Intralesional Methotrexate for the Treatment of Advanced Keratinocytic Tumors: A Multi-Center Retrospective Study

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ArticleTitle _{Intralesional Methotrexate for the Treatment of Advanced Keratinocytic Tumors: A Multi-Center} Retrospective Study

Article Sub-Title

Article CopyRight The Author(s)

(This will be the copyright line in the final PDF)

Journal Name Dermatology and Therapy

Corresponding Author Family Name Caravello

Particle

Given Name Simone

Suffix

Division Department of Dermatology, Spedali Civili di Brescia

Organization University of Brescia

Address Brescia, Italy

Phone Fax

Email caravello.simone29@gmail.com; s.caravello001@unibs.it

URL

ORCID http://orcid.org/0000-0001-7223-5480

Author Family Name Gualdi

Particle

Given Name Giulio

Suffix

Division Department of Medicine and Aging Science

Organization Dermatologic Clinic, University G. D’Annunzio

Address Chieti Pescara, Italy

Phone Fax Email URL ORCID

Author Family Name Frasci

Particle

Given Name Fabiano

Suffix Division

Organization University of Sassari

Address Sassari, Italy

Phone Fax Email URL

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ORCID

Author Family Name Giuliani

Particle

Given Name Federica

Suffix

Author Family Name Moro

Particle

Given Name Ruggero

Suffix

Division Escuela de Doctorado

Organization Universidad Católica de Valencia San Vicente Mártir

Address València, Spain

Division Dermatologia

Organization Clinica San Carlo

Address Paderno Dugnano, Italy

Author Family Name Fargnoli

Particle

Given Name Maria C.

Suffix

Division Dermatology, Department of Biotechnological and Applied Clinical Sciences

Organization University of L’Aquila

Address L’Aquila, Italy

Author Family Name Ciciarelli

Particle

Given Name Valeria

(4)

Division Dermatology, Department of Biotechnological and Applied Clinical Sciences

Organization University of L’Aquila

Address L’Aquila, Italy

Author Family Name Argenziano

Particle

Given Name Giuseppe

Suffix

Division Dermatology Unit

Organization University of Campania

Address Naples, Italy

Author Family Name Giorgio

Particle

Given Name Caterina M.

Suffix

Division Dermatology Unit

Organization University of Campania

Address Naples, Italy

Author Family Name Requena

Particle

Given Name Celia

Suffix

Division Department of Dermatology

Organization Instituto Valenciano de Oncología

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Author Family Name Nagore

Particle

Given Name Eduardo

Suffix

Division School of Medicine

Author Family Name Sanmartín

Particle

Given Name Onofre

Suffix

Division School of Medicine

Author Family Name Martorell

Particle

Given Name Antonio

Suffix

Organization Hospital de Manises

Author Family Name Fantini

Particle

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Suffix

Organization Ospedale “Manzoni” Lecco

Address Lecco, Italy

Author Family Name Calzavara-Pinton

Particle

Given Name Piergiacomo

Suffix

Division Department of Dermatology, Spedali Civili di Brescia

Organization University of Brescia

Address Brescia, Italy

Author Family Name Amerio

Particle

Given Name Paolo

Suffix

Phone Fax Email URL ORCID Schedule Received 10 April 2020 Revised Accepted Abstract Introduction:

Keratinocyte tumors (KT) are frequently observed. Surgery is the treatment gold standard. In some cases, a surgical approach might not be the best option. Radiotherapy (RT) and systemic treatments can frequently cause side effects or be contraindicated. Intralesional methotrexate (MTX) can be a conservative yet effective alternative. We decided to evaluate the effectiveness and safety of intralesional chemotherapy with MTX for the treatment of squamous cell carcinoma (SCC), keratoacanthoma (KA), and basal cell carcinoma (BCC).

Methods:

All patients had a histologically confirmed diagnosis of BCC, SCC, or KA and no indication to surgery or RT. MTX was injected subcutaneously proceeding from the periphery of the lesion toward the center. Different protocols in terms of dose, frequency, and length of treatment were used to compare them.

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Treatment efficacy was evaluated in terms of tumor size reduction. Patients were divided into three groups: responders (improvement of more than 50%), partial responders (< 50%), and non-responders (no

improvement or worsening). All data were analyzed using the chi-squared test (χ 2_).

