Immunologic and Angiogenic Effects of Open & Closed Abdominal

Surgery

Richard L. Whelan, MDSt. Luke’s Roosevelt Hospital

Columbia UniversityNew York, N.Y.

2012 MISS Meeting – Salt Lake City

Whelan Disclosures• Ethicon Endosurgery • Olympus Corporation • Atrium Corporation• Convatec• Rolex Watches (with 10 hour timer feature for long

laparoscopic colectomies)

• Trek Bicycle Corp. (off road surgical adventures)

• World Wrestling Association (for ongoing hand-assist vs laparoscopic debate with Morris Franklin & company)

Open Surgery & Immune Function

• Transient immunosuppression after major open surgery is well proven

• DTH studies• Blood protein studies• Lymphocyte proliferation & function

assays

Does it matter ??

Anergic vs Immunocompetent Patients

• Significantly higher rate of postop sepsis and mortality*

• Significantly lower resectability rates and higher recurrence rates in cancer patients

* Same is true for transplant patients & those on immunosuppressive drugs

In Immunocompetent Patients Does The Immunosuppression

Matter ?

• It is relative immunosuppression• Clinical manifestations are not clear• Significance is unclear

Immune Function After Laparoscopy: Summary of Results

- Laparoscopy is associated with less immunosuppression than open methods

- In most cases the differences are small and short lived

Less Immunosuppression after Laparoscopic Surgery*: Evidence

-Both animal and human studies - Lymphocytes more readily stimulated- Greater Th-1/Th-2 ratio post op -Macrophage & PMN marker studies:

- Systemic vs peritoneal - Results hard to interpret

* Short lived 1-2 day differences for most parameters

Immune Function Following Laparoscopic Procedures in Humans* ** + ++

• Lower serum levels of (closer to baseline):– IL-6– CRP – IL-8– IL-10 – granulocyte elastase

• Monocyte HLA-DR expression preserved • T cell expression CD 31 better preserved• Differences are short lived (often < 1 day)

*Ordemann et al. Surg Endosc DOI:1007/s004640090032 **Sietses et al. Dis Colon Rectum 2003 Feb;46(2):147-55. + Hildebrandt et al. Surg Endosc. 2003;17(2):242-6. ++ Kirman et al. Surg Endosc

Delayed-type Hypersensitivity Testing (DTH)

- Assesses cell-mediated immune function

- Most often used to establish anergy or immune competence

- Tests for prior exposure to specific infectious agent (TB, mumps, candida)

- Absence of DTH response defines the anergic patients

DTH Response After Open Colorectal Resection*

Open Group

0%

20%

40%

60%

80%

100%

120%

140%

160%

Preop POD 0 POD 3

Median

Mean

*

**

*P < 0.0005 vs. preop **P < 0.0003 vs preop

*

**

* Whelan and Franklin et al. Surg Endoscopy 2003;17(6):972-978

n=17 patients

Are

a o

f in

dura

tion

mm

2

n=18 n=18

* Kirman et al. Europ J Surg Oncol. 2007;33(10):1169-76.

Pre POD2 POD5 Pre POD2 POD5

Lymphocyte Microarray Studies

• Affymetrix oligonucleotide microarrays • 22,000 unique genes assessed• Murine study• Laparotomy, C02 pneumo, &

anesthesia alone• ½ animals sacrificed at 12 and 24 hours • Splenic T cells isolated & mRNA

extracted

12 Hour Results: Relative to Anesthesia Control Group*,+ ,**

# Genes # Genes Total Group Regulated Regulated # Genes

CO2 Pneumo 86 30 116

Laparotomy 362 36 398

* Threshold difference between groups > 2 X expression + Results validated with RT-PCR for 8 selected genes ** Sylla et al. Surgery. 2006 Jan;139(1):92-103.

