Prospective evaluation of percutaneous hepatic perfusion with melphalan as a treatment for unresectable liver metastases from colorectal cancer.

PURPOSE: Percutaneous hepatic perfusion with melphalan (M-PHP) is increasingly used in patients with liver metastases from various primary tumors, yet data on colorectal liver metastases (CRLM) are limited. The aim of this study was to prospectively evaluate the efficacy and safety of M-PHP in patients with CRLM.
MATERIALS AND METHODS: Prospective, single-center, single-arm phase II study of M-PHP with hemofiltration in patients with unresectable CRLM. Proven, extrahepatic metastatic disease was one of the exclusion criteria. Primary outcomes were overall response rate (ORR) and best overall response (BOR). Secondary outcomes were overall survival (OS), progression-free survival (PFS), hepatic PFS (hPFS), and safety.
RESULTS: A total of 14 M-PHP procedures were performed in eight patients between March 2014 and December 2015. All patients (median age 56 years, ranging from 46 to 68) had received (extensive) systemic chemotherapy before entering the study. The ORR was 25.0%, with two out of eight patients showing partial response as BOR. Median OS was 17.3 months (ranging from 2.6 to 30.9) with a one-year OS of 50.0%. Median PFS and hPFS were 4.4 and 4.5 months, respectively. No serious adverse events occurred. Grade 3/4 hematologic adverse events were observed in the majority of patients, though all were transient and well-manageable.
CONCLUSION: M-PHP is a safe procedure with only limited efficacy in patients with unresectable CRLM who already showed progression of disease after receiving one or more systemic treatment regimens.

Introduction

Colorectal cancer (CRC) is the third most common cancer type worldwide. In 2018, approximately 1.8 million new cases of CRC were diagnosed, which accounted for 9.2% (880.000) of all cancer-related deaths [1]. The most common site of distant metastases in CRC is the liver as the majority of venous blood from the colon drains into the hepatic portal vein via the superior and inferior mesenteric veins. Around 15–25% of patients will present with synchronous colorectal liver metastases (CRLM), and ultimately approximately 50% of patients with CRC will develop liver metastases at some point in the course of their disease [2,3].

Surgical resection is considered standard of care for patients with resectable CRLM with a median overall survival (OS) ranging from 36–56 months [4-6]. Despite the improvement of surgical techniques, expansion of the indications for surgery, and advances in neoadjuvant therapies, only about 25% of patients is eligible for surgery at the time of diagnosis [7]. In patients with unresectable CRLM, systemic therapy is considered to be the first treatment modality with a reported median OS of approximately 2.5 years [8]. Liver-directed therapies such as radioembolization, chemoembolization, hepatic arterial infusion pump chemotherapy, or isolated hepatic perfusion (IHP) may offer an alternative treatment with limited systemic side-effects, but are generally not considered as first-line therapy in patients with CRLM.

Percutaneous hepatic perfusion with melphalan (M-PHP) is a novel therapy that was developed as minimally invasive alternative to IHP. IHP is an invasive, complex surgical procedure in which the liver is isolated from the systemic circulation followed by infusion of a high dose of chemotherapy into the common hepatic artery and/or portal vein [9-13]. In patients with CRLM, hepatic response rates of 50–59% and a median OS of 24.8–28.8 months have been reported after IHP with a high-dose of melphalan [14,15]. A major drawback of IHP is that it is not repeatable and associated with considerable morbidity and mortality rates up to 7% as a result of the invasive surgical procedure [14,15].

M-PHP is a repeatable, well-tolerated procedure with an acceptable safety profile [16-18] that is able to prolong progression-free survival in patients with liver metastases from ocular melanoma [17,19-22]. Up to now, data on M-PHP in CRLM remain limited. Only a small number of patients with CRLM have been studied while they were part of a heterogeneous cohort of patients with liver metastases from different primary tumors [23-25]. Moreover, these studies did not report tumor response and survival in CRLM patients. The aim of this study was to prospectively evaluate the efficacy and safety of M-PHP in patients with unresectable CRLM.

Materials and methods

Patients selection and study design

The current study was designed as a prospective, single-arm, single-center phase II study and registered in advance at www.trialregister.nl (NTR4050). Ethical approval was obtained from the local ethics committee (Leiden University Medical Center) and the study was conducted in accordance with the Declaration of Helsinki, 2013 version. Written informed consent was obtained from all patients before inclusion.

