. The graph shows the mean ± SEM from diagnosis to progression or to the end of follow-up in two groups of patients treated with targeted therapies: progression-free patients (in blue, n = 12) or patients with progressive disease (in red, n = 16). (c, d) Individual changes in normalized serum IGF1 levels. Shown is paired comparison of serum IGF1 level at (c) the last progression-free follow-up or (d) at the time of progression, with serum IGF1 level 9 or 12 months earlier for each patient. Thick blue or red lines represent the mean change overall in patients. Percentages indicate the mean change in the IGF1 level. (e) Percentages of patients with progressive disease (denoted with PD) or progression-free patients (denoted with P-Free) with increase or decrease in serum IGF1 level. *P*-values were calculated using Fisher's exact test. (f) Distribution of serum IGF1 levels in progression-free patients (in blue) or patients with progressive disease (in red). Serum IGF1 levels were normalized at the indicated time to the IGF1 level at the last progression-free follow-up or at the time of progression for each patient. Statistical analysis with two-way ANOVA with Bonferroni's multiple comparisons posthoc test.
<table><caption>a</caption><thead><tr><th rowspan="2">IC<sub>50</sub></th><th colspan="2">cells</th><th colspan="2">spheroids</th></tr><tr><th>Vemurafenib<br>(µmol.L<sup>-1</sup>)</th><th>Cobimetinib<br>(nmol.L<sup>-1</sup>)</th><th>Vemurafenib<br>(µmol.L<sup>-1</sup>)</th><th>Cobimetinib<br>(nmol.L<sup>-1</sup>)</th></tr></thead><tbody><tr><td>A375-S</td><td>1.0 ± 0.2</td><td>4.0 ± 0.3</td><td>0.3 ± 0.0</td><td>4.0 ± 0.3</td></tr><tr><td>A375-R</td><td><strong>16.7 ± 1.1</strong></td><td><strong>709.0 ± 59.0</strong></td><td><strong>1.6 ± 0.3</strong></td><td><strong>22.0 ± 3.0</strong></td></tr><tr><td>SKMel28-S</td><td>1.4 ± 0.4</td><td>10.0 ± 3.0</td><td>1.8 ± 0.3</td><td>71.0 ± 9.0</td></tr><tr><td>SKMel28-R</td><td><strong>13.8 ± 0.8</strong></td><td><strong>54.0 ± 8.0</strong></td><td><strong>13.4 ± 3.2</strong></td><td><strong>28.0 ± 3.0</strong></td></tr><tr><td>451Lu-S</td><td>0.3 ± 0.0</td><td>6.0 ± 0.2</td><td>0.5 ± 0.1</td><td>12.0 ± 1.0</td></tr><tr><td>451Lu-R</td><td><strong>30.4 ± 3.3</strong></td><td><strong>8.0 ± 1.4</strong></td><td><strong>69.7 ± 15.5</strong></td><td><strong>18.0 ± 2.0</strong></td></tr></tbody></table>

<figure><img src="image_11.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_15.png" /></figure>

**Supplementary Figure S2. BRAFi/MEKi-resistant cells and spheroids show ERK and IGF1/IGF1R activation.** (a) Vemurafenib or cobimetinib concentrations required to inhibit cell growth by 50% (IC50) (mean ± SD, n ≥ 3) in parental (denoted with S) and resistant (denoted with R) cells or spheroids. (b–d, f) Cells or spheroids were treated with a combination of vemurafenib and cobimetinib (denoted with V+C) (vemurafenib: 4 µmol.l⁻¹ for A375 and SKMel28 and 8 µmol.l⁻¹ for 451Lu; cobimetinib: 1 nmol.l⁻¹). (b) Cell or spheroid viability after 72-h treatment (mean ± SEM, n ≥ 4). (c) Spheroid area after 72-h treatment. Data are normalized to the spheroid area in control (mean ± SEM, n ≥ 3). Representative pictures of spheroids are shown (bars = 400 µm). (d, e) Representative immunoblots with the indicated antibodies. GAPDH was used as the loading control. Histograms show the relative intensity of p-ERK1/2/ERK1/2 proteins (mean ± SEM, n = 3). (f) IGF1 concentrations measured in the medium after 72-h treatment. The levels of IGF1 detected per million cells are expressed as a rate of IGF1 detected in parental untreated cells. Dot denotes one measure; bars denote mean ± SEM (n = 6–8). \*P < 0.05, \*\*P < 0.01, and \*\*\*P < 0.001; Kruskal–Wallis with Dunn’s multiple comparisons posthoc test (for b–d) or Mann–Whitney U test (for f) was used for analysis. V denotes vemurafenib, and C denotes cobimetinib. BRAFi, BRAF inhibitor; ERK, extracellular signal-regulated kinase; h, hour; IC50, half-maximal inhibitory concentration; IGF1R, IGF1 receptor; MEKi, MAPK/extracellular signal-regulated kinase inhibitor; ns, not significant; NT, nontreated; p-ERK, phosphorylated extracellular signal-regulated kinase.
<figure><img src="image_16.png" /></figure>