24 Hour Results: Relative to Anesthesia Control Group*

# Genes # Genes Total Group Regulated Regulated # Genes

CO2 Pneumo 118 14 132

Laparotomy 133 24 157

* Threshold difference between groups > 2 X expression

What is the Clinical Significance ?

Cochrane Evidence Based Review: Short Term Benefits of Lap. vs Traditional Colectomy (All indications)* **

Main findings regarding laparoscopic methods– Lower blood loss – Lower intensity of pain– Shorter postoperative ileus– Pulmonary function improved

– Incidence post-op complications significantly lower (18.2%) vs open (23%) P=0.02

*Schwenk et al. Cochrane Database of Systematic Reviews 2006 Issue 3 ISSN 1464-780X ** 25 Randomized control trials reviewed.

Meta-Analysis of 17 Randomized Colectomy Trials (4013 Operations)*

• No difference in leak rate or overall morbidity found

• Significantly lower rate of wound complications noted in laparoscopic patients (odds ratio 0.65; p=0.01)

• Could better preserved immune function contribute to these findings ?

* Tjandra JJ et al. Colorectal Dis 2006;8:375-388.

Does Immune Function Impact Short Term Outcome Parameters ??

• Less pain ?• Better pulmonary function ?• Quicker resolution of ileus ?

What About Non-oncologic Long Term Outcome measures ??

• Fewer SBO admissions ?• Fewer Reops for SBO ?

What About Cancer ??

Cancer Recurrence & SurvivalTrial No. pts. Oncologic ResultsCOST 863 No DifferenceCOLOR 1248 No DifferenceCLASSIC 746 No DifferenceMeta-Analysis No Difference Lacy et al 208 Lap. Benefit

Current Conclusion: There is no difference.

Why is there no difference in survival ??

• The short term effects (7 days) clearly favor MIS methods

• In regards to some important late postoperative effects MIS and Open methods are very similar

• All the late effects concern angiogenesis

Angiogenesis

• Critical to wound healing• Wound healing and remodeling goes

on for weeks after major surgery• Also critical to cancer growth• Solid tumors cannot grow > 2mm

without new blood vessel formation

Late Postop Blood Sampling for Studies

• In past: Blood samples taken POD 1-3• Present: Get multiple blood samples during

first 5-8 weeks after surgery– Hard to get– Patients may refuse– Need to “bundle” specimens into 7 day periods &

consider as single time point

• Plasma protein & in vitro Endothelial Cell Cultures done

Cancer group: median VEGF levelsMIS Patients Only (n=69)

0

500

1000

1500

Median 150 192.7 246 380.6 572.2 611.1 512.1 210.4

OpDay POD1 POD3 POD5 POD7-13 POD14-20 POD21-27 POD28+

Pvalue 0.3644 0.009 0.0012 0.0008 0.0098 0.0117 0.4648

***

***^

^^

Ang 1 and Ang 2• Angiopoietin 1 (Ang 1) stabilizes mature blood

vessels and inhibits VEGF initiated early angiogenesis

• Angiopoietin 2 (Ang 2) facilitates the angiogenic response to VEGF by preventing the Ang 1 response (blocks Tie2 receptor)

• Ratio of Ang 1/ Ang 2:– ratio inhibits VEGF-related angiogenesis– ratio facilitates VEGF-related angiogenesis

Plasma Ang1/Ang2 – MIS Cancer Patients

0

1

2

3

4

5

6

7

8

9

PO POD1 POD3 POD 7-13

POD14-20

POD21-27

POD28+

An

g/A

ng

2

n = 96n = 85 n = 60 n = 42 n =10 n =17 n =24

+ ++^^

+ +

^

: p= 0.0001 vs. PO

: p= 0.002 vs. PO

: p= 0.041 vs. PO

: p- 0.0145 ns. PO+++

^^^

Other Proangiogenic Proteins Elevated Late After Colorectal Resection*

• Placental Growth Factor (PlGF)• Soluble Vascular Cell Adhesion Molecule

(sVCAM)• ANGPTL4

What is the net effect of these late protein changes on plasma function ??

