**Bacterial Immunotherapy -Antitumoral Potential of the Streptococcal Toxin Streptolysin S-**

Claudia Maletzki1, Bernd Kreikemeyer² , Peggy Bodammer³ , Joerg Emmrich³ and Michael Linnebacher1 *1Department of General, Vascular, Thoracic and Transplantation Surgery, Section of Molecular Oncology and Immunotherapy, ²Institute of Medical Microbiology, Virology and Hygiene, ³ Division of Gastroenterology Department of Internal Medicine University of Rostock, Rostock, Germany* 

#### **1. Introduction**

162 Pancreatic Cancer – Clinical Management

[161] Hingorani, S. R. *et al.* Preinvasive and invasive ductal pancreatic cancer and its early

[162] Olive, K. P. *et al.* Mutant p53 gain of function in two mouse models of Li–Fraumeni

detection in the mouse. *Cancer Cell* 4, 437–450 (2003).

syndrome. *Cell* 119, 847–860 (2004).

Chronic infections can lead to cancer. However, acute infection has beneficial effects often contributing to complete eradication of tumors. In the wake of this, bacteria and their related products were applied therapeutically for experimental immunotherapy. They exhibit direct antitumoral potential and are recognized by the host's immune system via Toll-like receptors (TLRs) finally promoting pro-inflammatory, often Th1-directed immune responses.

Recently, we described that local injection of live as well as lysed gram-positive Group A Streptococci (GAS) eradicates established pancreatic tumors in mice (Linnebacher et al., 2008; Maletzki et al., 2008). This antitumoral effect could be attributed to activation of immune response mechanisms including both the innate and even more important, the adaptive arm of the immune system. In the face of the vigorous immune attack induced by *S. pyogenes*, the identification of factors responsible for tumor disintegration might provide the basis for development of therapeutic approaches. Amongst other virulence factors delivered by *S. pyogenes*, the cytolysins Streptolysin O (SLO) and S (SLS) represent the most obvious therapeutically active candidates (Fraser & Proft, 2008; Hobohm et al., 2008; Nizet et al., 2008). SLO is an oxygen-labile, pore-forming toxin mediating cytolysis by disturbing the balance between influxes and effluxes across the cell membrane. While SLS is nonimmunogenic in the natural course of infection and can clinically be identified by betahaemolysis surrounding GAS colonies grown on blood agar. Besides their capacity to lyse erythrocytes, SLS also exerts cytolytic effects towards tumor cells and is by weight one of the most potent cytotoxins known (Ginsberg, 1999; Taketo & Taketo, 1966).

To address the question of the SLS contribution to the antitumoral effects observed in our previous studies, we performed a series of *in vivo* experiments in our murine syngeneic

Bacterial Immunotherapy-Antitumoral Potential of the Streptococcal Toxin Streptolysin S- 165

the incubation time, cell-free supernatants were transferred into a new 96-well plate and absorption was measured on a plate reader at 340 nm. Hemolytic activity was quantified by

Toxicity of bacteria towards tumor cells was quantified using a cytotoxicity detection kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. Quantification

For detection of apoptosis, activity of Caspase 3 was analyzed using the BD ApoAlert Caspase Assay plate system according to the manufacturer's instructions (BD Biosciences, Heidelberg, Germany). Cells were trypsinized and subsequently lysed. Quantification of Caspase 3 activity was performed by fluorometric detection on a Cytofluor 2300 (Millipore,

Under brief ether anaesthesia 1\*106 Panc02 were injected subcutaneously (s.c.) into the right hind leg. Tumor growth was routinely controlled at least twice a week and tumor volume was estimated according to the formula: V= width² \* length \* 0.52. After tumor establishment animals were subdivided into experimental groups. All treatments were performed by local, intratumoral application of bacterial preparations each dissolved in 50 µl of phosphate-buffered saline (PBS) according to the following treatment regimen: One group was given heat-inactivated *S. pyogenes* (8 x 107 cfu, four injections once a week, n=6). The second group received a single injection of the SLS-mu (ΔsagA). For control, mice were administered equivalent volumes of solvent alone (vehicle-treated controls, saline, n=6) or left without treatment (control, n=6). Tumor carrying mice (treatment, control) were sacrificed at day 28 or when they became moribund before the tumor volume reached 2000 mm³. At the end of each experiment, tumors as well as spleens, mesenteric lymph nodes, and blood samples were removed from selected animals for further analysis. For visualization of tumor-infiltrating leukocytes, frozen sections of resected tumor tissues (6 µm) were stained by the As-D chloroacetate esterase (As-D) technique. Numbers of leukocytes/mm² were determined in blinded counts by positive staining and morphology in

setting values of untreated cells as 1 and all other data were given as x-fold increase.

was performed by subsequent measurement on a plate reader at 492 nm.

**2.4 Cellular cytotoxicity assays** 

Schwalbach, Germany, ex/em: 380/460 nm).

20 consecutive high power fields (HPF).

**2.6 Phenotyping of lymphocyte subpopulations by flow cytometry** 

leukocytes (Mounting View, BD Biosciences). Relative numbers are given.

Leukocytes from treated and non-treated animals were labeled using the following FITCconjugated rat anti-mouse monoclonal antibodies (mAbs): CD3, CD19, NK1.1 (1 µg, Immunotools, Friesoythe, Germany), and Phycoerythrin (PE)-conjugated rat anti-mouse mAbs: CD4, CD8 (Miltenyi Biotec, Bergisch Gladbach, Germany). Afterwards, erythrocytes were lysed using FACS Lysing Solution (BD Pharmingen, Heidelberg, Germany). Negative controls consisted of blood lymphocytes stained with the appropriate isotypes (BD Pharmingen). Samples were analyzed on a FACSCalibur Cytometer (BD Biosciences). Data analysis was performed using CellQuest software (BD Biosciences) and gating on total

**2.5 Pancreatic tumor model & treatment regimen** 

Panc02 tumor model using different strategies of SLS-inactivation. Endpoints of the study were survival and tumor response. In a first series of experiments, a vital mutant strain, unable to produce SLS (ΔsagA) was injected into tumor-carrying mice. This ΔsagA mutant has been described to exhibit reduced cytotoxicity. *In vitro* and in a nude mouse model *in vivo*, effects were induced by minimal inflammation and lesser necrotic skin lesions than the isogenic wildtype strain (Datta et al., 2005). However, to circumvent the potential risk of unpleasant side effects of live bacteria such as systemic toxicity, another microbial preparation of SLS-inactivated bacteria (by heating) was employed. Our *in vivo* data show that local application of SLS-negative agents attenuates the antitumoral effects. Moreover, potent immune responses are only marginally induced, most likely because of reduced tumor cell impairment after infection, finally leading to an inhibition of vigorous antitumoral activity.