Results:

Thirty-five patients were included. Twenty-one patients suffered from SCC, 12 from KA, and 2 from BCC. KA showed a higher response rate than SCC and BCC. For AK, 92% of patients had a complete resolution; 8% were partial responders. For SCC, 47.6% of cases were responders and 14.3% partial responders, while 38% non-responders. All BCCs showed no improvement. A treatment protocol of weekly injections, performed for 4 to 6 weeks, was the most efficient. Doses of 25 mg/ml per session seemed to be most effective. About one third of our patients developed side effects with mild anemia being the most frequent.

Conclusions:

For selected cases, intralesional MTX can be a safe and effective option for the treatment of KT, especially in case of KA and, to a lesser extent, SCC.

Keywords (separated by '-') _{Basal cell carcinoma - Intralesional methotrexate - Kratinocytic tumors - Keratoacanthoma -} Non-melanoma skin cancer - Squamous cell carcinoma - Surgery

Footnote Information Digital Features To view digital features for this article go to https://doi.org/10.6084/m9.figshare.

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ORIGINAL RESEARCH 1

2

Intralesional Methotrexate for the Treatment

3

of Advanced Keratinocytic Tumors: A Multi-Center

4

Retrospective Study

5 Giulio Gualdi.Simone Caravello .Fabiano Frasci.Federica Giuliani.Ruggero Moro .Maria C. Fargnoli .

6 Valeria Ciciarelli . Giuseppe Argenziano . Caterina M. Giorgio . Celia Requena . Eduardo Nagore .

7 Onofre Sanmartı´n.Antonio Martorell.Fabrizio Fantini.Piergiacomo Calzavara-Pinton.Paolo Amerio

8

9 _{Received: April 10, 2020} 10 Ó_{The Author(s) 2020}

11

ABSTRACT

12 _{Introduction: Keratinocyte tumors (KT) are} 13 _{frequently observed. Surgery is the treatment} 14 _{gold standard. In some cases, a surgical} 15 _{approach might not be the best option.} Radio-16 _{therapy (RT) and systemic treatments can} fre-17 _{quently cause side effects or be contraindicated.}

18 Intralesional methotrexate (MTX) can be a

19 conservative yet effective alternative. We

deci-20 ded to evaluate the effectiveness and safety of

21 intralesional chemotherapy with MTX for the

22 treatment of squamous cell carcinoma (SCC),

23 keratoacanthoma (KA), and basal cell carcinoma

24 (BCC).

25 Methods: All patients had a histologically

con-26 ﬁrmed diagnosis of BCC, SCC, or KA and no

27 indication to surgery or RT. MTX was injected

28 subcutaneously proceeding from the periphery

Digital Features To view digital features for this article go tohttps://doi.org/10.6084/m9.ﬁgshare.12320330

G. Gualdi F. Giuliani P. Amerio

Department of Medicine and Aging Science, Dermatologic Clinic, University G. D’Annunzio, Chieti Pescara, Italy

S. Caravello (&) P. Calzavara-Pinton Department of Dermatology, Spedali Civili di Brescia, University of Brescia, Brescia, Italy e-mail: caravello.simone29@gmail.com; s.caravello001@unibs.it

F. Frasci

University of Sassari, Sassari, Italy

R. Moro

Escuela de Doctorado, Universidad Cato´lica de Valencia San Vicente Ma´rtir, Vale`ncia, Spain

R. Moro

Dermatologia, Clinica San Carlo, Paderno Dugnano, Italy

M. C. Fargnoli V. Ciciarelli

Dermatology, Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, L’Aquila, Italy

G. Argenziano C. M. Giorgio

Dermatology Unit, University of Campania, Naples, Italy

C. Requena E. Nagore O. Sanmartı´n

Department of Dermatology, Instituto Valenciano de Oncologı´a, Vale`ncia, Spain

E. Nagore O. Sanmartı´n

School of Medicine, Universidad Cato´lica de Valencia San Vicente Ma´rtir, Vale`ncia, Spain

A. Martorell

Department of Dermatology, Hospital de Manises, Vale`ncia, Spain

F. Fantini

Department of Dermatology, Ospedale ‘‘Manzoni’’ Lecco, Lecco, Italy

Dermatol Ther (Heidelb)

https://doi.org/10.1007/s13555-020-00400-z

Journal : Large 13555 Dispatch : 29-5-2020 Pages : 9

Article No. : 400 _{h LE} _{h TYPESET}

MS Code : DERM-D-20-00081R1 h CP4 h DISK4

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PROOF

29 _{of the lesion toward the center. Different} pro-30 _{tocols in terms of dose, frequency, and length of} 31 _{treatment were used to compare them.} Treat-32 _{ment efﬁcacy was evaluated in terms of tumor} 33 _{size reduction. Patients were divided into three} 34 _{groups: responders (improvement of more than} 35 _{50%), partial responders (\ 50%), and} non-re-36 _{sponders (no improvement or worsening). All} 37 _{data were analyzed using the chi-squared test} 38 _(v2_).

