Electrochemotherapy for Breast Cancer—Results From the INSPECT Database

Accepted Manuscript Electrochemotherapy for Breast Cancer – results from the INSPECT database Louise Wichmann Matthiessen, Mohammed Keshtgar, Pietro Curatolo, Christian Kunte, Eva-Maria Grischke, Joy Odili, Tobian Muir, David Mowatt, James P. Clover, Se Hwang Liew, Karin Dahlstroem, Jackie Newby, Valerie Letulé, Eva Stauss, Alison Humphreys, Shramana Banerjee, Annette Klein, Roberta Rotunno, Francesca de Terlizzi, Julie Gehl PII:

S1526-8209(17)30397-X

DOI:

10.1016/j.clbc.2018.03.007

Reference:

CLBC 775

To appear in:

Clinical Breast Cancer

Received Date: 26 June 2017 Revised Date:

4 March 2018

Accepted Date: 8 March 2018

Please cite this article as: Matthiessen LW, Keshtgar M, Curatolo P, Kunte C, Grischke E-M, Odili J, Muir T, Mowatt D, Clover JP, Liew SH, Dahlstroem K, Newby J, Letulé V, Stauss E, Humphreys A, Banerjee S, Klein A, Rotunno R, de Terlizzi F, Gehl J, Electrochemotherapy for Breast Cancer – results from the INSPECT database, Clinical Breast Cancer (2018), doi: 10.1016/j.clbc.2018.03.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Electrochemotherapy for Breast Cancer – results from the INSPECT database

RI PT

Louise Wichmann Matthiessen1, Mohammed Keshtgar2, Pietro Curatolo3, Christian Kunte4,5, EvaMaria Grischke6, Joy Odili7, Tobian Muir8,David Mowatt9, James P Clover10, Se Hwang Liew11, Karin Dahlstroem12, Jackie Newby13, Valerie Letulé5, Eva Stauss6, Alison Humphreys14, Shramana Banerjee2,15, Annette Klein4, Roberta Rotunno3, Francesca de Terlizzi16, Julie Gehl1,17,18 1

Department of Oncology, Copenhagen University Hospital Herlev and Gentofte, Herlev, Denmark Department of Breast Surgery, Royal Free London NHS Foundation Trust, London, UK 3 Department of Internal Medicine and Medical Specialties, Division of Dermatology, University of Rome “La Sapienza”, Rome, Italy 4 Department of Dermatologic surgery and Dermatology, Artemed Fachklinik München, Munich, Germany 5 Department of Dermatology and Allergology, Ludwig-Maximilian University Munich, Munich, Germany 6 Universitet Frauenklinik Tübingen, Tübingen, Germany 7 Department of Plastic Surgery, St. George’s University Hospitals NHS Foundation Trust, London, UK 8 Department of Reconstructive Plastic Surgery, James Cook University Hospital, Middlesbrough, UK 9 Department of Plastic Surgery, The Christie Hospital, Manchester, UK 10 Department of Plastic Surgery, Cork University Hospital and Cork Cancer Research Centre, University College Cork, Cork, Ireland 11 Merseyside Regional Burns and Plastic Surgery Unit, Whiston Hospital, Liverpool, UK 12 Department of Plastic Surgery, Copenhagen University Hospital Herlev and Gentofte, Herlev, Denmark 13 Department of Medical Oncology, Royal Free London NHS Foundation Trust, London, UK 14 Department of Oncology, James Cook University Hospital, Middlesbrough, UK 15 Division of Surgery and interventional Science, University College London, London, UK 16 IGEA S.p.a., Via Parmenide, 10/A Carpi, Italy 17 Center for Experimental Drug and Gene Electrotransfer (C*EDGE), Department of Clinical Oncology and Palliative Care, Zealand University Hospital, Roskilde, Denmark 18 Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

AC C

EP

TE D

M AN U

SC

2

Corresponding author: Louise Wichmann Matthiessen; [email protected]

1

ACCEPTED MANUSCRIPT

Abstract Background: Cutaneous recurrence from breast cancer can pose a clinical challenge. It might be the only disease site, or could be part of disseminated disease, and often profoundly impacts quality of life. Electrochemotherapy is a palliative treatment using

RI PT

electric pulses to locally permeabilise tumour cells and thereby increase bleomycin cytotoxicity significantly. Collaborating with the International Network for sharing Practice on ElectroChemoTherapy (INSPECT), we consecutively and prospectively accrued data on patients treated with electrochemotherapy for cutaneous metastases from breast cancer.

SC

Methods: Patients were treated with electrochemotherapy at ten European centers. Under either local or general anaesthesia patients were treated with either local injection (1000

M AN U

IU/ml intratumoural) or systemic infusion (15,000 IU/m2) of bleomycin.