In summary, when comparing with our earlier findings on using *S. pyogenes* as an active immunotherapeutic compound, the present data imply SLS as major antitumoral molecule both directly by acting on tumor cells and indirectly by activating the immune system.

### *2.* **Material & methods**

#### **2.1 Cell culture & mice**

All cell lines (Panc02, EL4, CMT-93, and MC3T3-E1) were maintained in DMEM/HamsF12 supplemented with 10% fetal calf serum (FCS), L-glutamine (2mmol/l) and antibiotics. All media and supplements were from PAA unless stated otherwise (Cölbe, Germany). Female 8–10-week-old C57Bl/6N mice were purchased from Charles River Inc. (Sulzfeld, Germany). Animals were exposed to cycles of 12 h light/12 h darkness and received standard food and water ad libitum. Upon approval by the local animal welfare committee, experiments were performed in accordance with the German legislation on protection of animals and the ''Guide for the Care and Use of Laboratory Animals''.

#### **2.2 Bacteria, toxin and culture conditions**

Bacteria (*S. pyogenes* serotype M49 strain 591; the ΔsagA mutant isogenic to strain 591 is a kind gift from Victor Nizet: A detailed description of how this mutant was generated can be found in (Datta et al., 2005)) were cultured in Todd-Hewitt (TH) broth or on TH agar (Oxoid Unipath, Wesel, Germany), both supplemented with 0.5 % yeast extract (THY) overnight to mid-log phase. Concentration was adjusted on the basis of an optical density reading at 600 nm and on plating analysis. Heat-inactivation of bacteria was obtained by one-hour incubation in a water bath at 75 °C. Inactivation was confirmed by plating samples on sheep blood agar followed by over night incubation at 37 °C and 5 % CO2. The purified toxin SLS (originally obtained from Sigma Aldrich, Munich, Germany) was dissolved in sterile PBS and supplemented with complete cell culture medium before treatment of cells.

#### **2.3 Hemolysis assay for SLS activity**

Hemolytic activity of SLS was determined by hemoglobin release from whole blood cells after 4 and 24 h incubation with bacteria or their related products. Briefly, whole blood of healthy donors was seeded in 96-well plates and supplemented with microbia. Following the incubation time, cell-free supernatants were transferred into a new 96-well plate and absorption was measured on a plate reader at 340 nm. Hemolytic activity was quantified by setting values of untreated cells as 1 and all other data were given as x-fold increase.

#### **2.4 Cellular cytotoxicity assays**

164 Pancreatic Cancer – Clinical Management

Panc02 tumor model using different strategies of SLS-inactivation. Endpoints of the study were survival and tumor response. In a first series of experiments, a vital mutant strain, unable to produce SLS (ΔsagA) was injected into tumor-carrying mice. This ΔsagA mutant has been described to exhibit reduced cytotoxicity. *In vitro* and in a nude mouse model *in vivo*, effects were induced by minimal inflammation and lesser necrotic skin lesions than the isogenic wildtype strain (Datta et al., 2005). However, to circumvent the potential risk of unpleasant side effects of live bacteria such as systemic toxicity, another microbial preparation of SLS-inactivated bacteria (by heating) was employed. Our *in vivo* data show that local application of SLS-negative agents attenuates the antitumoral effects. Moreover, potent immune responses are only marginally induced, most likely because of reduced tumor cell impairment after infection, finally leading to an inhibition of vigorous

In summary, when comparing with our earlier findings on using *S. pyogenes* as an active immunotherapeutic compound, the present data imply SLS as major antitumoral molecule both directly by acting on tumor cells and indirectly by activating the immune system.

All cell lines (Panc02, EL4, CMT-93, and MC3T3-E1) were maintained in DMEM/HamsF12 supplemented with 10% fetal calf serum (FCS), L-glutamine (2mmol/l) and antibiotics. All media and supplements were from PAA unless stated otherwise (Cölbe, Germany). Female 8–10-week-old C57Bl/6N mice were purchased from Charles River Inc. (Sulzfeld, Germany). Animals were exposed to cycles of 12 h light/12 h darkness and received standard food and water ad libitum. Upon approval by the local animal welfare committee, experiments were performed in accordance with the German legislation on protection of animals and the

Bacteria (*S. pyogenes* serotype M49 strain 591; the ΔsagA mutant isogenic to strain 591 is a kind gift from Victor Nizet: A detailed description of how this mutant was generated can be found in (Datta et al., 2005)) were cultured in Todd-Hewitt (TH) broth or on TH agar (Oxoid Unipath, Wesel, Germany), both supplemented with 0.5 % yeast extract (THY) overnight to mid-log phase. Concentration was adjusted on the basis of an optical density reading at 600 nm and on plating analysis. Heat-inactivation of bacteria was obtained by one-hour incubation in a water bath at 75 °C. Inactivation was confirmed by plating samples on sheep blood agar followed by over night incubation at 37 °C and 5 % CO2. The purified toxin SLS (originally obtained from Sigma Aldrich, Munich, Germany) was dissolved in sterile PBS

Hemolytic activity of SLS was determined by hemoglobin release from whole blood cells after 4 and 24 h incubation with bacteria or their related products. Briefly, whole blood of healthy donors was seeded in 96-well plates and supplemented with microbia. Following

and supplemented with complete cell culture medium before treatment of cells.

antitumoral activity.

*2.* **Material & methods 2.1 Cell culture & mice** 

''Guide for the Care and Use of Laboratory Animals''.