39 _{Results: Thirty-ﬁve patients were included.} 40 _{Twenty-one patients suffered from SCC, 12} 41 _{from KA, and 2 from BCC. KA showed a higher} 42 _{response rate than SCC and BCC. For AK, 92%} 43 _{of patients had a complete resolution; 8% were} 44 _{partial responders. For SCC, 47.6% of cases were} 45 _{responders and 14.3% partial responders, while} 46 _{38% non-responders. All BCCs showed no} 47 _{improvement. A treatment protocol of weekly} 48 _{injections, performed for 4 to 6 weeks, was the} 49 _{most efﬁcient. Doses of 25 mg/ml per session} 50 _{seemed to be most effective. About one third of} 51 _{our patients developed side effects with mild} 52 _{anemia being the most frequent.}

53 _{Conclusions: For selected cases, intralesional} 54 _{MTX can be a safe and effective option for the} 55 _{treatment of KT, especially in case of KA and, to} 56 _{a lesser extent, SCC.}

57 _{Keywords: Basal cell carcinoma; Intralesional} 58 _{methotrexate;} _Kratinocytic _tumors; 59 _{Keratoacanthoma; Non-melanoma skin cancer;} 60 _{Squamous cell carcinoma; Surgery}

61 62

Key Summary Points

63 65 66

67 _{Why carry out this study?}

68 _{Keratinocyte tumors are the most frequent} 69 _{human tumors found in medical pracice.} 70 _{A surgical approach is not always the best} 71 _{option, and radiotherapy, as well as} 72 _{systemic chemotherapy, comes with a} 73 _{high burden of costs and side effects} 74 _{Intralesional chemotherapy, in particular} 75 _{with MTX, could be a conservative yet} 76 _{effective treatment option}

77 79 80 What was learned from the study?

81 Keratoacanthoma (KA) response rate to

82 intralesional MTX is excellent. All KAs in

83 our study improved partially or

84 completely. About one half of squamous

85 cell carcinomas showed either

86 improvement or resolution, while basal

87 cell carcinoma had no improvement

88 A treatment protocol consisting of weekly

89 injections, performed for 4–6 weeks,

90 seemed to be the most efﬁcient. Dosages B

91 20 mg/ml per session showed little

92 efﬁcacy compared with 25 mg/ml doses.

93 The latter appeared more effective than

94 50 mg/ml as well

95 For selected cases, intralesional MTX can

96 be a safe and effective option for the

97 treatment of KT, especially in case of

98 keratoacanthomas and, to a lesser extent,

99 SCC 100 101

INTRODUCTION

102 Non-melanoma skin cancer (NMSC) is the most

103 common malignancy worldwide [1]. The vast

104 majority of NMSCs are keratinocyte tumors (KT).

105 In particular, squamous cell carcinoma (SCC)

106 and basal cell carcinoma (BCC) are the two most

107 frequent sub-types, with BCC accounting for

108 almost 80% of the cases of KT, while SCC for 20%

109 [2–3]. Sun exposure is the main risk factor for the

110 development of these tumors, especially in case

111 of SCC [4]. In 2002, the incidence of SCC in

112 Australia, where the highest number of cases is

113 registered, was 387 cases per 100,000, while in

114 Europe the highest rates were reported in Wales

115 (31.7 cases per 100,000 person/year) and

116 Switzerland (28.9 cases per 100,000 person/year)

117 [5–6]. In Italy, AIRTUM (Associazione Italiana

118 Registro Tumori) reported 127,879 new cases of

119 skin cancer in a 10-year period (2005–2015) [7].

120 BCCs and SCCs are common, but fortunately the

121 prognosis is good in the vast majority of cases.