Patients with histologically proven and unresectable CRLM were eligible for the study. Exclusion criteria are listed in Table 1. Prior to inclusion, all patients were discussed at a multidisciplinary team (MDT) meeting. To achieve an acceptable inclusion rate few restrictive exclusion criteria were incorporated in the study protocol. Patients with unresectable CRLM were eligible for inclusion regardless of any prior systemic treatment. This allowed inclusion of patients who were intolerant to systemic chemotherapy or chemo-naïve and unwilling to undergo systemic therapy. However, our MDT always gave preference to first-line systemic chemotherapy over study inclusion in chemo-naïve patients.

Table 1

Exclusion criteria.

Laboratory test results	Other
APTT > 1.5 × ULN	Age < 18 or > 75 years
PT > 1.5 × ULN	Extrahepatic metastatic disease (on CECT or FDG-PET/CT)
Leukocytes < 3.0 × 109/L	WHO performance status ≥ 2
Thrombocytes < 100 × 109/L	Severe comorbiditya
Creatinine clearance < 40 ml/min	< 40% healthy liver tissue
AST > 2.5 × ULN	Vascular anatomy impeding M-PHP
ALT > 2.5 × ULN	Prior Whipple’s surgery
Serum bilirubin > 1.5 × ULN	Intracranial lesions with propensity to bleed (on CT/MRI)
ALP > 2.5 × ULN	Pregnancy

ALP, alkaline phosphatase; ALT, alanine aminotransferase; APTT, activated partial thromboplastin time; AST, aspartate aminotransferase; CECT, contrast-enhanced computed tomography of chest and abdomen; FDG-PET/CT, positron emission tomography with integrated non-contrast enhanced computed tomography and 18F-2-fluoro-2-deoxy-D-glucose as radiotracer; M-PHP, percutaneous hepatic perfusion with melphalan; PT, prothrombin time; ULN, upper limit of normal.

a e.g. cardiovascular or pulmonary disease precluding general anaesthesia, diabetes with nephropathy, active infections, other liver disease.

Treatment consisted of two M-PHP procedures at a 5–8 weeks interval. Patients with progressive disease (PD) or unacceptable adverse events (AEs) after the first M-PHP procedure, did not receive a second procedure. The melphalan dose was reduced with 20–25% if patients developed grade 3/4 hematologic toxicity after the first procedure. All patients received a subcutaneous injection of granulocyte-colony stimulating factor (pegfilgrastim 6 mg) within 72h after each M-PHP. M-PHP was scheduled at least one month after resection of the primary tumor to prevent gastro-intestinal bleeding complications as a result of per-procedural heparinization.

Procedure details

The M-PHP procedure has been described in greater detail previously [26]. Essential steps are discussed below.

Hepatic angiography was performed approximately one week prior to the first M-PHP and hepatico-enteric anastomoses were embolized if deemed necessary to prevent inadvertent flow of melphalan to the gastrointestinal tract.

All M-PHP procedures were performed under general anesthesia with continuously monitoring of vital signs. Per-procedural heparin was administrated to achieve an activated clotting time of ≥ 450 seconds. A 18-F sheath was placed percutaneously into the right common femoral vein (CFV) and through the sheath a 16-F double-balloon catheter (Isofuse Isolation Aspiration Catheter, Delcath Systems Inc, New York, NY, USA) was placed in the inferior vena cava (IVC) via the right CFV. The cranial balloon was inflated at the atriocaval junction and the caudal balloon at the infrahepatic portion of the IVC to prevent flow of melphalan into the systemic circulation. Melphalan 3 mg/kg (maximum dose 220 mg) was infused into the proper hepatic artery. Alternatively, the dose was split and infused into both the right and left hepatic artery. The chemosaturated blood was then aspirated through catheter fenestrations in a segment between the two balloons, pumped through an extracorporeal hemofiltration system (GEN 2 CHEMOSAT® filtration system, Delcath Systems Inc, New York, NY, USA) and returned to the patient via a 10-F sheath in the right internal jugular vein. Extracorporeal filtration was continued for 30 minutes after completion of melphalan infusion. Protamine sulphate 3 mg/kg was administrated at the end of the procedure. The arterial sheath in the left common femoral artery was removed and hemostasis was achieved using a closure device.

Follow-up

Contrast-enhanced computed tomography (CECT) of the chest and abdomen was performed at baseline, 4–8 weeks after each M-PHP, and then every 3 months in the first year and every 6 months thereafter until progression occurred.

Blood tests were performed daily during hospital admission and at several fixed time points after discharge. Adverse events were continuously monitored and reported according to the Common Terminology Criteria for Adverse Events version 4.03 (CTCAE v4.03).