**2.2 Bacteria, toxin and culture conditions** 

**2.3 Hemolysis assay for SLS activity** 

Toxicity of bacteria towards tumor cells was quantified using a cytotoxicity detection kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. Quantification was performed by subsequent measurement on a plate reader at 492 nm.

For detection of apoptosis, activity of Caspase 3 was analyzed using the BD ApoAlert Caspase Assay plate system according to the manufacturer's instructions (BD Biosciences, Heidelberg, Germany). Cells were trypsinized and subsequently lysed. Quantification of Caspase 3 activity was performed by fluorometric detection on a Cytofluor 2300 (Millipore, Schwalbach, Germany, ex/em: 380/460 nm).

#### **2.5 Pancreatic tumor model & treatment regimen**

Under brief ether anaesthesia 1\*106 Panc02 were injected subcutaneously (s.c.) into the right hind leg. Tumor growth was routinely controlled at least twice a week and tumor volume was estimated according to the formula: V= width² \* length \* 0.52. After tumor establishment animals were subdivided into experimental groups. All treatments were performed by local, intratumoral application of bacterial preparations each dissolved in 50 µl of phosphate-buffered saline (PBS) according to the following treatment regimen: One group was given heat-inactivated *S. pyogenes* (8 x 107 cfu, four injections once a week, n=6). The second group received a single injection of the SLS-mu (ΔsagA). For control, mice were administered equivalent volumes of solvent alone (vehicle-treated controls, saline, n=6) or left without treatment (control, n=6). Tumor carrying mice (treatment, control) were sacrificed at day 28 or when they became moribund before the tumor volume reached 2000 mm³. At the end of each experiment, tumors as well as spleens, mesenteric lymph nodes, and blood samples were removed from selected animals for further analysis. For visualization of tumor-infiltrating leukocytes, frozen sections of resected tumor tissues (6 µm) were stained by the As-D chloroacetate esterase (As-D) technique. Numbers of leukocytes/mm² were determined in blinded counts by positive staining and morphology in 20 consecutive high power fields (HPF).

#### **2.6 Phenotyping of lymphocyte subpopulations by flow cytometry**

Leukocytes from treated and non-treated animals were labeled using the following FITCconjugated rat anti-mouse monoclonal antibodies (mAbs): CD3, CD19, NK1.1 (1 µg, Immunotools, Friesoythe, Germany), and Phycoerythrin (PE)-conjugated rat anti-mouse mAbs: CD4, CD8 (Miltenyi Biotec, Bergisch Gladbach, Germany). Afterwards, erythrocytes were lysed using FACS Lysing Solution (BD Pharmingen, Heidelberg, Germany). Negative controls consisted of blood lymphocytes stained with the appropriate isotypes (BD Pharmingen). Samples were analyzed on a FACSCalibur Cytometer (BD Biosciences). Data analysis was performed using CellQuest software (BD Biosciences) and gating on total leukocytes (Mounting View, BD Biosciences). Relative numbers are given.

Bacterial Immunotherapy-Antitumoral Potential of the Streptococcal Toxin Streptolysin S- 167

Fig. 1. Lytic activity of different bacterial preparations towards erythrocytes in whole blood

In order to elucidate, if SLS also directly damages tumor cells, we applied the purified toxin in LDH release experiments. In particular, Panc02 tumor cells were treated with three increasing concentrations (25, 50, and 100 U/ml) for six hours. These analyses revealed a dose-dependent increase in cell damage with a maximum of up to 65% (100 U/ml). To a minor part, these effects could be attributed to the induction of apoptosis as the activity of the effector caspase 3

In line with these findings, a ΔsagA strain of *S. pyogenes* mediated no significant growth inhibition or killing activity at a multiplicity of infection of 25 for four and six hours (Figure

To further clarify the antitumoral potential of the toxin SLS a series of *in vivo* experiments using two different preparations of inactivated SLS was performed (Figure 3a). Panc02 tumors established subcutaneously in C57Bl/6 mice were infected with the ΔsagA strain (107 cfu). The intratumoral administration of ΔsagA did not affect pancreatic carcinoma growth within the first three weeks. Palpable tumors continued to grow and reached an average size of 723.8 ± 95.8 mm3, which was comparable to tumor sizes of control animals (saline: 841.4 ± 96.3 mm3). Thereafter, tumors in the infection group became frequently ulcerous and necrotized. This finally resulted in about 40 % reduced tumor volumes compared to saline-treated animals (day

**3.2 SLS-deficient preparations of S. pyogenes have minor antitumoral potential** 

samples. Following treatment with increasing concentrations of bacteria, cell-free supernatants were harvested and absorption was measured on a plate reader at 340 nm. Lytic activity was quantified by setting values of untreated cells as 1, and all other data were given as x-fold increase. Results show data from at least three separate experiments each performed with two healthy volunteer's blood samples. Values are given as the mean±SEM.

slightly increased after treatment with the 100 U/ml doses of SLS (Figure 2).

2). Also, activity of Caspase 3 was not altered post infection.

28: 689.2 ± 119.8 mm3 vs. saline: 1228.1 ± 220.2 mm3, p < 0.05).

#### **2.7 ELISPOT assay for Interferon-γ–secreting lymphocytes**

IFN-γ–specific, mAb (Mabtech, Hamburg, Germany) –coated, 96-well microtiter plates were filled with 1 x 104 target cells/well (Panc02, EL4, CMT-93, MC3T3-E1, and peripheral blood mononuclear cells (PBMC)) and incubated for 2 h. Splenocytes (105) were given to the targets and co-cultured overnight. Finally, bound antibody was visualized by BCIP/NBT (KPL, Gaithersburg, Maryland, USA), and spots were counted using a dissection microscope (Zeiss, Oberkochen, Germany). Presented are the numbers of IFN-γ–secreting cells per 105 effector cells corrected for background levels counted in the absence of target cells, which usually was between 10–50 spots/105 cells. Target cells without effector cells showed no background level.