122 While risk of BCC metastasizing is extremely low,

123 SCC is more aggressive, yet it is associated with a

124 5-year median survival rate [ 90%. Metastases

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125 _{develop in 2–5% of SCC cases [}₆_{]. If metastatic} 126 _{lesions develop, the median survival is\2 years.} 127 _{NMSC is the ﬁfth most expensive cancer for the} 128 _{US health care system. This economic burden} 129 _{depends on the high incidence of KT, recurrence} 130 _{rates and cost of individual treatments [}₈_]. 131 _{Nowadays, the gold standard treatment for} 132 _{NMSC is surgery. However, in particular cases,} 133 _{surgery may not be the optimum choice. Several} 134 _{considerations have to be taken into account} 135 _{when deciding whether to perform a surgical} 136 _{procedure: ﬁrst, patient features such as age and} 137 _{comorbidities, then tumor characteristics such} 138 _{as lesion size and anatomic location. Finally,} 139 _{the surgical procedure itself has to be} consid-140 _{ered as well: the risk of recurrence, infection, or} 141 _{excessive bleeding must guide the surgeon} 142 _{while deciding about the best treatment option} 143 _{for each patient.}

144 _{When surgery is not indicated, few effective} 145 _{therapeutic options are available. Radiotherapy} 146 _{(RT) is widely used and seems to be quite effective} 147 _{as well. Unfortunately, RT is time consuming and} 148 _expensive. _Frequent _treatments _are _often 149 _{demanding for the patient, who has to come to} 150 _{the hospital many days in a few weeks. Moreover,} 151 _{RT can determine the secondary development of} 152 _{radiation dermatits. For larger tumors, RT is used} 153 _{as neoadiuvant therapy, but it is not indicated for} 154 _{the treatment of extensive areas, especially on} 155 _{prominent sites such as the face.}

156 _{Systemic treatments directed against} molec-157 _{ular targets have recently given promising} 158 _{results. Smoothened (Smo) receptor inhibitors,} 159 _{such as vismodegib and sonidegib, can be} 160 _effective _therapeutic _options _for _locally 161 _{advanced BCC. Likewise, anti-PD-1 drugs such} 162 _{as cemiplimab—and to a lesser extent} anti-163 _{EGFr—were proven to be effective in treating} 164 _{SCC. However, these treatments are only able to} 165 _{completely eradicate the tumor in a minority of} 166 patients. Other subjects show a partial response, 167 _{which can be held only with a prolonged} 168 _{maintenance treatment. These long periods of} 169 _{CHT are associated with a high risk of toxicity,} 170 _{often unbearable for many patients. In} addi-171 _{tion, CHT has to be frequently discarded} 172 _{because of contraindications.}

173 _{To avoid invasive surgery approaches or the} 174 _{frequent side effects related to systemic drugs,}

175 intralesional chemotherapy (iCHT) can be a

176 conservative yet effective alternative.

177 Methotrexate (MTX) is widely used in

oncol-178 ogy as an anti-tumoral agent. Its mechanisms of

179 action include the competitive inhibition of the

180 folic acid reductase enzyme, which is essential to

181 supply methyl donor groups for DNA, RNA, and

182 protein synthesis. This inhibition enables the

183 conversion of dihydrofolic to tetrahydrofolic

184 acid, blocking the production of thymidylic acid,

185 a pyrimidine metabolite. Without thymidylic

186 acid, DNA synthesis is impossible and cell

divi-187 sion is therefore arrested [9].

188 The aim of the study was to retrospectively

189 evaluate the effectiveness and safety of

intrale-190 sional chemotherapy with MTX for the treatment

191 of squamous cell carcinoma (SCC),

keratoacan-192 thoma (KA), and basal cell carcinoma (BCC).

193 MTX was only administered to patients who

194 had contraindications to surgery, RT, surgery, or

195 systemic drug therapy. The secondary objective

196 of the study was to compare different treatment

197 protocols in terms of dose and duration of

198 treatment.

199

METHODS

200 This is a multicenter retrospective study: the

201 Departments of Dermatology of Brescia, Chieti,

202 L’Aquila, Lecco, and Napoli in Italy performed

203 this study together with the Instituto

Valen-204 ciano de Oncologı´a of Valencia in Spain. All

205 patients, included in our study between 2017

206 and 2019, had a histologically conﬁrmed

diag-207 nosis of BCC, SCC, or KA (exclusively eruptive

208 squamous cell carcinoma with

keratoacan-209 thoma-like features). Surgery was considered as

210 the ﬁrst option followed by RT. Considering the

211 parameters previously mentioned (age,

comor-212 bidities, lesion size and anatomic location, risk

213 of recurrence, infection, etc.), an alternative

214 treatment was needed for each patient.