Results: 119 patients were included at ten institutions in the INSPECT network. Primary location was the chest (89%), median diameter of the cutaneous metastases being 25mm. 90 patients were available for response evaluation after 2 months. Complete response was observed in 45 (50%) patients, partial response in 19 (21%), stable disease in 16 (18%), and progressive disease in 7 (8%). 3 patients were not evaluable. Common side

TE D

effects were ulceration, long-lasting hyperpigmentation, and low grade pain. No serious adverse events were observed.

Conclusion: Electrochemotherapy showed high response rates after a single treatment.

EP

Electrochemotehrapy has few side effects and can be used as an adjunct to systemic therapies or as a solo treatment. We therefore recommend considering electrochemotherapy for patients with cutaneous metastases.

AC C

Keywords:

Electrochemotherapy, cutaneous metastases, breast cancer, palliative care, bleomycin.

2

ACCEPTED MANUSCRIPT

Introduction Breast cancer may behave in many ways and can disseminate to various sites including the skin. Cutaneous metastases may present as isolated disease or as part of multiple organ disease. Cutaneous metastases may cause bleeding, oozing, disfigurement, and

RI PT

pain, as well as having a negative aesthetic impact. Treatment of cutaneous metastases could reduce or eliminate these symptoms and improve quality of life [1]. Different

treatment options include chemotherapy, radiotherapy and targeted therapies. Among local therapies electrochemotherapy is gaining importance [2, 3].

Electrochemotherapy is a local treatment for cutaneous metastases of any histology.

SC

Electrochemotherapy is a technique of combining local electric pulses with non-permeant chemotherapeutic agents (figure 1). Electric pulses cause a transient permeabilisation of

M AN U

the cell membrane allowing non-permeable drugs to enter the cell interior and thus increasing the potency of the chemotherapeutic agent. The greatest enhancements in cytotoxic effect have been found for the chemotherapeutic drug bleomycin. Bleomycin is a large, hydrophilic, and charged molecule; consequently there is no cellular uptake mechanism available for this drug. Combined with electric pulses the effect of bleomycin is enhanced by a factor 300 or more. For drugs that can diffuse freely across the cell

TE D

membrane no enhancement in effect is observed. Bleomycin administered without electric pulses has no effect on cutaneous metastases [4]. Electrochemotherapy has proven an efficient treatment for cutaneous metastases of

EP

various cancers including breast cancer [5]. Several small studies have shown electrochemotherapy to be an effective treatment for breast cancer patients [6, 7, 8, 9, 10, 11].

AC C

The INSPECT database (International Network on Sharing Practices for ElectroChemoTherapy) was formed to investigate the effectiveness and utility of electrochemotherapy, by collecting prospective data and to share experience and good practice, regarding the use of electrochemotherapy. Here we present data collected on breast cancer patients within the INSPECT collaboration.

Patients & Methods Patients Patients were recruited and treated at institutions in the INSPECT network. Centres

3

ACCEPTED MANUSCRIPT uploaded patient data prospectively to the same database. Approval from ethics committee, and data protection authority was according to guidelines of each institution. Patients eligible for inclusion had histologically proven breast cancer with measurable cutaneous or subcutaneous metastases suitable for application of electric pulses. Patients had been offered standard treatment options, were ≥ 18 years old, had ECOG

RI PT

performance status ≤ 2, had life expectancy of at least 3 months and, where appropriate, were using adequate contraception. A platelet count ≥ 50 mia/l was required, with a prothrombin time ≤ 40 sec and an activated partial thromboplastin time in the normal range.

SC

Patients were ineligible if they previously had allergic reactions to bleomycin or to any of the components required for anaesthesia, if the cumulative dose of 250mg (400,000 IU)

M AN U

bleomycin/m2 had previously been exceeded, and in case of chronic renal dysfunction (serum creatinine >150µmol/l) or acute lung infection.

Clinical information retrieved by the database included: demographics, number of treated lesions, site and size of the largest lesion, previous irradiation, duration of follow-up. Data on histology, hormone receptor status, HER2 receptor status and previous systemic treatment was also collected. Follow up was planned for up to 6 months. Patients who

TE D

started new systemic antineoplastic treatment after electrochemotherapy were censored from further analysis at the time the new treatment started. Procedure

EP

The electrochemotherapy sessions were performed based on the standard operating procedures [12]. Anaesthesia - either local or general anaesthesia depending on size of the area to be treated - is used as insertion of needles in the tissue and delivery of pulses

AC C

causes brief pain. Bleomycin is administered, and afterwards electric pulses are delivered using either non-invasive plate electrodes or invasive needle electrodes to deliver the electric pulses (figure 1).