#### **2.8 LDH cytotoxicity assay**

The colorimetric CytoTox-One Homogeneous Membrane Integrity Assay (Promega, Madison, WI) was used evaluating lactate dehydrogenase (LDH) release from lysed cells. Experiments were performed as described before (Maletzki et al., 2008).

#### **2.9 Bio-plex protein array system**

A panel of serum cytokines was measured in duplicate using the Bio-Plex Protein Array system (BioRad, Munich, Germany), according to the manufacturer's instructions. With the Bio-Plex cytokine assay kit in combination with the Bio-Plex Manager Software, serum IFNγ, TNF-α, IL-6, IL-10, GM-CSF (granulocyte macrophage colony stimulating factor), and G-CSF (granulocyte-colony stimulating factor) levels were assessed. Values of the respective serum cytokine levels of untreated control mice were set as 1, and all other data were given as x-fold increase.

#### **2.10 Statistical analysis**

All values are expressed as mean ± SEM. After proving the assumption of normality, differences between saline and treated animals were determined by using the unpaired Student's *t*-test. If normality failed, the nonparametric Mann-Whitney *U*-Test was applied. Similarly, differences between treated and non-treated cell *in vitro* were calculated by using the nonparametric Mann-Whitney *U*-Test. Data were recruited from experiments which had been done in triplicates and replicated at least three times. The tests were performed by using Sigma-Stat 3.0 (Jandel Corp, San Rafael, CA). The criterion for significance was set to p < 0.05.

#### **3. Results**

#### **3.1 In vitro analysis**

First, activity of SLS from different bacterial preparations was analyzed in a simple hemoglobin release assay (Figure 1). As expected, the purified toxin mediated substantial lysis of erythrocytes within a few hours. In contrast, lysis was completely absent post infection with the ΔsagA strain or the heat-inactivated form of the M49wt, thereby confirming inactivation of the toxin. As a control, vital M49 bacteria and the lysate, used in our previous studies (Maletzki et al., 2008), were employed. Both preparations mediated nearly complete lysis of erythrocytes.

IFN-γ–specific, mAb (Mabtech, Hamburg, Germany) –coated, 96-well microtiter plates were filled with 1 x 104 target cells/well (Panc02, EL4, CMT-93, MC3T3-E1, and peripheral blood mononuclear cells (PBMC)) and incubated for 2 h. Splenocytes (105) were given to the targets and co-cultured overnight. Finally, bound antibody was visualized by BCIP/NBT (KPL, Gaithersburg, Maryland, USA), and spots were counted using a dissection microscope (Zeiss, Oberkochen, Germany). Presented are the numbers of IFN-γ–secreting cells per 105 effector cells corrected for background levels counted in the absence of target cells, which usually was between 10–50 spots/105 cells. Target cells without effector cells showed no

The colorimetric CytoTox-One Homogeneous Membrane Integrity Assay (Promega, Madison, WI) was used evaluating lactate dehydrogenase (LDH) release from lysed cells.

A panel of serum cytokines was measured in duplicate using the Bio-Plex Protein Array system (BioRad, Munich, Germany), according to the manufacturer's instructions. With the Bio-Plex cytokine assay kit in combination with the Bio-Plex Manager Software, serum IFNγ, TNF-α, IL-6, IL-10, GM-CSF (granulocyte macrophage colony stimulating factor), and G-CSF (granulocyte-colony stimulating factor) levels were assessed. Values of the respective serum cytokine levels of untreated control mice were set as 1, and all other data were given

All values are expressed as mean ± SEM. After proving the assumption of normality, differences between saline and treated animals were determined by using the unpaired Student's *t*-test. If normality failed, the nonparametric Mann-Whitney *U*-Test was applied. Similarly, differences between treated and non-treated cell *in vitro* were calculated by using the nonparametric Mann-Whitney *U*-Test. Data were recruited from experiments which had been done in triplicates and replicated at least three times. The tests were performed by using Sigma-Stat 3.0 (Jandel Corp, San Rafael, CA). The criterion for significance was set to p < 0.05.

First, activity of SLS from different bacterial preparations was analyzed in a simple hemoglobin release assay (Figure 1). As expected, the purified toxin mediated substantial lysis of erythrocytes within a few hours. In contrast, lysis was completely absent post infection with the ΔsagA strain or the heat-inactivated form of the M49wt, thereby confirming inactivation of the toxin. As a control, vital M49 bacteria and the lysate, used in our previous studies (Maletzki et al., 2008), were employed. Both preparations mediated

Experiments were performed as described before (Maletzki et al., 2008).

**2.7 ELISPOT assay for Interferon-γ–secreting lymphocytes** 

background level.

as x-fold increase.

**3. Results** 

**3.1 In vitro analysis** 

nearly complete lysis of erythrocytes.

**2.10 Statistical analysis** 

**2.8 LDH cytotoxicity assay** 

**2.9 Bio-plex protein array system** 

Fig. 1. Lytic activity of different bacterial preparations towards erythrocytes in whole blood samples. Following treatment with increasing concentrations of bacteria, cell-free supernatants were harvested and absorption was measured on a plate reader at 340 nm. Lytic activity was quantified by setting values of untreated cells as 1, and all other data were given as x-fold increase. Results show data from at least three separate experiments each performed with two healthy volunteer's blood samples. Values are given as the mean±SEM.

In order to elucidate, if SLS also directly damages tumor cells, we applied the purified toxin in LDH release experiments. In particular, Panc02 tumor cells were treated with three increasing concentrations (25, 50, and 100 U/ml) for six hours. These analyses revealed a dose-dependent increase in cell damage with a maximum of up to 65% (100 U/ml). To a minor part, these effects could be attributed to the induction of apoptosis as the activity of the effector caspase 3 slightly increased after treatment with the 100 U/ml doses of SLS (Figure 2).

In line with these findings, a ΔsagA strain of *S. pyogenes* mediated no significant growth inhibition or killing activity at a multiplicity of infection of 25 for four and six hours (Figure 2). Also, activity of Caspase 3 was not altered post infection.