Endpoints

All images were reviewed by independent radiologists using the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria [27]. Primary endpoint was the overall response rate (ORR). ORR was defined as the percentage of patients with complete response (CR) or partial response (PR). Best overall response (BOR) was used to determine ORR. BOR was defined as the best response at any time point after the first M-PHP and prior to the start of any other anti-cancer therapy. Secondary endpoints included best hepatic response, OS, progression-free survival (PFS), hepatic PFS (hPFS), and safety.

Statistical analyses

OS was defined as time of first M-PHP until death due to any cause or censoring. PFS was defined as time of first M-PHP until progressive disease (PD), death due to any cause or censoring. hPFS was defined as time of first M-PHP until progression of liver disease, death due to any cause, or censoring.

The sample size calculations were based on the primary outcome measure of response after two M-PHP procedures. In a previous clinical trial where patients were treated with one IHP procedure, an ORR was observed in over 50% of patients [14]. Treatment with two M-PHP perfusions was expected to increase this response percentage. We choose a sample size that allows the response percentage to be determined with sufficient accuracy, i.e. with a sufficiently narrow confidence interval. Assuming a true response percentage of 60%, a sample size of 34 patients will yield a two-sided confidence interval of length 0.33 (± 16.5% around the observed proportion).

Median OS, median PFS, and hPFS were measured in days and subsequently converted into months. Graphs shown in this study were generated with dedicated software (SPSS 23.0, SPSS Inc., Chicago, IL, USA) using the Kaplan-Meier method.

Results

Our study was terminated prematurely due to slow recruitment. Several factors contributed to the slow recruitment, such as the availability of alternative therapies (e.g. systemic chemotherapy or radioembolization), competing trials with systemic drugs or intra-arterial therapies, and ineligibility of screened patients.

Between March 2014 and December 2015, a total of 16 patients with unresectable CRLM were assessed for eligibility (Fig 1). Of these, eight patients were excluded for the following reasons: extrahepatic disease (n = 4) or because systemic chemotherapy was preferred during the MDT meeting as standard first-line therapy (n = 4). Thus, eight patients with a median age of 56 years (range 46–68) participated in this study. Baseline patient characteristics are summarized in Table 2. All patients received some form of systemic therapy before entering the study and half of the patients received prior surgical resection of CRLM. The median interval between diagnosis of CRLM and first M-PHP was 23.7 months (ranging from 8.3 to 35.1).

Fig 1

Patient flow diagram.

Table 2

Baseline characteristics.

				Liver metastases		Previous therapya
Pt	Gender	Age	Type CRC	Type	No.	Systemic	Local
1	M	57	Sigmoid	Synchr.	> 10	Bevacizumab/CAPOX; irinotecan/bevacizumab; panitumumab; CAPOX	Segmentectomy S8
2	M	46	Sigmoid	Synchr.	2–5	CAPOX; panitumumab	Metastasectomy S6 & S8, single RFA
3	M	64	Sigmoid	Synchr.	> 10	Bevacizumab/CAPOXb; CAPOX	Metastasectomy S5 & S6, multiple RFAs
4	F	57	Rectum	Synchr.	> 10	CAPOXc; CRT with capecitabine; irinotecan; cetuximab	Metastasectomy S5-7
5	M	55	Cecum	Synchr.	> 10	CAPOX; capecitabine	-
6	M	51	Cecum	Synchr.	> 10	Bevacizumab/CAPOX; FOLFOX	-
7	F	49	Rectum	Metachr.	> 10	CRT with capecitabine; 5FU/irinotecan/bevacizumab; FOLFOX/panitumumab	-
8	F	68	Transverse colon	Synchr.	2–5	CAPOX	-

Pt, patient; CRC, colorectal cancer; CRT, chemoradiation therapy; S, liver segment; Synchr., synchronous; Metachr., metachronous; M-PHP, percutaneous hepatic perfusion with melphalan; No., number; RFA, radiofrequency ablation; CAPOX, capecitabine + oxaliplatin; FOLFOX, oxaliplatin + leucovorin + 5FU; -, no previous local therapy.

a Besides resection of the primary tumor.

b Neoadjuvant treatment.

c Induction therapy.

A total of 14 M-PHPs were performed in eight patients. Six patients received two M-PHP procedures as per protocol and the other two patients received only one M-PHP procedure due to PD after the first procedure. Median melphalan dose was 220 mg (ranging from 190 to 220) for the first cycle and 220 mg (ranging from range 160 to 220) for the second cycle. In all procedures, hospital length-of-stay was two or three days.

There was no loss to follow-up. Tumor response and survival outcomes are reported in Table 3. ORR was 25% with two out of eight patients showing partial response (PR) (Fig 2). Three patients (38%) showed stable disease (SD) as BOR and the other three patients (38%) showed PD.