Local anaesthesia was used for small or few metastases, whereas general anaesthesia was preferred for multiple or large metastases (>3 cm), metastases adhering to the periosteum or located in sensitive regions (e.g. scalp), and in accordance with patient preference.

4

ACCEPTED MANUSCRIPT Bleomycin was administrated either intratumourally (i.t.) using 1000 IU/ml or intravenously (i.v) using 15,000 IU/m2. Route of administration was chosen depending on the number of cutaneous metastases to be treated and the size of metastases. Depending on the clinician's choice, one of the following electrodes was used: 1) Type I electrodes: two plates with a 6 mm gap between the plates; 2) Type II electrodes: two

RI PT

parallel rows of needles with 4 mm between rows; 3) Type III electrodes: a hexagonal array with 7.9 mm between the needles (figure 1).

Electric pulses (eight pulses of 100 µs duration) were delivered using a square wave electroporator (IGEA, Carpi, Italy). The applied voltage relative to distance between

SC

electrodes was 1.3 kV/cm for plate electrodes and 1.0 kV/cm for needle electrodes, i.e. for the type II needle electrode with a 4 mm gap between the needles the applied voltage was

M AN U

400 V. For type I and II electrodes, the pulses are applied with 1 Hz or 5 kHz, whereas for type III electrodes, pulses can only be applied with 5 kHz. Electric pulses are generated by a pulse generator and applied using handheld electrodes. Pulses are quickly delivered and the electrode can be moved around to cover the whole tumour. After electrochemotherapy, the treated metastases were covered with standard dressings

Response evaluation

TE D

where necessary.

Evaluation of the tumour response was by measurement of the dimensions of the treated metastases. This was documented using digital photography. The response was

EP

registered for each target lesion at follow up two months after electrochemotherapy. The response rate was evaluated similarly to the Response Evaluation Criteria in Solid Tumours (RECIST version 1.0) [13]: Complete response (CR) was defined as

AC C

disappearance of the target lesion; partial response (PR) with at least 30% decrease in the diameter of the target lesion; progressive disease (PD) with at least 20% increase in the diameter of the target lesion and stable disease (SD) with neither sufficient shrinkage to qualify for PR or sufficient increase to qualify for PD. In some cases with ulcerated tumours evaluation was not possible due to crust formation.

Safety Safety was reported in the form of adverse events using Common Toxicity Criteria version 3.0. Patients were asked if they would potentially agree for another session as a measure of how patients felt about the treatment procedure. Pain assessment was performed at

5

ACCEPTED MANUSCRIPT four time points: before treatment, within 24 hours after treatment, thereafter within 45 days from treatment and after more than 45 days from treatment. Pain intensity was evaluated using the Numeric Rating Scale (NRS) for pain [14]. NRS is a uni-dimensional 11-point numeric scale in which the patient is asked to indicate a whole number between “0” as “no pain” and “10” as “worst pain”. Respondents were asked to indicate their

RI PT

average pain intensity related to cutaneous metastases irrespectively the use of pain medication. We used a previously published cut-off on NRS score (26): 0 – 2 mild pain, 3 – 4 moderate pain, 5 – 10 severe pain [15]. Pain medication was registered as ‘none’, ‘sometimes’, ‘controlled by non-opioids’, ‘controlled by opioids’, ‘uncontrolled’, or

SC

‘unknown’.

Statistical consideration

M AN U

Descriptive methods were employed for statistical analysis using NCSS 9 [16]. Continuous variables were described by median value and range, categorical variables by absolute number and percentage. Analysis of response for different lesions’ sizes have been reported on 2 by k contingency table and analysed with Chi Square test for trend. Chi Square value (and degree of freedom) has been reported together with p value. Multivariable analysis was performed by logistic regression model for objective response

TE D

(CR+PR), using as covariates only significant variables observed in the univariable analysis (lesions size, coverage of margins, ulceration, current delivered, previous treatment with trastuzumab, histology).

EP

Local tumour control was expressed as local progression free survival which was the time from ECT up to the date to relapse or progression or last follow-up. Survival curves were calculated by the Kaplan–Meier test. Curves were calculated for patients with objective

AC C

response (CR+PR) and for CR and PR separately. Patients were excluded from further evaluation if new antineoplastic treatment was started. All patients treated with ECT were included for evaluation of efficacy and safety.