#### **3.2 SLS-deficient preparations of S. pyogenes have minor antitumoral potential**

To further clarify the antitumoral potential of the toxin SLS a series of *in vivo* experiments using two different preparations of inactivated SLS was performed (Figure 3a). Panc02 tumors established subcutaneously in C57Bl/6 mice were infected with the ΔsagA strain (107 cfu). The intratumoral administration of ΔsagA did not affect pancreatic carcinoma growth within the first three weeks. Palpable tumors continued to grow and reached an average size of 723.8 ± 95.8 mm3, which was comparable to tumor sizes of control animals (saline: 841.4 ± 96.3 mm3). Thereafter, tumors in the infection group became frequently ulcerous and necrotized. This finally resulted in about 40 % reduced tumor volumes compared to saline-treated animals (day 28: 689.2 ± 119.8 mm3 vs. saline: 1228.1 ± 220.2 mm3, p < 0.05).

Bacterial Immunotherapy-Antitumoral Potential of the Streptococcal Toxin Streptolysin S- 169

when comparing to controls, tumors showed a trend towards growth retardation, suggesting only reduced but not completely abolished antitumoral potential of the heatinactivated bacteria. Hence, the final tumor volume at day 28 was significantly lower than in

Animals were sacrificed at day 28 post start of therapy. Infection with the ΔsagA strain was well tolerated by most animals, with only one animal displaying signs of systemic bacterial infection (i.e. weight loss, ataxia). Because of the severity of infection, this animal was euthanized and related data excluded. As expected, the heat-inactivated bacterial

**Tumor volume [mm³]**

Fig. 3. **Data of** *in vivo* **analysis of Panc02-tumor carrying C57Bl/6N mice.** (A) Tumor growth kinetics and (B) survival curve after i.t. application of bacteria. Treatment regimens comprised the vital SLS-deficient ΔsagA strain (1 x 107 cfu,), which is isogenic to the M49 wildtype and heat-inactivated preparations of the M49 wildtype (8 x 107 cfu, four injections once a week, n=6). Control mice were administered equivalent volumes of solvent alone (saline, n=6) or left without treatment (tumor, n=6). Animals were sacrificed at day 28 or when they became moribund before the tumor volume reached 2000 mm³. Tumor growth was only at later stages affected and when comparing with our previous studies on using vital as well as lysed wild type bacteria, to a lesser extend (graphical presentation adopted from Linnebacher et al., 2008

To further validate the potential of SLS to influence tumor growth, we analyzed systemic parameters of animals treated with ΔsagA in more detail. Analysis of blood samples from ΔsagA infected animals revealed no alteration regarding the number of circulating leukocytes. However, thrombocyte as well as hematocrit levels were found to be decreased post infection (Figure 4a). Similarly, activities of the plasma enzymes ASAT and LDH were reduced, while levels of ALAT were not affected by bacteria (Figure 4b). In this case, the infection-mediated decrease in blood LDH activities might correlate with the retardation of

and Maletzki et al., 2008). \*p<0.05 vs. saline, \*\*p<0.001 vs. saline *U*-Test.

**3.4 Gross findings & hematological alterations post ∆sagA infection** 

A

**Saline M49wt vital M49wt lysate M49wt HI sagA**

**start of therapy**

**Time [days] 0 10 20 30**

\*

\*\* \*\* \*\* \*\*

saline-treated mice (693.2 ± 63.0 mm3 vs. 1154.5 ± 277.1 mm3, p < 0.05).

preparation had no negative impact on survival (Figure 3b).

**Time [days] 0 10 20 30**

**Saline M49wt vital M49wt lysate M49wt HI DsagA**

**∆sag A**

tumor growth observed *in vivo*.

**3.3 Survival** 

**Survival [%]**

B

**80**

**100**

**0**

Fig. 2. *In vitro* **analysis on direct effects of SLS on tumor cells.** (A) Quantitative analysis of SLS cytotoxicity towards Panc02 cells as assessed by either LDH release or caspase activity following a 6 h incubation period. These analyses revealed a dose-dependent increase in cell damage. (B) Quantitative analysis of the ΔsagA effects on Panc02 tumor cells. Infection with the ΔsagA strain mediated no significant cytotoxicity, detected by LDH release. Results show data from three separate experiments. Values are given as the mean±SEM. \*p<0.05 vs. control, *U*-Test.

To validate these findings, tumor-carrying animals were repeatedly treated with a bacterial preparation which was preheated to inactivate SLS. Similar to what has been observed after i.t. infection with the ΔsagA strain, injection of heat-inactivated streptococci mediated no significant alteration of Panc02 tumor growth until day 14 post start of therapy. However, when comparing to controls, tumors showed a trend towards growth retardation, suggesting only reduced but not completely abolished antitumoral potential of the heatinactivated bacteria. Hence, the final tumor volume at day 28 was significantly lower than in saline-treated mice (693.2 ± 63.0 mm3 vs. 1154.5 ± 277.1 mm3, p < 0.05).

#### **3.3 Survival**

168 Pancreatic Cancer – Clinical Management

Fig. 2. *In vitro* **analysis on direct effects of SLS on tumor cells.** (A) Quantitative analysis of SLS cytotoxicity towards Panc02 cells as assessed by either LDH release or caspase activity following a 6 h incubation period. These analyses revealed a dose-dependent increase in cell damage. (B) Quantitative analysis of the ΔsagA effects on Panc02 tumor cells. Infection with the ΔsagA strain mediated no significant cytotoxicity, detected by LDH release. Results show data from three separate experiments. Values are given as the mean±SEM. \*p<0.05 vs.

To validate these findings, tumor-carrying animals were repeatedly treated with a bacterial preparation which was preheated to inactivate SLS. Similar to what has been observed after i.t. infection with the ΔsagA strain, injection of heat-inactivated streptococci mediated no significant alteration of Panc02 tumor growth until day 14 post start of therapy. However,

control, *U*-Test.

Animals were sacrificed at day 28 post start of therapy. Infection with the ΔsagA strain was well tolerated by most animals, with only one animal displaying signs of systemic bacterial infection (i.e. weight loss, ataxia). Because of the severity of infection, this animal was euthanized and related data excluded. As expected, the heat-inactivated bacterial preparation had no negative impact on survival (Figure 3b).