Table 3

Tumor response and survival.

Pt	Time between CRLM and 1st M-PHP (mo)	No. procedures	Tumor response	PFS (mo)	hPFS (mo)	OS (mo)	Treatments after PD
1	35.1	1	PDa	1.1	3.2	7.2	Capecitabine
2	28.1	2	PR	5.7	7.1	28.7	FOLFIRI/bevacizumab; capecitabine
3	30.0	2	PR	5.9	5.9	25.2	Capecitabine/bevacizumab; panitumumab; irinotecan
4	19.3	1	PDb	1.1	1.1	2.6	-
5	8.3	2	PDc	3.1	3.1	9.5	RTe; bevacizumab/irinotecan
6	8.7	2	SD	7.3	7.3	33.3	BMS-986156/nivolumabd; TAS 102
7	9.5	2	SD	2.9	2.9	9.1	-
8	32.1	2	SD	23.6	23.6	30.9	Capecitabine

BOR, best overall response; FOLFIRI, folinic acid (leucovorin) + fluorouracil (5FU) + irinotecan; hPFS, hepatic progression-free survival; mo, months; M-PHP, percutaneous hepatic perfusion with melphalan OS, overall survival; PD, progressive disease; PFS, progression-free survival; PR, partial response; Pt, patient; RT, radiation therapy; SD, stable disease; TAS 102, trifluridine/tipiracil; -, no treatment after PD.

a Although liver disease was stable, there was a new lymph node metastasis.

b Progression of liver metastases and development of extrahepatic disease.

c After the 1st M-PHP, patient showed SD. After the 2nd M-PHP, patient showed PD.

d Phase I/II study of BMS-986156 (i.e. a glucocorticoid-induced tumor necrosis factor receptor–related protein) with our without nivolumab.

e RT for bone metastases.

Fig 2

M-PHP in a 46-year-old male with a solitary liver metastasis from colorectal carcinoma in the left liver lobe after previous radiofrequency ablation in liver segment 2, metastasectomy of liver segment 4, and a right hepatectomy.

(a-b) Postero-anterior and lateral images during venography, performed by manual injection of non-diluted contrast medium through side holes of the double-balloon catheter. The cranial balloon (black arrowhead) was inflated at the atriocaval junction and the caudal balloon (dotted black arrow) in the infrahepatic portion of the inferior vena cava. Note the opacification of both the right hepatic vein (white arrow in a) and middle hepatic vein (white arrow in b), while there was no leakage alongside the balloons. A microcatheter (white arrowhead) was placed into the hepatic artery proper for the infusion of melphalan. Note also the coils after successful embolization of the right gastric artery and gastroduodenal artery. (c) Axial CT image before treatment showing a solitary hypovascular lesion (white arrowhead). (d) Axial CT image after two cycles of M-PHP showing reduction in size of the lesion (white arrowhead) corresponding with partial response.

At the time of study termination, all patients had passed away. Median OS was 17.3 months (ranging from 2.6 to 30.9) (Fig 3). The one-, two-, and three-year OS was 50.0%, 50.0%, and 0% respectively. Median PFS was 4.4 months (ranging from 1.1 to 23.6) and median hPFS was 4.5 months (ranging from 1.1 to 23.6). Six out of eight patients received some form of subsequent treatment after progression of disease occurred (Table 3).

Fig 3

Kaplan-Meier estimate of OS for all included patients (n = 8).

Safety

No deaths or other serious AEs occurred. All AEs are listed in Table 4. Grade 3/4 thrombocytopenia, anemia, leukocytopenia, and lymphocytopenia was observed in 75.0% (6/8), 37.5% (3/8), 87.5% (7/8) and 100.0% (8/8) of patients, respectively. Grade 4 neutropenia was observed in 50.0% (4/8) of patients. Grade 3 elevation of AST was observed in 25.0% of patients (2/8). The most common non-hematologic and non-hepatic AE was grade 1/2 post-procedural fever without any infection focus; this was observed in 50.0% of patients (4/8).

Table 4

Adverse events.