Results Patient population A total of 119 patients with cutaneous metastases from breast cancer were included between June 2007 and March 2015. Table 1 illustrates patient characteristics at baseline and table 2 previous treatments. Ten institutions in the INSPECT network accrued patients

6

ACCEPTED MANUSCRIPT (Herlev (43 patients), London Royal Free (17 patients), Rome (17 patients), Munich (15 patients), Tubingen (9 patients), London St. George (8 patients), Middlesbrough (6 patients), Manchester (2 patients), Cork (1patients), Liverpool (1 patient). 117 patients underwent electrochemotherapy for 244 cutaneous metastases. One patient did not receive electrochemotherapy due to poor lung function and one patient did not receive

RI PT

electrochemotherapy due to pneumonia. Data on 47 patients are previously reported [17, 18].

104 patients were evaluable for safety and toxicity. 90 patients with 207 cutaneous

metastases had a follow up of at least two months. The reasons for not having follow up at

SC

two months were death (10 patients), systemic disease progression (7 patients), lost to follow up (5 patients), new systemic treatment (4 patients), and unwilling to attend the

Tumour Size and Treatment

M AN U

clinic (1 patient).

The median diameter of the cutaneous metastases was 25 mm ranging from 1 mm to 550 mm. The metastasis with a diameter of 550 mm was a flat confluent mass of tumour nodules impossible to separate into smaller metastases – therefore the diameter was measured for the whole area. Locations of the cutaneous metastases are presented in

TE D

table 3.

As presented in table 3, most patients (84%) were treated with general anaesthesia and a type III (hexagonal) electrode was mostly used (80%), which is in accordance with many

EP

patients presenting with numerous or large cutaneous metastases.

Treatment response

27 patients were lost to follow up before evaluation at two months after

AC C

electrochemotherapy. 90 patients with 207 treated cutaneous metastases were available for response evaluation at two months after electrochemotherapy. Responses are presented in table 4 and duration of response is shown in figure 3. CR was observed in 45 (50 %) patients, PR was observed in 19 (21 %) patients, SD was observed in 16 (18 %) patients and PD in 7 (8 %) patients. Response was not evaluable in 3 (3%) patients with 9 nodules. This was due to crust formation in the treated area making it impossible to measure size of the nodules. In multivariate analysis response was correlated to size of the treated metastases with an objective response rate (CR + PR) of 83.3 % of metastases less than 3 cm in diameter and 70.7% of metastases with a diameter of 3 cm

7

ACCEPTED MANUSCRIPT or more (p=0.05). In multivariate analysis previous treatments did not influence response rates. There was a tendency towards less oozing and suppuration from the cutaneous metastases. 31 patients received another session or more of electrochemotherapy due to either new nodules or progression of treated nodules.

Safety

RI PT

No serious adverse events (SAE) were observed. Symptoms before electrochemotherapy, within 45 days after electrochemotherapy, and more than 45 days after

electrochemotherapy are presented in table 5. The only significant change was observed for hyperpigmentation which increased in the skin after electrochemotherapy. Temporary

SC

ulceration, flu-like symptoms and occasional nausea within 45 days after electrochemotherapy were noted. In patients receiving more than one

M AN U

electrochemotherapy session an increase in side effects was not observed.

Discussion

Electrochemotherapy is a therapeutic procedure which is increasingly used to treat cutaneous metastases from different tumour types including breast cancer. In this study

TE D

data from the INSPECT collaboration is presented.

Patients with cutaneous metastases from breast cancer have often been exposed to various treatments – including systemic therapy, surgery and radiotherapy with various effects on cutaneous metastases before being referred to electrochemotherapy.

EP

Electrochemotherapy will treat only cutaneous metastases and have no effect on systemic disease, the main focus is therefore relief of symptoms from cutaneous metastases.

AC C

In this study patients had previously been treated with systemic chemotherapy (87%), radiotherapy (81%) and if relevant, with antihormone treatment and HER2 targeted treatment (shown in table 2) before receiving electrochemotherapy. In multivariate analysis these previous treatments did not influence response rates which indicate that electrochemotherapy is effective despite previous treatments. CR was obtained in 50% of patients and PR in 21% of patients (table 4) which is in accordance with previous studies reporting objective response rates between 16-92 % [6, 7, 9, 10, 11]. This confirms the high efficacy of electrochemotherapy despite progression on previous therapy. Electrochemotherapy is in general well tolerated with hyper pigmentation being the most common side effect.

8

ACCEPTED MANUSCRIPT There have been reports of treatment resistant pain when treating large (> 3cm in diameter) cutaneous metastases [7], but for most patients pain decreases after 45 days following treatment [18]. Patients at risk for posttreatment pain include pain before treatment, prior radiotherapy, large treated areas [18] and repeated treatments [6]. Patients with risk of increased pain after electrochemotherapy can be identified at the

RI PT

pretreatment visit and intensified pain treatment can be instituted. In this study there is no significant difference in pain score before and after treatment (table 5). Although some patients experience increase in pain score after treatment, electrochemotherapy has also

as a treatment which can improve quality of life [1, 6].