Fig. 3. **Data of** *in vivo* **analysis of Panc02-tumor carrying C57Bl/6N mice.** (A) Tumor growth kinetics and (B) survival curve after i.t. application of bacteria. Treatment regimens comprised the vital SLS-deficient ΔsagA strain (1 x 107 cfu,), which is isogenic to the M49 wildtype and heat-inactivated preparations of the M49 wildtype (8 x 107 cfu, four injections once a week, n=6). Control mice were administered equivalent volumes of solvent alone (saline, n=6) or left without treatment (tumor, n=6). Animals were sacrificed at day 28 or when they became moribund before the tumor volume reached 2000 mm³. Tumor growth was only at later stages affected and when comparing with our previous studies on using vital as well as lysed wild type bacteria, to a lesser extend (graphical presentation adopted from Linnebacher et al., 2008 and Maletzki et al., 2008). \*p<0.05 vs. saline, \*\*p<0.001 vs. saline *U*-Test.

#### **3.4 Gross findings & hematological alterations post ∆sagA infection**

To further validate the potential of SLS to influence tumor growth, we analyzed systemic parameters of animals treated with ΔsagA in more detail. Analysis of blood samples from ΔsagA infected animals revealed no alteration regarding the number of circulating leukocytes. However, thrombocyte as well as hematocrit levels were found to be decreased post infection (Figure 4a). Similarly, activities of the plasma enzymes ASAT and LDH were reduced, while levels of ALAT were not affected by bacteria (Figure 4b). In this case, the infection-mediated decrease in blood LDH activities might correlate with the retardation of tumor growth observed *in vivo*.

Bacterial Immunotherapy-Antitumoral Potential of the Streptococcal Toxin Streptolysin S- 171

Next, flow cytometric phenotyping of circulating leukocytes was performed. As shown in Table 1, we observed higher numbers of circulating NK cells in animals treated with the ΔsagA strain (34.6 ± 7.1% vs. saline 16.5 ± 2.7%). Similarly, levels of Gr1+ granulocytes were also raised (32.5 ± 4.6% vs. saline 25.0 ± 3.0%). Regarding the number of T cells, no significant differences were obtained between the infection and control groups. Likewise, numbers of circulating CD19+ pre B cells showed no alteration after therapy with values remaining similar to controls. Thus, in our experimental system, microbial therapy with SLS-deficient or heat-inactivated *S. pyogenes* preparations seemed to affect exclusively the

Control Tumor Saline ΔsagA

**CD3+CD4+** 19.5 ± 1.9 15.7 ± 1.4 8.6 ± 1.4 14.1 ± 0.9

**CD3+CD8+** 10.9 ± 0.7 9.5 ± 1.3 6.9 ± 1.7 10.3 ± 0.5

**CD19+** 56.5 ± 1.9 42.2 ± 4.1 31.4 ± 2.5 41.6 ± 4.2

**NK1.1+** 14.8 ± 2.6 15.8 ± 1.8 16.5 ± 2.7 34.6 ± 7.1

**Gr1+** 9.5 ± 2.4 19.8 ± 3.1 25.0 ± 3.0 32.5 ± 4.6

Values are given as mean ± SEM. ΔsagA-infected animals (n=7); saline-treated animals (n=6); tumor

Table 1. Flow cytometric phenotyping of whole blood in control groups (control, tumor,

Despite raised levels of infiltrating granulocytes, especially in the boundary areas of treated tumors (Figure 5a), we did not observe any significant difference in the number of CD4+ and CD8+ lymphocytes infiltrating tumors between control and treatment groups (data not

In a first series of experiments, reactivity against syngeneic tumor cell lines was tested in ELISpot assays using lymph node derived lymphocytes as effector cells from control as well as ΔsagA-treated animals. Surprisingly, recognition of tumor cells was restricted to the syngeneic colorectal carcinoma cell line CMT-93. Other tested cells (i.e. Panc02, EL4) did not

In a more functional cytotoxicity assay, splenocytes were used as effectors. Again, Panc02 tumor cells were only ineffectively lysed by immune cells from infected mice (22.1 ± 2.5 % vs. saline 6.7 ± 1.7 %). Similar results were obtained with the non-cancerous MC3T3-E1 fibroblasts and with PBMCs (Figure 5c), indicating only little specific killing activity of effector cells. In contrast, we again observed most lytic activity against the syngeneic tumor cell line CMT-93 (35.8 ± 12.7 % vs. saline: 22.2% ± 7.5 %) and additionally against EL4 (38.6 ±

induce release of IFN-γ from lymphocytes of ΔsagA treated animals (Figure 5b).

saline) and post infection with the ΔsagA strain (% positive cells).

**3.6 Analysis of antitumoral immune responses** 

**3.5 Flow cytometric phenotyping of circulating leukocytes** 

innate arm of the immune system.

control animals (n=7); *U*-Test.

11.4 % vs. saline: 18.2% ± 3.0 %).

shown).

As these data indicated an inflammatory reaction in the ΔsagA treated animals, we subsequently determined plasma cytokine levels from infected and non-infected animals (Figure 4c). Analysis of the neutrophile chemotaxis polypeptide G-CSF and the GM-CSF showed slight increases post infection which were, however not statistically significant. Levels of both cytokines displayed 2-fold rises post infection (p=0.256 vs. saline (G-CSF) and p= 0.007 vs. saline (GM-CSF), *t*-test). A similar pattern was seen for IL6 (p=0.62), while the Th1 cytokines TNF-α and IFN-γ showed no alterations at all.

Fig. 4. **Assessment of systemic blood parameters.** (A) Numbers of leukocytes, thrombocytes, and hematocrit levels as well as (B) plasma enzyme activities of ALAT, ASAT and LDH from ΔsagA-infected and control animals at day 28. (C) Levels of Th1 and Th2 cytokines in serum of treated and untreated mice. Mice received a single i.t. infection of the ΔsagA strain (1x 107 cfu). Control animals received equivalent volumes of PBS (saline) or no injection (tumor). Values are given as the mean±SEM. *U*-Test.