<table><thead><tr><th rowspan="2">IC50</th><th colspan="2">Linsitinib (µmol.l<sup>-1</sup>)</th></tr><tr><th>cells</th><th>spheroids</th></tr></thead><tbody><tr><td>A375-S</td><td>5.6 ± 0.6</td><td>20.9 ± 1.7</td></tr><tr><td>A375-R</td><td>5.6 ± 0.6</td><td>11.4 ± 1.2</td></tr><tr><td>SKMel28-S</td><td>7.8 ± 0.6</td><td>22.1 ± 1.1</td></tr><tr><td>SKMel28-R</td><td>15.2 ± 1.3</td><td>20.0 ± 1.7</td></tr><tr><td>451Lu-S</td><td>29.7 ± 1.1</td><td>38.6 ± 1.3</td></tr><tr><td>451Lu-R</td><td>26.6 ± 0.9</td><td>35.9 ± 1.2</td></tr></tbody></table>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

**Supplementary Figure S3.** Linsitinib acts synergistically with BRAFi/MEKi to inhibit resistant cell proliferation and Akt activation. (a) Wound-healing assay in parental (denoted with S) and resistant (denoted with R) cells on fibronectin and treated without (-SVF) or with 50 ng IGF1 for 12 h. Wound closure was calculated relative to the wound distance at time 0 and normalized to the control. Data are expressed as mean ± SEM at 12 h (n ≥ 3). (b) Linsitinib concentrations required to inhibit cell growth by 50% (IC50) (mean ± SD, n ≥ 3) in cells or spheroids. (c) Representative immunoblots with p-IGF1R and total IGF1R in resistant cells or spheroids after 72-h treatment with 5 µmol.l⁻¹ linsitinib (denoted with Lins). GAPDH was used as the loading control. (d) Dose-response matrices with mean inhibition ± SD (n ≥ 3). Summary synergy scores >10 indicated that the interaction between drugs is likely to be synergistic. (e–h) Resistant cells or tumors were treated with a combination of vemurafenib and cobimetinib (denoted with V+C) and/or linsitinib (denoted with L) (vemurafenib: 4 µmol.l⁻¹ for A375-resistant and SKMel28-resistant cells and 8 µmol.l⁻¹ for 451Lu-resistant cell; cobimetinib: 1 nmol.l⁻¹; L: 5 µmol.l⁻¹). (e) Cell viability is expressed as the mean ± SD (n = 3). (f) Representative immunoblots with the indicated antibodies. GAPDH was used as the loading control. Histograms show the relative intensity of phosphorylated/total proteins (mean ± SEM, n = 2–3). (g) Schematic representation of the in ovo assay principle. (h) Toxicity of vemurafenib/cobimetinib and linsitinib in chicken embryos. The number of dead and surviving embryos for the different experimental groups at the end of the treatments is shown. *P* < 0.05, **P* < 0.01, and ***P* < 0.001; unpaired *t*-test with Welch's correction (for a) and Kruskal-Wallis with Dunn's multiple comparisons posthoc test (for e and f) were used for analysis. Akt, protein kinase B; BRAFi, BRAF inhibitor; ERK, extracellular signal-regulated kinase; h, hour; IC50, half-maximal inhibitory concentration; IGF1, IGF1 receptor; MEKi, MAPK/extracellular signal-regulated kinase inhibitor; NT, nontreated; p-Akt, phosphorylated protein kinase B; p-ERK, phosphorylated extracellular signal-regulated kinase; p-IGF1R, phosphorylated IGF1 receptor; SVF, fetal calf serum.
