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In Vivo Lung Perfusion (IVLP) for Cancer Therapy and Gene Therapy

of the Lung

Marcelo Cypel MD, MSc, FRCSCCanada Research Chair in Lung Transplantation

Surgical Director ECLS Program UHNAssociate Professor of SurgeryDivision of Thoracic Surgery

University of Toronto

The ProblemThe lung is a very common site for metastatic disease

Often is the only site of spread

Recurrence after initial surgical resection is exceedingly high: - Colorectal 50%

- Sarcoma 70%- Melanoma 88%

World J Surg. 2013 Jun;37(6):1315-21Ann Surg Oncol. 2007 Oct;14(10):2847-53

Prognostic GroupsMST (mos.)

group I single and DFI > 35 mos. 61II single or DFI > 35 mos. 34III multiple, DFI < 36 mos. 24IV incomplete resection 14

J Thorac Cardiovasc Surg 1997; 113:37-49

More than 3 lesions or < 1 year DFI = no survivors at 5 years (osteosarcoma)

4

Annals of Oncology 20:1136-1141, 2009

Toronto data

Current Limitations• 5 years survival after surgery is only 15-45%(Including recent series with modern chemotherapy and targeted therapies)

• Median disease free survival is short (12-18mos)

• Reoperations are feasible, but often patients become inoperable

• Systemic therapy with variable grades of response and excessive toxicity

Micrometastases

6

J Vis Exp. 2012 Aug 21;

How about giving anti-cancer treatment only to the lung?

In Vivo Lung Perfusion (IVLP)

Thoracic Surgery Latner Labs

TORONTO EX VIVO LUNG PERFUSION (EVLP) SYSTEM

Perfusion : 40% COVentilation: 7cc/kg, 7BPM, PEEP 5, FiO2 = 21%

Cypel/Keshavjee J Heart Lung Transplant 2008; 27(12):1319-25Cypel/Keshavjee NEJM 2011; 14;364(15):1431-40

NEJM 2011

J Thorac Cardiovasc Surg. 2014 774-81

• Dose escalation study• 75mg/m2 well tolerated• 150mg/m2 moderate injury• 225mg/m2 severe injury leading to animal death

12

Annals of Thoracic Surgery 2016

Doxorubicin 75 mg/m2

200x

Pre IVLP Post Reperf

200x

IVLP clinical trial1) To determine the safety of IVLP with Dox.

2) To determine the maximal tolerated dose by using a titration design.

3) To determine the time from treatment to recurrence in the ipsilateral treated lung versus the non - IVLP treated contralateral lung.

IVLP

15

Recent Patient: DC home in 6 days

Dox concentrationPt02

Sample Doxorubicin

(ng/assay)serum T3 IVLPO2 T3S (200 µL) NDserum T4 IVLPO2 T4S (200 µL) NDPOD1 IVLPO2 POD1 (200 µL) NDPOD2 IVLPO2 POD2 (200 µL) NDPOD3 IVLPO2 POD3 (200 µL) NDPOD4 IVLPO2 POD4 (200 µL) ND

POD5 IVLPO2POD5 (200 µL) NDT0.5 IVLPO2 T0.5 perfusate (20 µL) 143.4T1 IVLPO2 T1 perfusate (20 µL) 76.4T2 IVLPO2 T2 perfusate (20 µL) 31.8

T3 IVLPO2 T3 perfusate (20 µL) 20.2

T4 IVLPO2 T4 lung (15 mg) 104.9

Evaluating Efficacy of Novel Treatments

18

Establishment of colorectal and sarcoma lung metastases model

Day 28 Evaluation

Day 0 InjectionSarcoma cells (5 x 10^6 cells) Colorectal cells (2.5 x 10^6 cells)

Micrometastases (Day 4)

Sarcoma model(Day 28)

Colorectal ca model(Day 28)Day 4

Establishment of colorectal and sarcoma lung metastases model

Day 28 Evaluation

Day 0 InjectionSarcoma cells (5 x 10^6 cells) Colorectal cells (2.5 x 10^6 cells)

Micrometastases (Day 4)

Sarcoma model(Day 28)

Colorectal ca model(Day 28)Day 4

Treatment IV or IVLP

IVLP only IV (Dox)

IVLP (Dox)

Sarcoma model

Left lung weights at day 25

mg *

Kruskal-Wallis statistic (p = 0.0002)(Non-parametric ) and

Dunn's Multiple Comparison Test

*

IVLP only IV FOLFOX

IVLP with FOLFOX

Colorectal model

Lung-specific gene delivery to treat/prevent pulmonary metastases recurrences

24

Lentivirus-Luciferase Distribution

Day 8

Day 28

IVLP (4 weeks) at sacrifice

Left lungRight lung

Heart

Liver Spleen

kidney

Left lung

Targeted gene: Interleukin 12 (Localized immunetherapy)

Control Group (Shamgroup)

Group 1: IT LV-IL12 (LD)

Group 2: IT LV-IL12 (HD)

Group 3: IV Oxaliplatin

Group 4: IV Oxaliplatin+ IT LV-IL12 (LD)

Group 5: IV Oxaliplatin+ IT LV-IL12 (HD)

DAY 28

Sacrifice:Histological

analysis

+ Analysis of the number of

nodules (Weksler

Technique)

+ Measurement of

IL12INF-γTNF-α

DAY 0N=5 for each

group

Injection 2.5 × 106

Cells

RCN9 colorectal cancer cell

line

Left Jugularvein

Fischer F344 250g

DAY 7

Assessment of LV-IL12 efficiency

• India ink staining + Fekete’s Solution

Slides with H&E

staining

IL-12 Lung expression

At Day 28

Efficiency of LV-IL12

Number of lung nodules/Slides

One way Anova p<0.0001

* Mann-Whitney test p<0.05

IT IL-12 group Control

Combination treatment increased lung NK cell content

Combination treatment increased IFNγ+

and polyfunctional CD4+ T cells

Conclusions

• IVLP is a new platform for treatment of pulmonarymetastases

• Phase I clinical trial IVLP Dox for sarcoma is underway(starting oxaloplatin for colorectal ca in 2018)

• Preliminary studies in small animals have shownlocalized IL-12 gene therapy to be effective

• Future directions:Complete the small animals studiescombining IVLP and IL-12. Localized IL-12 delivery in large animals (safety studies).

marcelo.cypel@uhn.cawww.dotscanada.ca