**3.5 Flow cytometric phenotyping of circulating leukocytes** 

170 Pancreatic Cancer – Clinical Management

As these data indicated an inflammatory reaction in the ΔsagA treated animals, we subsequently determined plasma cytokine levels from infected and non-infected animals (Figure 4c). Analysis of the neutrophile chemotaxis polypeptide G-CSF and the GM-CSF showed slight increases post infection which were, however not statistically significant. Levels of both cytokines displayed 2-fold rises post infection (p=0.256 vs. saline (G-CSF) and p= 0.007 vs. saline (GM-CSF), *t*-test). A similar pattern was seen for IL6 (p=0.62), while the

Th1 cytokines TNF-α and IFN-γ showed no alterations at all.

**thrombocytes [x109/l]**

**control**

**cytokine level [x-fold increase vs. saline]**

**0**

C

**2**

**4**

**6**

**leukocytes [x109/l]**

A

**tumor**

**G-CSF**

**0**

**1**

**2**

**3**

**4**

**5**

**GM-CSF**

**TNF-a**

**TNF-**

**IFN-g**

injection (tumor). Values are given as the mean±SEM. *U*-Test.

**IFN-**

**IL6**

**IL10**

Fig. 4. **Assessment of systemic blood parameters.** (A) Numbers of leukocytes,

thrombocytes, and hematocrit levels as well as (B) plasma enzyme activities of ALAT, ASAT and LDH from ΔsagA-infected and control animals at day 28. (C) Levels of Th1 and Th2 cytokines in serum of treated and untreated mice. Mice received a single i.t. infection of the ΔsagA strain (1x 107 cfu). Control animals received equivalent volumes of PBS (saline) or no

**saline**

**1**

**control**

**0**

**200**

**400**

**600**

**800**

**tumor**

**saline**

B

**plasma enzymes [U/l]**

**1**

**control**

**0**

**200**

**600**

**800**

**tumor**

**saline**

**1**

**sagA**

**control**

**0**

**10**

**20**

**30**

**40**

**50**

**hematocrit [%]**

**tumor**

**saline**

**1**

**sagA**

**sagA**

**sagA**

Next, flow cytometric phenotyping of circulating leukocytes was performed. As shown in Table 1, we observed higher numbers of circulating NK cells in animals treated with the ΔsagA strain (34.6 ± 7.1% vs. saline 16.5 ± 2.7%). Similarly, levels of Gr1+ granulocytes were also raised (32.5 ± 4.6% vs. saline 25.0 ± 3.0%). Regarding the number of T cells, no significant differences were obtained between the infection and control groups. Likewise, numbers of circulating CD19+ pre B cells showed no alteration after therapy with values remaining similar to controls. Thus, in our experimental system, microbial therapy with SLS-deficient or heat-inactivated *S. pyogenes* preparations seemed to affect exclusively the innate arm of the immune system.


Values are given as mean ± SEM. ΔsagA-infected animals (n=7); saline-treated animals (n=6); tumor control animals (n=7); *U*-Test.

Table 1. Flow cytometric phenotyping of whole blood in control groups (control, tumor, saline) and post infection with the ΔsagA strain (% positive cells).

#### **3.6 Analysis of antitumoral immune responses**

Despite raised levels of infiltrating granulocytes, especially in the boundary areas of treated tumors (Figure 5a), we did not observe any significant difference in the number of CD4+ and CD8+ lymphocytes infiltrating tumors between control and treatment groups (data not shown).

In a first series of experiments, reactivity against syngeneic tumor cell lines was tested in ELISpot assays using lymph node derived lymphocytes as effector cells from control as well as ΔsagA-treated animals. Surprisingly, recognition of tumor cells was restricted to the syngeneic colorectal carcinoma cell line CMT-93. Other tested cells (i.e. Panc02, EL4) did not induce release of IFN-γ from lymphocytes of ΔsagA treated animals (Figure 5b).

In a more functional cytotoxicity assay, splenocytes were used as effectors. Again, Panc02 tumor cells were only ineffectively lysed by immune cells from infected mice (22.1 ± 2.5 % vs. saline 6.7 ± 1.7 %). Similar results were obtained with the non-cancerous MC3T3-E1 fibroblasts and with PBMCs (Figure 5c), indicating only little specific killing activity of effector cells. In contrast, we again observed most lytic activity against the syngeneic tumor cell line CMT-93 (35.8 ± 12.7 % vs. saline: 22.2% ± 7.5 %) and additionally against EL4 (38.6 ± 11.4 % vs. saline: 18.2% ± 3.0 %).

Bacterial Immunotherapy-Antitumoral Potential of the Streptococcal Toxin Streptolysin S- 173

Different observations indicate that exposure by vaccination or infection to pathogenassociated molecular patterns (PAMP) can have beneficial effects on neoplastic diseases (Hobohm et al., 2008). PAMP recognizing TLRs serve as a first line of defense for the immune system, inducing soluble and cellular mediators of innate immunity and initiating key steps of the adaptive immune response. In recent years, manipulating the immune response via TLR stimulation has gained therapeutic and/or prophylactic value for cancer. In particular, administration of the synthetic TLR9 agonist CpG-ODN is being developed for cancer vaccines and cancer therapy, due to its described capacity to stimulate Th1-like innate and adaptive anti-tumor responses in numerous preclinical models (Krieg 2007; Jacobs et al., 2010). Another potent inducer of tumor-directed cellular immune responses is the TLR7 agonist imiquimod. In recent years, several studies proved enhancement of antigen-specific T cell activation followed by tumor eradication (Rechtsteiner et al., 2005; Prins et al., 2006; Xiong & Ohlfest, 2011). Very recently, we provided evidence for tumor growth control by avitalized grampositive bacteria. In these experiments, therapeutic application of avitalized bacteria effectively delayed tumor growth accompanied by increased numbers of tumor-infiltrating immune cells