* Patient population: MIS colorectal resections

In Vitro Endothelial Cell (EC) Culture Studies Hold the Answer

• Endothelial Cell is the main target of angiogenesis

• Goal: Determine the impact of pre & postop plasma on EC behavior

• Parameters: – Proliferation (microtubule) formation– Invasiveness and migration

PreOp POD 7-130

100

200

300

Mea

n m

igra

ted

cel

lsIn vitro Endothelial Cell Migration Assay:

Preop vs POD 7-13

n =24Median: 98.7

n =24Median: 132.1

+ :p=0.005 vs. PreOp.

+

In vitro EC Culture Assays: Preop Vs Postop Significantly Greater Results Postop ?

+

Parameter BPF Migration Invasion

POD 7-13 (n=30)* + + +

POD 14-20 (n=26)** + + +

* Mean sampling day = 11 ** Mean sampling day = 17.5 + Extent of change between pre and post op results is 17 - 25 %

0

20

40

60

80

100

120

140

160

BP INV MIG

Me

an

Bra

nc

h p

oin

ts(B

P),

Inv

as

ive

ce

lls(I

NV

), o

r M

igra

ted

ce

lls (

MIG

)

PreOp

Day 7-13

* **[* ]: P=0.0001

Open Colectomy Endothelial Cell Culture Study

POD 7-13 vs Preop Plasma Samples Results Branch Point Proliferation Assay

Summary: Late Effects of Surgery• Proangiogenic• Last for up to 1 month• Likely more influential than the 1-3 day effects• May stimulate the growth of residual tumor

deposits & circulating tumor cells• Open and MIS methods have similar late

changes & effects• Suggests that safe anti-cancer therapy should

be given during 1st postop month

Immune Function Summary

• MIS methods are less immunosuppressive

• Duration is brief• May account for;

– Lower wound infection rates– Lower morbidity– Other short term benefits (? Pain, LOS, etc)

Where the Same Genes Effected ? *

# Genes # Genes Group 12 hours 24 hours

Altered expression 60 (13%) 77 (39%) in both groups

Increased expression 338 80 in Open Group only

Increased expression 59 41 in CO2 Group only * Threshold difference between groups > 2 X expression

Surgery in Immunocompromised Patients

- Population: - Transplant patients - Pts. on immunosuppresive drugs - Disease-related immunosuppression

- Higher complication rates- Higher mortality

0

20

40

60

80

100

120

BP INV MIG

Me

an

Bra

nc

h p

oin

ts(B

P),

Inv

as

ive

ce

lls(I

NV

), o

r M

igra

ted

ce

lls (

MIG

)

PreOp

Day 14-20

Open Colectomy Endothelial Cell Culture Study

**

*[* ]: P=0.0001

POD 14-20 vs Preop Plasma Samples Results Branch Point Proliferation Assay

In vitro Endothelial Branch PointFormation: Preop vs POD 7-13

n =24Median: 51.15

n =24Median: 62.950

+ :p=0.039 vs. PreOp.

PreOpO POD 7-13 0

102030405060708090

100110

BP

+

Clinical Significance of Immune Function Differences is Uncertain

• Further studies needed• Need to document clinical benefits• There is no conclusive evidence,

however, …• There is some suggestive evidence

PreOp POD 7-130

100

200

300

Mea

n m

igra

ted

cel

ls

In vitro Endothelial Cell Migration Assay: Preop vs POD 7-13

n =24Median: 98.7

n =24Median: 132.1

+ :p=0.005 vs. PreOp.

+

In vitro Endothelial Branch Point Formation: Preop vs POD 7-13

n =24Median: 51.15

n =24Median: 62.950

+ :p=0.039 vs. PreOp.

PreOpO POD 7-13 0

102030405060708090

100110

BP

+