SC

in some cases proven efficient in pain score reduction [4, 18, 22] and could be considered

As expected, response lasted longer in patients obtaining CR than in patients obtaining

M AN U

only PR (figure 2). In patients with PR or SD treatment can be repeated several times in the same area and thereby achieve CR or can be applied when progression is observed. This was performed in 31 of the 119 patients in this study. Electrochemotherapy may therefore be applied when needed instead of following a standardized scheme. In this study, and in accordance with previous studies [1, 17, 19], smaller metastases (≤3 cm) have a higher response rate compared to larger metastases (>3cm). In a recent study

TE D

it was shown that coverage of margins also influence response rate [20]. Margins are easier to cover when cutaneous metastases are small. A suggestion would be to consider electrochemotherapy when cutaneous metastases appear small, making coverage of margins easier and thereby getting better responses and longer lasting control. Repeated

EP

sessions of electrochemotherapy can then be applied when necessary as a supplement to systemic treatment. Side effects are not increased with repeated sessions [6] but

AC C

cumulated doses of bleomycin should not exceed 400,000 IU as this will increase the risk of bleomycin induced pulmonary fibrosis [21]. Electrochemotherapy has up to now mainly been used for breast cancer patients as a last resort. As electrochemotherapy has few side effects and is a local treatment with, as far as we know, no effect on systemic disease, it could be considered a supplement to systemic therapy, where visceral disease is stable but progression is observed in cutaneous metastases. It should be applied individually when needed and not when all other treatment options have been tried. In this way cutaneous metastases could be treated

9

ACCEPTED MANUSCRIPT when small and thereby obtaining better response rates offering the patient improved quality of life. Many of the patients in this study were referred to a center offering electrochemotherapy from other centers when no further treatment options where available at the center referring the patient. However other treatment options should be considered and

expected results and least impact on daily life.

RI PT

discussed in a multidisciplinary team as it is important to choose treatment with best

Palliative surgery can in selected cases provide local control but is not always possible in previously irradiated or operated areas and also depends on the number, location, and

SC

size of metastases.

Palliative re-irradiation is feasible and may in carefully selected cases relieve symptoms

M AN U

[23], but often radiotherapists are reluctant due to low response rates are expected and possible severe side effects. Hyperthermia added to radiotherapy gives higher response rates compared to radiotherapy alone [24]. The method is technically demanding and only offered at selected radiotherapy centres.

Laser and photodynamic therapies are in selected cases used for treatment of cancer located in the skin. Few side-effects are reported, but the methods are limited by their

TE D

limited penetration into the tissue (up to 1 cm) [25].

Conclusion

EP

In conclusion this study confirms a high local response to electrochemotherapy and should be considered an option for cutaneous metastases in breast cancer patients. Metastases should be treated when small as better responses are expected, local anaesthesia can be

AC C

used, less pain is observed and patient distress due to large cutaneous metastases is avoided. Therefore early referral is recommended.

Acknowledgement

The authors are grateful to all the women who received electrochemotherapy. We would also like to thank local co-workers who made the implementation of electrochemotherapy possible at different centers.

10

ACCEPTED MANUSCRIPT

Disclosures IGEA (Carpi, Italy) hosts the INSPECT database, but the database is controlled by an independent board, and the uploaded data are contractually belonging to the investigators involved.

RI PT

Francesca de Terlizzi (FT) is an employee at IGEA. Christian Kunte (CK) received honoraria for lectures and was invited to meetings on Electrochemotherapy by IGEA

The other authors declare that they have no competing interests.

Treatment costs were covered by local institutions. Database assistance and funding for

AC C

EP

TE D

M AN U

SC

database meetings were provided by IGEA (Carpi, Italy).

11

ACCEPTED MANUSCRIPT

References

1. Campana LG, Mocellin S, Basso M, et al. Bleomycin based electrochemotherapy: clinical outcome from a single institution's experience with 52 patients. Ann Surg

RI PT

Oncol 2009; 16:191-9.

2. Spratt DE, Gordon Spratt EA, Wu S, et al. Efficacy of skin directed therapy for cutaneous metastases from advanced cancer. J Clin Oncol 2014; 32:1-12. 3. National Institute for Health and Care Excellence (NICE) guideance.

SC

https://www.nice.org.uk/guidance/ipg446. Accessed 2 February 2017 4. Gehl J, Skovsgaard T, Mir LM. Enhancement of cytotoxicity by

1998; 9(4):319-25.