Patient		1	2	3	4	5	6	7	8
Hematologic events
	Thrombocytopenia (gr)	4	3	1	3	2	3	4a	4
	Leukopenia (gr)	4	4	4	4	1	4	4	4
	Anemia (gr)	3	2	2	2	2	2	3b	3b
	Lymphopenia (gr)	3	3	3	4	3	4	3	3
	Neutropenia (gr)	4	4	4	n/a	-	4	n/a	n/a
Non-hematologic events
	Elevated AST (gr)	2	2	1	3	3	1	2	-
	Elevated ALT (gr)	1	1	-	n/a	n/a	2	-	-
	Elevated bilirubin (gr)	2	-	-	n/a	n/a	2	-	-
	Fever, treatment related (gr)	2	1	-	-	1	1	-	-
	Nausea (gr)	-	1	2	-	1	1	2	-
	Alopecia (gr)	1	-	-	-	-	-	-	1
	Other	c	-	d	-	e	-	-	f

Note: All patients received a subcutaneous injection of granulocyte-colony stimulating factor within 72h after each M-PHP procedure.

AST, aspartate transaminase; ALT, alanine transaminase; Bili, bilirubin; Gr, grade; n/a, not available; -, no adverse event.

a Treated with platelet transfusion.

b Treated with red blood cell transfusion.

c Haemorrhage groin, treated with a tight pressure dressing.

d Peripheral edema due to periprocedural overhydration, treated with diuretics.

e Lower urinary tract infection, treated with oral antibiotics.

f Aneurysma spurium, successfully treated with a thrombin injection.

Discussion

To date, published series including patients with CRLM that were treated with M-PHP have predominantly reported on hemodynamic and metabolic changes, pharmacokinetics and toxicity [23-25]. The current study was designed to prospectively investigate the efficacy and safety of M-PHP in patients with unresectable CRLM. The ORR of 25% and median OS of 17.3 months were lower than expected based on studies on IHP in patients with CRLM [14,15].

Tumor response in our study was unfavorable compared to the prospective study by Rothbarth et al. (ORR 59%) in which 71 patients with CRLM were treated with IHP using a high dose of melphalan [15]. This appears to be largely attributable to a difference in baseline characteristics between both study populations. Whereas 37% of patients in the study by Rothbarth et al. had received a previous treatment for liver metastases (36% systemic therapy and 1% resection) prior to study inclusion, this was 100% in our study population (100% systemic therapy and 50% resection). This was also reflected in a substantial difference in median interval from diagnosis of CRLM to treatment; this was 4 months in the study by Rothbarth et al. versus 23.7 months in the current study. As all patients that were included in our study had already shown disease progression following systemic chemotherapy, it seems plausible that this limited the a priori probability of M-PHP being effective in these patients.

Fluoropyrimidines, i.e. 5-fluorouracil (5-FU) and capecitabin which is the orally administered pro-drug of 5-FU, are the backbone of systemic chemotherapy for metastatic colorectal cancer and are often combined with oxaliplatin (FOLFOX) or irinotecan (FOLFIRI). Although percutaneous hepatic perfusion (PHP) with 5-FU has been investigated, currently available hemofiltration systems are only intended for the use of melphalan. Given the disease progression under systemic therapy with fluoropyrimidines in our study population, it seems questionable to treat patients suffering from CRLM with PHP using 5-FU.

We were able to confirm the findings of prior studies that M-PHP is well-tolerated and has an acceptable safety profile [15-18]. This was also the case for one patient with a dihydropyrimidine dehydrogenase (DPD)-deficiency in whom systemic chemotherapy was stopped early due to severe 5-FU-associated toxicity. In a pharmacological study we demonstrated that the mean extraction rate of the GEN 2 hemofiltration system, which is also used in the current study, is 86% [18]. As a result, only a small fraction of melphalan that is administered through the hepatic artery will eventually reach the systemic circulation where it can cause hematologic adverse events. Moreover, all study patients received a G-CSF injection following each M-PHP procedure to further limit systemic side-effects.

This study had some limitations. Most notably, the study was terminated early because of a slow recruitment and therefore the patient number was too low to draw definitive conclusions. Second, the study was a single center study. In relation to this problem, the importance of multi-center recruitment, as performed in other locoregional chemotherapy trials must be emphasized [20,28,29]. Third, there was no control arm.

Conclusion

We were able to confirm earlier findings that M-PHP is a well-tolerated and safe procedure. The outcomes on tumor response and survival, however, did not meet our expectations and imply that there currently is no clear role for M-PHP in patients with CRLM outside of clinical trials.

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12 Apr 2021

PONE-D-21-02466

Prospective evaluation of percutaneous hepatic perfusion with melphalan as a treatment for unresectable liver metastases from colorectal cancer

PLOS ONE

Dear Dr. Meijer,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

ACADEMIC EDITOR:

This is a prospective study on a complex locoregional chemotherapy procedure named percutaneous hepatic perfusion. Percutaneous hepatic perfusion was performed with melphalan (high-dosage - approximately 200 mg) and hemofiltration.