There-215 fore, it was decided to treat them with

intrale-216 sional MTX. Patients refusing surgery and RT,

217 and subjects previously treated with such

tech-218 niques that later showed a recurrence or disease

219 progression, were included as well. Before

220 including a patient in the study, each case was

221 subjected to a multidisciplinary board

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222 _{consultation. Exclusion criteria included: bone} 223 _{marrow and hepatic failure; HIV, HBV, and} 224 _{HCV infection; and metastasis. Information} 225 _{about age, sex, and tumor size and site was} 226 _{collected, along with information about} treat-227 _ment _protocols _(cumulative _dose, _length, 228 _{number of procedures). All patients were} thor-229 _{oughly informed and gave their consent to} 230 _{treatment. Moreover, the study was approved} 231 _{by the Internal Scientiﬁc Board of Dermatology} 232 _{of the Department of Medicine and Aging} Sci-233 _{ence, University G. D’Annunzio, Chieti Pescara,} 234 _{Italy. This study was performed in accordance} 235 _{with the Helsinki Declaration of 1964 and its} 236 _{later amendments. The patients also provided} 237 _{their written informed consent for the} publica-238 _{tion of their images.}

239 _{A physical examination was performed} 240 _{before each administration of MTX to assess the} 241 _{presence of side effects and the response to} 242 _{treatment, which was evaluated through the} 243 _{measurement of the main axis of the tumor. All} 244 _{patients underwent a laboratory workup with} 245 _{hematology, liver and kidney biochemistries,} 246 _{and urine analysis before and a month after the} 247 _{treatment. Folic acid supplementation was} 248 _{administered in accordance with guidelines to} 249 _{avoid the risk of development of hematologic} 250 _{side effects. MTX was injected subcutaneously,} 251 _{using a syringe with a 30-gauge needle. The} 252 _{tumor was divided into four equal parts. The} 253 _{drug was equally distributed in the four parts,} 254 _{treating the periphery of each area with a single} 255 _{injection (four injections per lesion per} treat-256 _{ment session). At the following visit, if the} 257 _{tumor showed a decrement is size, injections} 258 _{were performed at the new periphery of the} 259 _{lesion, proceeding in this way from the outer} 260 _{parts toward the center. As there is no} stan-261 _{dardized protocol available at the moment,} 262 _{each physician used a different protocol in} 263 terms of dose, frequency, and length of treat-264 _{ment. The chosen dose and time interval} 265 _{between each administration were kept the} 266 _{same for each patient. This allowed us to} com-267 _{pare different regimens. Treatment efﬁcacy was} 268 _{evaluated in terms of reduction in size of the} 269 _{tumor at different time points: 3 and 6 months.} 270 _{Regarding the response to therapy, patients} 271 _{were divided into three groups: responders}

272 (improvement [ 50%), partial responders

(im-273 provement \ 50%), and non-responders (no

274 improvement or worsening of the lesion). All

275 data were analyzed using the chi-squared test

276 (v2) to search for a statistically relevant

correla-277 tion between the different factors considered.

278

RESULTS

279 Thirty-ﬁve patients were included in this study.

280 The average age was 79.57 ± 11.02 years.

281 Seventeen patients were male; 18 were female.

282 Twenty-one patients suffered from SCC, 12

283 from KA, and 2 from BCC. The average

dimen-284 sion before treatment (major axis) was

285 3.6 ± 2.61 cm. Two patients had been

previ-286 ously treated with cryotherapy and one with

287 RT. The average number of treatments was

288 4.46 ± 2.15, while its duration ranged from 3 to

289 16 weeks, averaging 6.54 ± 2.84 weeks.

Cumu-290 lative average dose was 133.29 ± 99.24 mg/ml,

291 while average dose for a single treatment was

292 22.15 ± 15.6 mg/ml. Twenty-one patients were

293 responders, four had a partial response, and ten

294 patients were non-responders (Table1).

295 The initial dimension of the tumor does not

296 seem to inﬂuence the efﬁcacy of the treatment.