M AN U

electropermeabilization: an improved method for screening drugs. Anticancer Drugs

5. Marty M, Sersa G, Garbay JR, et al. Electrochemotherapy, An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. EJC Supplements 2006; 4:3-13.

TE D

6. Campana LG, Valpione S, Falci C, et al. The activity and safety of electrochemotherapy in persistent chest wall recurrence from breast cancer after mastectomy. Breast Cancer Res Treat 2012; 134:1169-1178. 7. Matthiessen LW, Johannesen HH, Hendel HW, Moss T, Kamby C, Gehl J.

EP

Electrochemotherapy for large cutaneous recurrence of breast cancer: a phase II clinical trial. Acta Oncol 2012; 51:713–721

AC C

8. Sersa G, Cufer T, Paulin SM, Cemazar M, Snoj M. Electrochemotherapy of chest wall breast cancer recurrence. Cancer Treat Rev 2012; Aug38:379-86. 9. Benevento R, Santorello A, Perna G, Canonico S. Electrochemotherapy of cutaneous metastases from breast cancer in elderly patients. BMC Surg 2012; 12: S6.

10. Campana LG, Galuppo S, Valpione S, et al., Bleomycin electrochemotherapy in elderly metastatic breast cancer patients: clinical outcome and management considerations. J Cancer Res Clin Oncol 2014; 140(9):1557-65.

12

ACCEPTED MANUSCRIPT 11. Cabula C, Campana LG, Grilz G, et al. Electrochemotherapy in the treatment of cutaneous metastases from breast cancer: a multicenter cohort analysis. Ann Surg Oncol 2015; 22:442–450 12. Mir LM, Gehl J, Sersa G, et al. Standard operating procedures of the lectrochemotherapy: Instructions for the use of bleomycin or cisplatin administered

RI PT

either systemically or locally and electric pulses delivered by the Cliniporator (TM) by means of invasive or non-invasive electrodes. EJC Supplements 2006; Nov4(11):14-25.

13. Therasse P, Arbuck SG, Eisenhauer EA, et al. New guidelines to evaluate the

SC

response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National

M AN U

Cancer Institute of Canada. J Natl Cancer Inst 2000; 92(3):205-16. 14. Hawker GA, Mian S, Kendzerska T, French M. Measures of adult pain: Visual Analog Scale for Pain (VAS Pain), Numeric Rating Scale for Pain (NRS Pain), McGill Pain Questionnaire (MPQ), Short-Form McGill Pain Questionnaire (SFMPQ), Chronic Pain Grade Scale (CPGS), Short Form-36 Bodily Pain Scale (SF-36 BPS), and Measure of Intermittent and Constant Osteoarthritis Pain (ICOAP).

TE D

Arthritis Care Res (Hoboken) 2011; Nov(63) Suppl 11:240-252. 15. Gerbershagen HJ, Rothaug J, Kalkman CJ, Meissner W. Determination of moderate-to-severe postoperative pain on the numeric rating scale: a cut-off point analysis applying four different methods. Br J Anaesth 2011; Oct107(4):619-626.

EP

16. NCSS 9 Statistical Software (2013). NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/ncss.

AC C

17. Matthiessen LW, Chalmers RL, Sainsbury DCG, et al. Management of cutaneous metastases using electrochemotherapy. Acta Oncol 2011; 50:621-629. 18. Quaglino P, Matthiessen LW, Curatolo P et al. Predicting patients at risk for pain associated with electrochemotherapy. Acta Oncol 2015; 54:298-306. 19. Larkin JO, Collins CG, Aarons S, et al. Electrochemotherapy: aspects of preclinical development and early clinical experience. Ann Surg 2007; 24:469-79. 20. Kunte C, Letulé V, Gehl J, et al. Electrochemotherapy in the treatment of metastatic malignant melanoma: A prospective cohort study by InspECT. Br J Dermatol 2017; Jan 24.

13

ACCEPTED MANUSCRIPT 21. Sleijfer S. Bleomycin-induced pneumonitis. Chest 2001; Aug;120 (2):617-24. 22. Mir LM, Glass LF, Sersa G, et al. Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy. Br J Cancer 1998; Jun 77(12):2336-42. 23. Wurschmidt F, Dahle J, Petersen C, et al. Reirradiation of recurrent breast cancer

RI PT

with and without concurrent chemotherapy. Radiat Oncol 2008; 3:28.

24. Jones EL, Oleson JR, Prosnitz LR, et al. Randomized trial of hyperthermia and radiation for superficial tumors. J Clin Oncol 2005; 23(13):3079-3085.