<table><caption>Supplementary Table S1. Clinical and Biological Characteristics of all Patients with Melanoma according to the Gender at the Time of Study Enrolment</caption><thead><tr><th>Characteristics</th><th>Total (n = 205) n (%)</th><th>Male (n = 98) n (%)</th><th>Female (n = 107) n (%)</th><th>P-Value</th></tr></thead><tbody><tr><td>Age, y, median (range)</td><td>64 (28–97)</td><td>64 (35–91)</td><td>63 (27–96)</td><td>0.21</td></tr><tr><td>Breslow, mm, median (range)</td><td>1.8 (0–20)</td><td>1.9 (0–20)</td><td>1.7 (0–11)</td><td>0.77</td></tr><tr><td>missing</td><td>4</td><td>2</td><td>2</td><td></td></tr><tr><td>Ulceration</td><td></td><td></td><td></td><td>0.24</td></tr><tr><td>Yes</td><td>73 (37%)</td><td>39 (41%)</td><td>34 (32%)</td><td></td></tr><tr><td>No</td><td>127 (63%)</td><td>56 (59%)</td><td>71 (68%)</td><td></td></tr><tr><td>missing</td><td>5</td><td>3</td><td>2</td><td></td></tr><tr><td>Mitotic rate</td><td></td><td></td><td></td><td>0.18</td></tr><tr><td>0 or &lt;1</td><td>36 (19%)</td><td>17 (19%)</td><td>19 (19%)</td><td></td></tr><tr><td>1</td><td>35 (19%)</td><td>17 (19%)</td><td>18 (18%)</td><td></td></tr><tr><td>2–3</td><td>69 (37%)</td><td>38 (43%)</td><td>31 (32%)</td><td></td></tr><tr><td>4–10</td><td>39 (21%)</td><td>12 (14%)</td><td>27 (28%)</td><td></td></tr><tr><td>&gt;10</td><td>7 (4%)</td><td>4 (5%)</td><td>3 (3%)</td><td></td></tr><tr><td>unknown</td><td>19</td><td>10</td><td>9</td><td></td></tr><tr><td>Primary tumor (T)</td><td></td><td></td><td></td><td>0.16</td></tr><tr><td>Tis</td><td>5 (3%)</td><td>3 (3%)</td><td>2 (2%)</td><td></td></tr><tr><td>T1</td><td>33 (16%)</td><td>18 (18%)</td><td>15 (14%)</td><td></td></tr><tr><td>T2</td><td>80 (39%)</td><td>32 (32%)</td><td>48 (45%)</td><td></td></tr><tr><td>T3</td><td>42 (20%)</td><td>25 (26%)</td><td>17 (16%)</td><td></td></tr><tr><td>T4</td><td>41 (20%)</td><td>18 (18%)</td><td>23 (21%)</td><td></td></tr><tr><td>Tx</td><td>4 (2%)</td><td>2 (2%)</td><td>2 (2%)</td><td></td></tr><tr><td>Regional lymph nodes (N)</td><td></td><td></td><td></td><td>0.68</td></tr><tr><td>N<sub>0</sub></td><td>141 (69%)</td><td>66 (67%)</td><td>75 (70%)</td><td></td></tr><tr><td>N<sub>+</sub></td><td>62 (30%)</td><td>31 (32%)</td><td>31 (29%)</td><td></td></tr><tr><td>N<sub>x</sub></td><td>2 (1%)</td><td>1 (1%)</td><td>1 (1%)</td><td></td></tr><tr><td>Distant metastases (M)</td><td></td><td></td><td></td><td>0.63</td></tr><tr><td>M<sub>0</sub></td><td>184 (90%)</td><td>89 (91%)</td><td>95 (89%)</td><td></td></tr><tr><td>M<sub>+</sub></td><td>21 (10%)</td><td>9 (9%)</td><td>12 (11%)</td><td></td></tr><tr><td>Stage</td><td></td><td></td><td></td><td>0.31</td></tr><tr><td>0 (Tis)</td><td>5 (2%)</td><td>3 (3%)</td><td>2 (2%)</td><td></td></tr><tr><td>I</td><td>79 (40%)</td><td>32 (33%)</td><td>47 (44%)</td><td></td></tr><tr><td>II</td><td>50 (24%)</td><td>26 (26%)</td><td>24 (22%)</td><td></td></tr><tr><td>III</td><td>50 (24%)</td><td>28 (29%)</td><td>22 (21%)</td><td></td></tr><tr><td>IV</td><td>21 (10%)</td><td>9 (9%)</td><td>12 (11%)</td><td></td></tr><tr><td>BRAF status</td><td></td><td></td><td></td><td>0.57</td></tr><tr><td>Wild type</td><td>36 (47%)</td><td>19 (50%)</td><td>17 (44%)</td><td></td></tr><tr><td>BRAF<sup>V600E</sup> variant</td><td>41 (53%)</td><td>19 (50%)</td><td>22 (56%)</td><td></td></tr><tr><td>unknown</td><td>128</td><td>60</td><td>68</td><td></td></tr><tr><td>Progressive disease</td><td></td><td></td><td></td><td>0.87</td></tr><tr><td>Yes</td><td>45 (22%)</td><td>22 (22%)</td><td>23 (21%)</td><td></td></tr><tr><td>No</td><td>160 (78%)</td><td>76 (78%)</td><td>84 (79%)</td><td></td></tr><tr><td>IGF1 (ng/ml), median (range)</td><td>136 (32–299)</td><td>140 (32–299)</td><td>131 (66–256)</td><td>0.60</td></tr></tbody></table>