The sample size is small (8 patients) and the primary endpoint is response. Considering the small sample size, it is not possible to investigate about the effect of selected variables on response (logistic regression analysis). Secondary endpoints include adverse events that resulted very limited (probably in relation to the use of hemofiltration and the skill of the Authors).

The scientific contribution is important but, in my opinion, the Authors should support with more emphasis the reasons to perform this locoregional chemotherapy procedure.

The indications for M-PHP are still not defined in the international scientific literature.

International guidelines consider locoregional chemotherapy as third line for unresectable CRCLM.

The Authors protocol (as reported in the supporting information file), considers M-PHP for the CRCLM patients for whom “No other standard systemic treatment options (are) available”, but, at same time, include patients “Candidate for neoadjuvant chemotherapy as determined during the multidisciplinary meeting to down size the tumor.” and “Patients (they had) received no, one or more courses of systemic therapy”.

Abstract page 3, line 6: please, added “with hemofiltration”.

Materials and Methods, Procedure details, page 7, line 12: please, clarify if the 16-F double-balloon catheter was positioned percutaneously or by surgical preparation of the femoral (or saphenous) vein.

Table 4: please, precise if patients with grade 3/4 leukopenia/neutropenia received granulocyte colony-stimulating factors.

Table 4: please, re-format line 4 (gr).

In Discussion, the Authors should clarify with more details the reasons for believing and choosing a complex procedure such as M-PHP. In particular the Authors should clarify the reasons to propose percutaneous hepatic perfusion in CRCLM patients not previously submitted to systemic therapy or after failure of the first line.

In Discussion, the Authors should elaborate on the reasons of the very limited adverse events.

In Discussion, as concerning the limitations paragraph, page 12, line 8, please added: “…definitive conclusions. In relation to this problem, the importance of multi-center recruitment, as performed in other locoregional chemotherapy trials […please cite this reference… Mambrini A, Sanguinetti F, Pacetti P, Caudana R, Iacono C, Guglielmi A, Guadagni S, Del Freo A, Fiorentini G, Cantore M. Intra-arterial infusion of 5-fluorouracil, leucovirin, epirubicin, and carboplatin (FLEC regimen) in unresectable pancreatic cancer: results of a ten-year experience. In vivo. 2006; 20: 751-6.], must be emphasized.

==============================

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1 Jun 2021

Respone to the reviewers

Thank you for reviewing our manuscript. We have appreciated your comments and tried to incorporate them in our manuscript.

Academic editor:

This is a prospective study on a complex locoregional chemotherapy procedure named percutaneous hepatic perfusion. Percutaneous hepatic perfusion was performed with melphalan (high-dosage - approximately 200 mg) and hemofiltration.

The sample size is small (8 patients) and the primary endpoint is response. Considering the small sample size, it is not possible to investigate about the effect of selected variables on response (logistic regression analysis). Secondary endpoints include adverse events that resulted very limited (probably in relation to the use of hemofiltration and the skill of the Authors).

The scientific contribution is important but, in my opinion, the Authors should support with more emphasis the reasons to perform this locoregional chemotherapy procedure.

Reply: Although systemic therapy is currently considered the first-line treatment modality in patients with unresectable CRLM, it can be accompanied by serious side-effects. We hypothesised that M-PHP would be a good alternative with limited systemic side-effects for metastatic CRC patients with liver-only disease. It has already been demonstrated that M-PHP is a safe, well-tolerated procedure in patients with liver metastases from ocular melanoma (Cardiovasc Intervent Radiol. 2019;42(6):841–852). Unfortunately, the current study was terminated prematurely due to slow recruitment. Several factors such as the availability of alternative therapies (e.g. systemic chemotherapy or radioembolization) and competing trials with systemic drugs or intra-arterial therapies probably contributed to this slow recruitment. This, together with a tumor response rate and median OS that did not meet our expectations (unfavorable compared to the study by Rothbarth et al. in which patients with CRLM were treated with isolated hepatic perfusion using a high dose of melphalan (Br J Surg. 2003;90(11):1391–1397)) implies that there is currently no clear role for M-PHP in patients with CRLM outside of clinical trials.

The indications for M-PHP are still not defined in the international scientific literature.

International guidelines consider locoregional chemotherapy as third line for unresectable CRCLM.

The Authors protocol (as reported in the supporting information file), considers M-PHP for the CRCLM patients for whom “No other standard systemic treatment options (are) available”, but, at same time, include patients “Candidate for neoadjuvant chemotherapy as determined during the multidisciplinary meeting to down size the tumor.” and “Patients (they had) received no, one or more courses of systemic therapy”.