25. Karrer S, Szeimies RM, Hohenleutner U, et al. Role of lasers and photodynamic

SC

therapy in the treatment of cutaneous malignanct. Am J Clin Dermatol 2001;

AC C

EP

TE D

M AN U

2(4):229-237

14

ACCEPTED MANUSCRIPT

Figure legends Figure 1: Electrochemotherapy procedure In the top right (A) bleomycin is injected at the tumour site – either intratumourally or intravenously (not shown). In the top middle (B) the electric pulses are applied by inserting

RI PT

the electrode into the cutaneous metastases. After pulse delivery the electrode is moved around until the whole tumour is covered. In the top left (C) the handheld electrodes used for the procedure are shown; hexagonal needle arrangement (right), linear needle arrangement (middle) and plates electrode (left).

SC

Bottom right (D) bleomycin is injected but unable to freely diffuse across the cell

membrane, in the bottom middle (E) cells are permeabilised by the electric pulses and bleomycin enters the cell interior. In bottem left (F) the cells reseal after a few minutes and

M AN U

the extracellular drug is washed out while the internalized bleomycin molecules remain trapped intracellularly.

Figure 2: Local disease control over time

The duration of local response have been calculated for all patients obtaining complete

TE D

response and partial response (CR+PR) with censored patients marked, and for complete response (CR) alone and partial response (PR) alone. The median duration of local disease control was 92 days (range of 60-844 days). Data on local disease control has

EP

been reported for all patients with at least one follow-up and the cumulative local progression free survival curve was calculated. We calculated for each patient the time since ECT session and start of local progression of disease (event). Patients who

AC C

maintained the local control of the disease up to their last follow-up have been censored at last follow-up. The median time to progression was 3.05 months (min 0.33- max 28.1). Many patients were lost to follow up; mainly for reasons such as death, systemic disease progression and new systemic treatment. Patients were censored if lost to follow up.

Figure 3: Pictures of breast cancer patients treated with electrochemotherapy. Top row shows patient with cutaneous metastases from breast cancer on the chest wall and abdomen before treatment (A), 23 days after treatment (B), and 135 days after treatment (C) where complete response was reported. The patient developed bone

15

ACCEPTED MANUSCRIPT metastases after last follow up and started new systemic treatment. Middle row shows patients with ulcerated metastases from breast cancer in the axilla before treatment (D), 19 days after treatment (E), and 65 days after treatment (F) where partial response was reported. The patient died 9 days after last follow up. Bottom row shows patient with cutaneous metastases related to the mastectomy cicatrice on the chest wall, before

RI PT

treatment (G), 29 days after treatment (H), and 116 days after treatment (I) where

AC C

EP

TE D

M AN U

SC

complete response was reported.

16

ACCEPTED MANUSCRIPT

Table 1: Patients’ characteristics at baseline

Age distribution 30-39 40-49 50-59 60-69 70-79 80-89 90-99 Unknown

5 11 18 33 26 11 1 14

4% 9% 15 % 28 % 22 % 9% 1% 12 %

Histology Invasive Ductal Carcinoma Invasive Lobular Carcinoma Inflammatory Breast Cancer Unknown

87 22 4 6

73 % 17 % 4% 6%

32

27 %

AC C

EP

Median years from diagnosis to electrochemotherapy in years (range) Presence of other metastasis Skin only Brain Soft Tissue Visceral Bone and other places Bone only

SC

M AN U

TE D

Receptor Status1 Hormone receptor positive and HER2 normal expression Hormone receptor positive and HER2 over expression Hormone receptor negative and HER2 over expression Hormone receptor negative and HER2 normal expression Unknown2

RI PT

Patients (%)

Median age in years (range)

Patients Total (N=119) 65 (32-91)

14

12 %

19

16 %

34

29 %

20

17 %

4.9 (0.7-33.7)

40 4 41 19 11 4

34 % 3% 34 % 16 % 9% 3%

ACCEPTED MANUSCRIPT 1

AC C

EP

TE D

M AN U

SC

RI PT

Hormone receptor measured as estrogen receptor positive, of the 48 patients positive for the estrogen receptor (2 having unknown HER2 status) 33 were progesterone receptor positive, 8 were progesterone negative and 7 had unknown progesterone receptor. 2 Electrochemotherapy is often performed at centres different from where patients are diagnosed with and treated for breast cancer, therefore a relative large number of patients receptor status is unknown as that data was collected retrospectively.