297 Indeed, partially improved lesions had a larger

298 initial size than lesions that showed no

299 response. A treatment protocol consisting of

300 weekly injections, performed for 4–6 weeks,

301 seemed to be the most efﬁcient. After the 6th

302 week, the treatment appeared to lose its

efﬁ-303 cacy, and no further beneﬁcial effect was

304 observed. Dosages B 20 mg/ml per session

305 showed little efﬁcacy compared with doses of

306 25 mg/ml. The latter appeared more effective

307 than 50 mg/ml as well (Table2). No statistically

308 signiﬁcant differences were found when

com-309 paring the efﬁcacy of the treatment for tumors

310 in different areas (p = 0.580). Similarly, there

311 was no difference in efﬁcacy between the

312 immune-suppressed patient and the others.

313 Previous treatments also did not seem to

inﬂu-314 ence the efﬁcacy of this therapy. All patients

315 who had already been treated with other

tech-316 niques showed either a complete or partial

317 response.

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318 _{KA showed a higher response to intralesional} 319 _{MTX compared with SCC and BCC. All KAs} 320 _{improved after therapy: 92% had a complete} 321 _{resolution while 8% were partial responders. For} 322 _{SCC, 47.6% of patients were responders; 14.3%} 323 _{had a partial response, while 38% showed either} 324 _{no improvement or progression of the disease.} 325 _{All BCCs in our study showed no improvement} 326 _(Figs.₁_,₂_,₃_).

327 All patients showing a partial response, as

328 the size of the tumor decreased, were then

suc-329 cessfully treated with surgery. As each patient

330 included in the study was at least initially

331 unsuitable for surgery, a surgical approach was

332 still not an option for non-responders.

There-333 fore, non-responders were addressed to the

334 oncologist to undergo systemic chemotherapy.

335 Follow-up duration was 6 months. Neither Table 1 Different responses of KA, SCC and BCC to different treatment protocols

Variable N Overall NR (n = 10) RP (n = 4) R (n = 21) p value Mean – SD Age 35 79.57 ± 11.01 80.8 ± 11.05 75.5 ± 13.33 79.76 ± 10.96 0.590 Sex Male 35 17.00 4 (0.24) 3 (0.18) 10 (0.59) 0.802 Female 18.00 6 (0.33) 1 (0.06) 11 (0.61) Weeks 35 6.54 ± 2.84 5.5 ± 2.37 10 ± 5.83 6.38 ± 1.77 0.168 Diameter 35 3.6 ± 2.61 3.86 ± 3.49 6.5 ± 3 2.92 ± 1.6 0.433 No. of treatments 35 4.46 ± 2.15 3.4 ± 1.51 6.25 ± 1.5 4.62 ± 2.29 0.670 Cumulative dosage 35 133.29 ± 99.24 96.5 ± 67.78 198.75 ± 63.29 138.33 ± 111.86 0.442 Dosage for treatment 35 22.15 ± 15.6 20.93 ± 16.87 30 ± 23.18 21.23 ± 13.83 0.141 KT types

SCC 35 21.00 8 (0.38) 3 (0.14) 10 (0.48) 0.018 KA 12.00 0 (0) 1 (0.08) 11 (0.92)

BCC 2.00 2 (1) 0 (0) 0 (0)

Table 2 Response to different single doses

0 2 4 6 8 10 12 14 16 18 15 mg/ml 20mg/ml 25mg/ml 50mg/ml

Dosage for single treatment

Responder+ParƟal Responder non Responder

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Fig. 1 Keratoacanthoma before treatment (a) showing a complete response after therapy (b)

Fig. 2 Basal cell carcinoma before therapy (a) showing progression despite therapy (b)

Fig. 3 Squamous cell carcinoma before therapy (a) showing a partial response to therapy (b)

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336 _{patients showing a complete response nor the} 337 _{ones with partial response subsequently treated} 338 _{with surgery showed recurrence at 6 months.} 339 _{No data are available regarding non-responders} 340 _{as they were referred to another unit.}

341 _{About one third of our patients developed} 342 _{side effects (34.3%). Anemia (Hb \ 12 g/dl) was} 343 _{the most frequent (5 patients), but only one} 344 _{patient developed severe anemia and required a} 345 _transfusion. _Other _common _side _effects 346 _{observed were: leukopenia (1 patient), elevation} 347 _of _liver _{transaminases} ₍₄ _patients), _and 348 _{increased nitrogen levels (1 patient).} Non-re-349 _{sponders developed side effects in 30% of cases,} 350 _{responders in 33%, showing no difference} 351 _{between the two categories. Efﬁcacy of} treat-352 _{ment does not seem to have a correlation with} 353 _{the development of side effects. Similarly,} 354 _{cumulative dose and duration of treatment did} 355 _{not seem to play a role. Two patients developed} 356 _{metastasis during treatment.}