Abbreviation: Tis, melanoma in situ.

n denotes the number of subjects and (percentage). P-value denotes the comparison between male and female patients with melanoma and was calculated by contingency table for qualitative data, whereas Mann-Whitney U test was employed for quantitative data.
<table><caption>Supplementary Table S2. Main Characteristics of the 28 Patients with Melanoma Treated with BRAFi/MEKi-Targeted Therapies at the Beginning of the Treatment</caption><thead><tr><th rowspan="2">Characteristics</th><th>Total (n = 28)</th><th>Male (n = 15)</th><th>Female (n = 13)</th><th rowspan="2">P-Value</th></tr><tr><th>n (%)</th><th>n (%)</th><th>n (%)</th></tr></thead><tbody><tr><td>Age, y, median (range)</td><td>67 (35–87)</td><td>65 (35–81)</td><td>69 (35–87)</td><td>0.28</td></tr><tr><td>Breslow, mm, median (range)</td><td>2.2 (0.22–29)</td><td>4.2 (0.22–29)</td><td>1.4 (0.22–12)</td><td>0.65</td></tr><tr><td>missing</td><td>9</td><td>5</td><td>4</td><td></td></tr><tr><td>Ulceration</td><td></td><td></td><td></td><td>0.65</td></tr><tr><td>Yes</td><td>12 (63%)</td><td>7 (70%)</td><td>5 (56%)</td><td></td></tr><tr><td>No</td><td>7 (37%)</td><td>3 (30%)</td><td>4 (44%)</td><td></td></tr><tr><td>missing</td><td>9</td><td>5</td><td>4</td><td></td></tr><tr><td>Mitotic rate</td><td></td><td></td><td></td><td>0.59</td></tr><tr><td>0 or &lt;1</td><td>3 (16%)</td><td>1 (11%)</td><td>2 (20%)</td><td></td></tr><tr><td>1</td><td>0 (0%)</td><td>0 (0%)</td><td>0 (0%)</td><td></td></tr><tr><td>2–3</td><td>8 (42%)</td><td>5 (56%)</td><td>3 (30%)</td><td></td></tr><tr><td>4–10</td><td>7 (37%)</td><td>3 (33%)</td><td>4 (40%)</td><td></td></tr><tr><td>&gt;10</td><td>1 (5%)</td><td>0 (0%)</td><td>1 (10%)</td><td></td></tr><tr><td>unknown</td><td>9</td><td>6</td><td>3</td><td></td></tr><tr><td>LDH level, UI/l, median (range)</td><td>241 (134–558)</td><td>248 (134–558)</td><td>207 (176–345)</td><td>0.39</td></tr><tr><td>Less than standard, 214 UI/l</td><td>12 (43%)</td><td>4 (27%)</td><td>8 (62%)</td><td>0.06</td></tr><tr><td>Higher than standard, 214 UI/l</td><td>16 (57%)</td><td>11 (73%)</td><td>5 (38%)</td><td></td></tr><tr><td>ECOG status</td><td></td><td></td><td></td><td>0.07</td></tr><tr><td>0</td><td>23 (83%)</td><td>13 (87%)</td><td>10 (77%)</td><td></td></tr><tr><td>1</td><td>3 (11%)</td><td>0 (0%)</td><td>3 (23%)</td><td></td></tr><tr><td>2</td><td>2 (7%)</td><td>2 (13%)</td><td>0 (0%)</td><td></td></tr><tr><td>Primary tumor (T)</td><td></td><td></td><td></td><td>0.74</td></tr><tr><td>T1</td><td>1 (4%)</td><td>1 (7%)</td><td>0 (0%)</td><td></td></tr><tr><td>T2</td><td>7 (25%)</td><td>3 (20%)</td><td>4 (31%)</td><td></td></tr><tr><td>T3</td><td>4 (14%)</td><td>2 (13%)</td><td>2 (15%)</td><td></td></tr><tr><td>T4</td><td>7 (25%)</td><td>4 (27%)</td><td>3 (23%)</td><td></td></tr><tr><td>Tx</td><td>9 (32%)</td><td>5 (33%)</td><td>4 (31%)</td><td></td></tr><tr><td>Regional lymph nodes (N)</td><td></td><td></td><td></td><td>0.74</td></tr><tr><td>N<sub>0</sub></td><td>6 (21%)</td><td>3 (20%)</td><td>3 (24%)</td><td></td></tr><tr><td>N<sub>+</sub></td><td>12 (43%)</td><td>7 (47%)</td><td>5 (38%)</td><td></td></tr><tr><td>N<sub>x</sub></td><td>10 (36%)</td><td>5 (33%)</td><td>5 (38%)</td><td></td></tr><tr><td>Distant metastases (M)</td><td></td><td></td><td></td><td>0.25</td></tr><tr><td>M<sub>0</sub></td><td>3 (12%)</td><td>2 (13%)</td><td>1 (9%)</td><td></td></tr><tr><td>M<sub>+</sub></td><td>25 (88%)</td><td>13 (87%)</td><td>12 (91%)</td><td></td></tr><tr><td>M<sub>1a</sub></td><td>2 (8%)</td><td>1 (7%)</td><td>1 (8%)</td><td></td></tr><tr><td>M<sub>1b</sub></td><td>5 (20%)</td><td>4 (31%)</td><td>1 (8%)</td><td></td></tr><tr><td>M<sub>1c</sub></td><td>13 (52%)</td><td>8 (62%)</td><td>5 (42%)</td><td></td></tr><tr><td>M<sub>1d</sub></td><td>3 (12%)</td><td>0 (0%)</td><td>3 (25%)</td><td></td></tr><tr><td>M<sub>+</sub> (not defined)</td><td>2 (8%)</td><td>0 (0%)</td><td>2 (17%)</td><td></td></tr><tr><td>Stage</td><td></td><td></td><td></td><td>1.00</td></tr><tr><td>III</td><td>3 (11%)</td><td>2 (13%)</td><td>1 (8%)</td><td></td></tr><tr><td>IV</td><td>25 (89%)</td><td>13 (87%)</td><td>12 (92%)</td><td></td></tr><tr><td>BRAF status</td><td></td><td></td><td></td><td>0.49</td></tr><tr><td>BRAF<sup>V600E</sup></td><td>23 (85%)</td><td>13 (87%)</td><td>10 (84%)</td><td></td></tr><tr><td>BRAF<sup>V600K</sup></td><td>3 (11%)</td><td>2 (13%)</td><td>1 (8%)</td><td></td></tr><tr><td>BRAF<sup>COSM30370</sup></td><td>1 (4%)</td><td>0 (0%)</td><td>1 (8%)</td><td></td></tr><tr><td>unknown</td><td>1</td><td>0</td><td>1</td><td></td></tr><tr><td>Therapeutic line</td><td></td><td></td><td></td><td>-</td></tr><tr><td>Adjuvant</td><td>3 (11%)</td><td>2 (13%)</td><td>1 (8%)</td><td></td></tr><tr><td>First line</td><td>16 (57%)</td><td>9 (60%)</td><td>7 (54%)</td><td></td></tr><tr><td>Second line</td><td>4 (14%)</td><td>1 (7%)</td><td>3 (23%)</td><td></td></tr><tr><td>Third line</td><td>5 (18%)</td><td>3 (20%)</td><td>2 (15%)</td><td></td></tr></tbody></table>