Reply: It is correct that relatively few exclusion criteria were incorporated in the study protocol whereby most patients with histologically proven, unresectable CRLM were indeed eligible for inclusion. As was shown in Fig. 1, in 4/16 patients that were discussed during a multidisciplinary team meeting prior to possible inclusion, were excluded from participation because first-line systemic therapy was preferred. Table 2 shows that all patients in the current study had received one or more types of systemic therapy before receiving M-PHP.

The ability to perform PHP may increase the role of hepatic perfusion for treatment of multiple tumor histologies since this procedure allows for sequential treatments to be delivered with lower morbidity. The management of unresectable liver metastases is a significant clinical problem that requires the combined efforts of multiple providers to develop an integrated approach for each patient. Continued evaluation of hepatic perfusion in these patients is necessary so that its role in this integrated approach can be more clearly defined.

Minor changes

Abstract page 3, line 6: please, added “with hemofiltration”.

Reply: We added these words.

Materials and Methods, Procedure details, page 7, line 12: please, clarify if the 16-F double-balloon catheter was positioned percutaneously or by surgical preparation of the femoral (or saphenous) vein.

Reply: We made alterations accordingly.

Table 4: please, precise if patients with grade 3/4 leukopenia/neutropenia received granulocyte colony-stimulating factors.

Reply: All patients received a subcutaneous injection of granulocyte-colony stimulating factor within 72h after each M-PHP procedure. We added a sentence in the caption of Table 4.

Table 4: please, re-format line 4 (gr).

Reply: We re-formatted Table 4.

In Discussion, the Authors should clarify with more details the reasons for believing and choosing a complex procedure such as M-PHP. In particular the Authors should clarify the reasons to propose percutaneous hepatic perfusion in CRCLM patients not previously submitted to systemic therapy or after failure of the first line.

Reply: See also one of the replies to the academic editor. We believe that there is currently no clear role for M-PHP in patients with CRLM outside of clinical trials. In an attempt to clarify this, some changes were made to the manuscript.

In Discussion, the Authors should elaborate on the reasons of the very limited adverse events.

Reply: In a pharmacological study that was conducted in 2017, de Leede et al. demonstrated that the mean extraction rate of the GEN 2 hemofiltration system is 86%. (Cardiovasc Intervent Radiol. 2017;40:1196–1205). This means that only a small fraction of melphalan that is administered in the hepatic artery will eventually reach the systemic circulation. Additionally, all patients receive a subcutaneous injection with granulocyte colony-stimulating factor after each M-PHP procedure in an attempt to limit any systemic side-effects as much as possible. We made some changes to the Discussion section.

In Discussion, as concerning the limitations paragraph, page 12, line 8, please added: “…definitive conclusions. In relation to this problem, the importance of multi-center recruitment, as performed in other locoregional chemotherapy trials […please cite this reference… Mambrini A, Sanguinetti F, Pacetti P, Caudana R, Iacono C, Guglielmi A, Guadagni S, Del Freo A, Fiorentini G, Cantore M. Intra-arterial infusion of 5-fluorouracil, leucovirin, epirubicin, and carboplatin (FLEC regimen) in unresectable pancreatic cancer: results of a ten-year experience. In vivo. 2006; 20: 751-6.], must be emphasized.

Reply: We made alterations accordingly.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

29 Jun 2021

PONE-D-21-02466R1

Prospective evaluation of percutaneous hepatic perfusion with melphalan as a treatment for unresectable liver metastases from colorectal cancer

PLOS ONE

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Reviewer #1: The manuscript entitled ‘Prospective evaluation of percutaneous hepatic perfusion with melphalan as a treatment for unresectable liver metastases from colorectal cancer’ with the aim to prospectively evaluate the efficacy and safety of M-PHP in patients with unresectable CRLM.

The manuscript can be further improved.

Sample size calculation

Page 9. the write up on the sample size requires further improvement. Information on margin of error, power of study to be stated. The statement of ‘16.5% around the observed proportion’ not clear.

Page 10, what range refers to, min – max or IQR to be clearly stated.

All percentage figures to be at least 1 decimal point.

For Figure 1, more information could be added such as intervention, assessment, outcome variables, duration/period etc.

For Table 2, 3, 4, dash (-) to be denoted in the table footnote.

For Table 3, days to be stated as the initial measurements which then converted to months. This needs to be stated in the methodology. mo to be denoted in table footnote.

In the Reference list, reference number 28 and 29 to conform with the journal format i.e journal name.

Reviewer #2: The paper is very interesting but this is a very complex type of locoregional chemotherapy for hepatic metastases.