357

DISCUSSION

358 _{KTs are the most frequent human tumors} 359 _{observed in medical practice. Surgery is the gold} 360 _{standard for treating such lesions. As discussed} 361 _{before, in some cases a surgical approach might} 362 _{not be the best option. Patient and tumor} fea-363 _{tures, along with cost and duration of the} 364 _{therapy, should be considered.}

365 _{Even if the percentage of patients with KT} 366 _{who have some contraindication to surgery is} 367 _{low, the high frequency of KT determines a} 368 _{large number of patients for whom an} alterna-369 _{tive to surgery is needed. RT, which shows a} 370 _{high efﬁcacy rate in the literature (up to 92%), is} 371 _an _expensive _option _and _is _often _quite 372 _demanding _and _time _consuming _for _the 373 _{patient. To the best of our knowledge, no study} 374 _{comparing surgery with RT is currently present} 375 _{in the literature. RT is mostly used as} neoadju-376 _{vant therapy before surgery (to decrease the size} 377 _{of the tumor before surgical intervention) or in} 378 _{case of relapse. The risk of inducing a} radio-379 _{dermatitis, and therefore potentially another} 380 _{neoplastic lesion, and the complexity of} treat-381 _{ing a relapse if developed on an area previously} 382 _{treated with RT are the main critical issues when}

383 approaching this technique. Systemic CHT can

384 be effective in treating advanced KT. However,

385 complete eradication of the tumor is rare, and

386 patients have to go through prolonged

main-387 tenance treatment, which carries a high risk of

388 toxicity.

389 Intralesional MTX for the treatment of KT is

390 described in the literature for KA, often on a low

391 number of patients. In our study, the efﬁcacy of

392 intralesional MTX was evaluated on 35 patients

393 affected by different kinds of KT and not just

394 KA. Several treatment protocols were compared

395 as well. Our sample consisted of elderly, and

396 therefore fragile, patients (average age

397 79.5 years). Most of the lesions developed on

398 high-risk areas, such as the head or hands and

399 feet. Our data show great efﬁcacy for KAs, as all

400 patients improved or were completely healed.