ACCEPTED MANUSCRIPT

Surgery Breast Conserving Surgery Mastectomy No Surgery Unknown

19 % 62 % 7% 12 %

Endocrine treatment if relevant (n=66)1 Yes 54 No 6 Unknown 6

82 % 9% 9%

Chemotherapy Yes No Unknown

104 6 9

87 % 5% 8%

Radiotherapy Yes No Unknown

86 5 13

83 % 5% 13 %

TE D

M AN U

SC

23 74 8 14

RI PT

Table 2: Previous treatments before electrochemotherapy

AC C

EP

Trastuzumab if relevant (n=54)2 Yes 23 43 % No 12 22 % Unknown 19 35 % 1 Endocrine therapy is only applicable if patients are hormone receptor positive. The percentage of patients receiving previous endocrine treatment is therefore given as percentage of the 66 patients who were either hormone receptor positive or unknown hormone receptor status. 2 HER2 targeted therapy is only relevant if the HER2 receptor is overexpressed. The percentage of patients receiving previous trastuzumab is therefore given as percentage of the 54 patients who had either HER2 receptor overexpression or unknown HER2 receptor status. All patients received HER2 targeted therapy received trastuzumab – some received additional other HER2 targeted therapy e.g. lapatinib.

ACCEPTED MANUSCRIPT Table 3: Treatment data All Patients (n=117) All Patients (%) Median size of treated metastases in mm (range)

25 (1-550)

217 14 4 4 4 1

Chemotherapy administration Bleomycin I.T. Bleomycin I.V.

16 101

Anaesthesia Local General

19 98

16 % 84 %

Electrodes used2 Plate electrodes (type 1) Needle row electrodes (type 2) Needle hexagonal electrodes (type 3) Plate and needle hexagonal electrodes Needle row and hexagonal electrodes More than 2 electrodes

12 21 195 8 7 1

5% 9% 80 % 3% 3% 0%

149 70 25

61 % 29 % 10 %

SC

89 % 5% 2% 2% 2% 0%

14 % 86 %

M AN U

TE D

EP

1

AC C

Metastases previously irradiated Yes No Unknown

RI PT

Site of treated metastases1 Chest Head and neck Back Upper limbs Abdomen Lower limbs

A total of 244 metastases were treated as some patients had more than one cutaneous metastases. Electrodes used are registered per metastases treated. As some metastases are large more than one electrode can be used in the same treatment session in order to cover the metastases sufficiently.

2

ACCEPTED MANUSCRIPT

Table 4a: Response at 2 months after electrochemotherapy

45 19 16 7 3

50 % 21 % 18 % 8% 3%

According to RECIST 1.0 Guidelines

SC

1

All Patients (%)

RI PT

Overall response1 Complete response Partial response Stable disease Progressive disease Not evaluable

All Patients (n=90)

M AN U

Table 4b: Response at 2 months after electrochemotherapy according to size Cutaneous Metastases Cutaneous Cutaneous smaller metastases metastases than <3 cm (n=207) (%) (n=113)

58 % 21 % 14 % 3% 4%

TE D

According to RECIST 1.0 Guidelines

AC C

1

121 42 28 7 9

EP

Overall response1 Complete response Partial response Stable disease Progressive disease Not evaluable

81 16 9 1 6

Cutaneous Metastases smaller than <3 cm (%)

Cutaneous Metastases bigger than ≥3 cm (n=94)

Cutaneous Metastases bigger than ≥3 cm (%)

72 % 14 % 8% 1% 5%

40 26 19 6 3

43 % 28 % 20 % 6% 3%

ACCEPTED MANUSCRIPT

Table 5: Side effects Before electrochemothe rapy

9% 11 % 40 % 47 %

0.64 0.15 0.17 <0.0001

3% 32 % 6%

8% 30 % 9%

3% 19 % 3%

0.07 0.17 0.20

65 % 23 % 12 %

54 %

60 %

25 %

19 %

21 %

21 %

EP

TE D

NRS: Numeric Ratio Scale [14]

RI PT

6% 17 % 55 % 46 %

AC C

1

P-Value

6% 23 % 42 % 10 %

SC

Mild pain (NRS1 0-2) Moderate pain (NRS 3-5) Severe pain (NRS 5-10)

More than 45 days after electrochemotherapy

M AN U

Bleeding Oozing Ulceration Hyperpigentation Flu-like symptoms Suppuration Nausea

Within 45 days after electrochemotherapy

0.69

B

D

E

C

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

A

F

CR

M AN U

Percentage of patients with local control

SC

RI PT

ACCEPTED MANUSCRIPT

TE D

CR+PR

AC C

EP

PR

Time (months)

#pts 106

83

46

34

18

13

9

B

C

D

E

F

G

H

I

AC C

EP

TE D

M AN U

SC

A

RI PT

ACCEPTED MANUSCRIPT