In 8 patients the procedure has been performed percutaneously. The sample size is too small to demonstrate that the introduction of a 16F catheter is always possible without complications in all types of femoral arteries (also arteries with a diameter lower than 7 mm).

Minor changes suggested:

The authors should elaborate the previous concepts in Material and Methods and in Discussion.

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Clementi Review.docx

Click here for additional data file.

1 Nov 2021

Response to the reviewers

Thank you for reviewing our manuscript. We have appreciated your comments and tried to incorporate them in our manuscript. Trying to keep things comprehensive, we responded to the academic editor and reviewer(s) in blue.

----------------------------------------------------------------------------------------------------------------

Reviewer #1: The manuscript entitled ‘Prospective evaluation of percutaneous hepatic perfusion with melphalan as a treatment for unresectable liver metastases from colorectal cancer’ with the aim to prospectively evaluate the efficacy and safety of M-PHP in patients with unresectable CRLM.

The manuscript can be further improved.

Sample size calculation

Page 9. the write up on the sample size requires further improvement. Information on margin of error, power of study to be stated. The statement of ‘16.5% around the observed proportion’ not clear.

Reply: Unfortunately, no standard power calculation was performed since no clear null-hypothesis could be formulated. We choose a sample size that allows the response percentage to be determined with sufficient accuracy, i.e. with a sufficiently narrow confidence interval. Assuming a true response percentage of 60%, a sample size of 34 patients will yield a two-sided confidence interval of length 0.33 (± 16.5% around the observed proportion). Adjustments were made to our manuscript to clarify this.

Page 10, what range refers to, min – max or IQR to be clearly stated.

Reply: We changed made alterations accordingly, e.g. see page 10: ‘Median melphalan dose was 220 mg (ranging from 190 to 220) for the first cycle and 220 mg (ranging from range 160 to 220) for the second cycle.’

All percentage figures to be at least 1 decimal point.

Reply: We made alterations accordingly. e.g. see page 10: ‘The one-, two-, and three-year OS was 50.0%, 50.0%, and 0% respectively.

For Figure 1, more information could be added such as intervention, assessment, outcome variables, duration/period etc.

Reply: Figure 1 is in our opinion to show the enrolment of patients, and why patients were not eligible for study inclusion. This is all provided in the figure. Elaborating on for instance intervention or outcome variables obtained distracts, in our opinion, from the information currently given in Figure 1.

For Table 2, 3, 4, dash (-) to be denoted in the table footnote.

Reply: Changes were made accordingly. See Table 2, 3 and 4.

For Table 3, days to be stated as the initial measurements which then converted to months. This needs to be stated in the methodology. mo to be denoted in table footnote.

Reply: Changes were made accordingly. See page 9: ‘Median OS, median PFS, and hPFS were measured in days and subsequently converted into months.’ Also changes were made accordingly in the footnote of Table 3.

In the Reference list, reference number 28 and 29 to conform with the journal format i.e journal name.

Reply: Changes were made accordingly. See reference 28 and 29

Reviewer #2: The paper is very interesting but this is a very complex type of locoregional chemotherapy for hepatic metastases.

In 8 patients the procedure has been performed percutaneously. The sample size is too small to demonstrate that the introduction of a 16F catheter is always possible without complications in all types of femoral arteries (also arteries with a diameter lower than 7 mm).

Minor changes suggested:

The authors should elaborate the previous concepts in Material and Methods and in Discussion.

Reply: This study aimed to evaluate the outcome in patients with colorectal liver metastases treated with percutaneous hepatic perfusion with Melphalan. The safety of this procedure has already been reported by Meijer et al. (Cardiovasc Intervent Radiol. 2019;42(6):841-852.) and is referred to in the last paragraph of the discussion. Until now, arterial access has not been a reason for exclusion of patients for this procedure in our hospital. Therefore, we do not elaborate extensively on this topic in the current paper.

Additional changes:

Figure one has been replace. The first box had a textual error, it said: ‘accessed for eligibility.’ It has now been changed to: assessed for eligibility.’

Submitted filename: Response to reviewers - 2 docx.docx

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15 Dec 2021

Prospective evaluation of percutaneous hepatic perfusion with melphalan as a treatment for unresectable liver metastases from colorectal cancer

PONE-D-21-02466R2

Dear Dr. Meijer,

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Stefano Guadagni

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Good revision addressing all my concerns.

Reviewers' comments:

6 Jan 2022

PONE-D-21-02466R2

Prospective evaluation of percutaneous hepatic perfusion with melphalan as a treatment for unresectable liver metastases from colorectal cancer

Dear Dr. Meijer:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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PLOS ONE