401 About half of the patients with cSCC responded

402 to therapy, while no patient affected by BCC

403 showed any improvement. This difference

404 might be explained by the mechanism through

405 which MTX dispatches its pharmacologic

406 properties. As MTX inhibits DNA synthesis

407 during cell replication, rapidly growing tumors,

408 such as KA and cSCC, are more sensitive to such

409 therapy compared with slow-growing tumors

410 such as BCC. This general consideration must

411 take into account the reduced signiﬁcance of

412 the BCC data, given the low number of cases

413 included. Our data regarding the correlation

414 between the efﬁcacy of intralesional MTX and

415 the size of the tumor seem to conﬁrm this

416 hypothesis as well. As larger tumors likely

417 replicate faster, they show a higher sensitivity to

418 MTX than smaller lesions, which do not

repli-419 cate at such a pace. A review from 2019

420 demonstrated a high efﬁcacy of intralesional

421 MTX for the treatment of KA (94%) [10]. Our

422 study conﬁrms these data. In another study,

423 Moss and Weber treated 157 KAs with a

com-424 plete resolution in 88% of the cases [11]. There

425 are few studies in the literature on the efﬁcacy

426 of MTX for treating cSCC. Moreover, most of

427 them evaluate MTX efﬁcacy as adjuvant

ther-428 apy, but not as a solitary treatment. In 2016,

429 Salido-Vallejo et al. compared 43 patients

trea-430 ted with surgery alone with 43 patients treated

431 with intralesional MTX before a surgical

432 approach. All surgical procedures were

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433 _{performed within 30 days of the diagnosis. In} 434 _{the first group, the average increment in lesion} 435 _{size was 19%. Patients treated with MTX} 436 _{showed a decrease in lesion size of 23%.} More-437 _{over, SCC of the lips seemed more responsive} 438 _{than tumors of other areas [}₁₂_{]. Similarly, in} 439 _{2018 Sendin and Perez described a group of} 440 _{patients affected by SCC of the lips, treated with} 441 _{two injections of MTX 50 days before surgery.} 442 _{The size of these tumors decreased significantly} 443 _{(68.18% decrease for the minor axis and 57.28%} 444 _{for the major) [}₁₃_{]. All the other studies on this} 445 _{matter are case reports in which MTX was again} 446 _{used as neoadjuvant therapy. Our study shows} 447 _{that MTX can be effective for about half of the} 448 _{cases of cSCC. This confirms that MTX can be a} 449 _{useful tool to treat tumors for which surgery} 450 _{could not be indicated. Regarding BCC, both} 451 _{lesions treated in our study showed an initial} 452 _{and mild improvement. There is only one study} 453 _{in the literature in which MTX was tested for} 454 _{the treatment of BCC. In this study, 11 patients} 455 _{were treated with a single dose of intralesional} 456 _{MTX; however, no improvement was observed} 457 _[₁₄_{]. One of the main issues regarding the use of} 458 _{intralesional MTX is that no specific} standard-459 _{ized treatment protocol has been established} 460 _{yet. Our data suggest that weekly} administra-461 _{tion of at least 25 mg/ml for a 4–6-week period,} 462 _{might be the best approach. No improvement} 463 _{was observed after the 6th week of treatment for} 464 _{any patient. No correlations between dose or} 465 _{duration of the treatment and side effects were} 466 _{found. Likewise, previous treatments, immune} 467 _{suppressive concomitant therapies, or site of the} 468 _{lesion do not seem to influence the efficacy of} 469 _{this therapy.}

470 _{The main limitation of the study is the low} 471 _{number of patients affected by BCC.}

472

CONCLUSION

473 _{For selected cases, intralesional MTX can be a} 474 _{safe and effective option for the treatment of} 475 _{KT, especially in case of KA and, to a lesser} 476 _{extent, SCC. Moreover, this procedure can also} 477 _{be considered as a pre-surgical option to reduce} 478 _{the tumor size and make the surgical procedure} 479 _{easier and safer for the patient.}

480

ACKNOWLEDGEMENTS

481 Funding. No funding or sponsorship was

482 received for this study or publication of this

483 article.

484 Authorship. All named authors meet the

485 International Committee of Medical Journal

486 Editors (ICMJE) criteria for authorship for this

487 article, take responsibility for the integrity of

488 the work as a whole, and have given their

489 approval for this version to be published.

490 Disclosures. Giulio Gualdi, Simone

Car-491 avello, Fabiano Frasci, Federica Giuliani,

Rug-492 gero Moro, Maria C. Fargnoli, Valeria Ciciarelli,

493 Giuseppe Argenziano, Caterina M. Giorgio,

494 Celia Requena, Eduardo Nagore, Onofre

San-495 martı´n, Fabrizio Fantini, and Paolo Amerio have

496 nothing to disclose. Piergiacomo

Calzavara-497 Pinton is a memeber of the journal’s Editorial

498 Board.

499 Compliance with Ethics Guidelines. All

500 patients were thoroughly informed and gave

501 their consent to treatment. Moreover, the study

502 was approved by the Internal Scientiﬁc Board of

503 Dermatology of the Department of Medicine

504 and Aging Science, University G. D’Annunzio,

505 Chieti Pescara, Italy. This study was performed

506 in accordance with the Helsinki Declaration of

507 1964 and its later amendments. The patient also

508 provided their written informed consent for the

509 publication of their images.

510 Data Availability. The datasets during and/

511 or analyzed during the current study are

avail-512 able from the corresponding author on

reason-513 able request.

514 Open Access. This article is licensed under a

515 Creative Commons

Attribution-Non-516 Commercial 4.0 International License, which

517 permits any non-commercial use, sharing,

518 adaptation, distribution and reproduction in

519 any medium or format, as long as you give

520 appropriate credit to the original author(s) and

521 the source, provide a link to the Creative

522 Commons licence, and indicate if changes were

AQ1

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523 _{made. The images or other third party material} 524 _{in this article are included in the article’s} 525 _{Creative Commons licence, unless indicated} 526 _{otherwise in a credit line to the material. If} 527 _{material is not included in the article’s Creative} 528 _{Commons licence and your intended use is not} 529 _{permitted by statutory regulation or exceeds the} 530 _{permitted use, you will need to obtain} permis-531 _{sion directly from the copyright holder. To view} 532 _a _copy _of _this _licence, _visit _http:// 533 _{creativecommons.org/licenses/by-nc/4.0/}_. 534

535

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