(continued)
**Supplementary Table S2. Continued**

<table><thead><tr><th rowspan="2">Characteristics</th><th>Total (n = 28)</th><th>Male (n = 15)</th><th>Female (n = 13)</th><th rowspan="2">P-Value</th></tr><tr><th>n (%)</th><th>n (%)</th><th>n (%)</th></tr></thead><tbody><tr><td>Progressive disease</td><td></td><td></td><td></td><td>0.63</td></tr><tr><td>Primary resistance</td><td>1 (4%)</td><td>1 (6%)</td><td>0 (0%)</td><td></td></tr><tr><td>Acquired resistance</td><td>14 (50%)</td><td>7 (47%)</td><td>7 (54%)</td><td></td></tr><tr><td>Partial response or stable disease</td><td>13 (46%)</td><td>7 (47%)</td><td>6 (46%)</td><td></td></tr><tr><td>Median time to progression, mo (range)</td><td>9 (3–24)</td><td>12 (3–24)</td><td>6 (3–14)</td><td>0.22</td></tr></tbody></table>

Abbreviations: BRAFi, BRAF inhibitor; ECOG, Eastern Cooperative Oncology Group; LDH, lactate dehydrogenase; MEKi, MAPK/extracellular signal-regulated kinase inhibitor.

n denotes the number of subjects with percentages. P-value denotes the comparison between male and female patients with melanoma. P-values were calculated by contingency table for qualitative data, whereas Mann-Whitney U test was employed for quantitative data.