In line with this, we previously observed that treatment with *S. pyogenes* leads to pancreatic tumor reduction or even cure by the orchestrated induction of innate and subsequent adaptive antitumoral immune responses (Linnebacher et al., 2008; Maletzki et al., 2008). However, little is known about the nature of active components responsible for this success. We hypothesized, that bacterial toxins are the most obvious candidate molecules to explain the antitumoral activity of *S. pyogenes*. Here, we explored the potential of the streptococcalelaborated oxygen-stable cytolysin Streptolysin S. In agreement with previous data referring to the broad cytolytic spectrum of SLS, we were able to confirm that it also efficiently kills tumor cells and in particular Panc02 mouse pancreatic carcinoma cells. To a minor part, these effects could be attributed to the induction of caspase-dependent apoptosis. In a first series of *in vivo* experiments, the SLS-deficient mutant *S. pyogenes* strain ΔsagA showed only impaired antitumoral activity. This strain has been described to exhibit strongly reduced epithelial cell killing compared with SLS-producing wildtype strains (Datta et al., 2005; Lin et al. 2009). Moreover, minimal evidence of necrosis and tissue injury is seen post infection with SLS-negative bacteria in murine models of skin lesions (Datta et al., 2005). Similarly, local infection with the ΔsagA strain exhibited antitumoral potential only at later timepoints post infection in our syngeneic murine Panc02 tumor model. This therapeutic success was significantly weaker than what we observed in our earlier studies using vital as well as lysed preparations of *S. pyogenes*. The hypothesis that SLS is a major antitumoral-acting molecule could be further substantiated in a second series of *in vivo* experiments. The application of streptococci depleted of active SLS by heating also resulted only in reduced

However, despite incomplete eradication of tumors, we want to strengthen the fact, that both preparations led to therapeutic responses as detected by significant reduction in tumor volumes. Thus, other factors are likely to contribute to the antitumoral effects of living and lysed *S. pyogenes* (e.g. M-Protein, superantigens, lipoteichoic acid) (Chau et al., 2009). Moreover, these findings hint towards a significant contribution of the immune system in

mainly belonging to the innate arm of the immune system (Klier et al., 2011).

antitumoral activity even after repetitive local treatments.

partial control of tumor growth.

**4. Discussion** 

Fig. 5. **Data of immune responses from Panc02 tumor-carrying mice.** (A) Quantitative analysis of tumor-infiltrating As-D chloroacetate esterase-positive leukocytes shown as number of positive cells per mm2. Leukocytes were found especially in boundary areas, and minor in the center of treated tumors. (B) Quantitative analysis of IFN-γ ELISpot assay. Reactivity of splenocytes against tumor targets was tested after co-incubation overnight at an E:T cell ratio of 30:1. Splenocytes from infected animals at 28 days after i.t. bacterial infection showed a marginally higher reactivity against target cells than those from control animals. (C) Quantitative analysis of cytotoxicity using LDH release assay. Lymphocytes were isolated from mesenteric lymph nodes and co-cultured with targets for 24 h at an E:T ratio of 30:1. Lymphocytes from infected animals at 28 days after i.t. bacterial infection lysed syngeneic tumor cell lines EL4 and CMT-93, but only to a minor extend Panc02 cells. Experiments were performed in triplicates. Values are given as the mean±SEM. \*p<0.05 vs. saline, *U*-Test.

#### **4. Discussion**

172 Pancreatic Cancer – Clinical Management

Fig. 5. **Data of immune responses from Panc02 tumor-carrying mice.** (A) Quantitative analysis of tumor-infiltrating As-D chloroacetate esterase-positive leukocytes shown as number of positive cells per mm2. Leukocytes were found especially in boundary areas, and minor in the center of treated tumors. (B) Quantitative analysis of IFN-γ ELISpot assay. Reactivity of splenocytes against tumor targets was tested after co-incubation overnight at an E:T cell ratio of 30:1. Splenocytes from infected animals at 28 days after i.t. bacterial infection showed a marginally higher reactivity against target cells than those from control animals. (C) Quantitative analysis of cytotoxicity using LDH release assay. Lymphocytes were isolated from mesenteric lymph nodes and co-cultured with targets for 24 h at an E:T ratio of 30:1. Lymphocytes from infected animals at 28 days after i.t. bacterial infection lysed

syngeneic tumor cell lines EL4 and CMT-93, but only to a minor extend Panc02 cells. Experiments were performed in triplicates. Values are given as the mean±SEM. \*p<0.05 vs.

saline, *U*-Test.

Different observations indicate that exposure by vaccination or infection to pathogenassociated molecular patterns (PAMP) can have beneficial effects on neoplastic diseases (Hobohm et al., 2008). PAMP recognizing TLRs serve as a first line of defense for the immune system, inducing soluble and cellular mediators of innate immunity and initiating key steps of the adaptive immune response. In recent years, manipulating the immune response via TLR stimulation has gained therapeutic and/or prophylactic value for cancer. In particular, administration of the synthetic TLR9 agonist CpG-ODN is being developed for cancer vaccines and cancer therapy, due to its described capacity to stimulate Th1-like innate and adaptive anti-tumor responses in numerous preclinical models (Krieg 2007; Jacobs et al., 2010). Another potent inducer of tumor-directed cellular immune responses is the TLR7 agonist imiquimod. In recent years, several studies proved enhancement of antigen-specific T cell activation followed by tumor eradication (Rechtsteiner et al., 2005; Prins et al., 2006; Xiong & Ohlfest, 2011). Very recently, we provided evidence for tumor growth control by avitalized grampositive bacteria. In these experiments, therapeutic application of avitalized bacteria effectively delayed tumor growth accompanied by increased numbers of tumor-infiltrating immune cells mainly belonging to the innate arm of the immune system (Klier et al., 2011).

In line with this, we previously observed that treatment with *S. pyogenes* leads to pancreatic tumor reduction or even cure by the orchestrated induction of innate and subsequent adaptive antitumoral immune responses (Linnebacher et al., 2008; Maletzki et al., 2008). However, little is known about the nature of active components responsible for this success. We hypothesized, that bacterial toxins are the most obvious candidate molecules to explain the antitumoral activity of *S. pyogenes*. Here, we explored the potential of the streptococcalelaborated oxygen-stable cytolysin Streptolysin S. In agreement with previous data referring to the broad cytolytic spectrum of SLS, we were able to confirm that it also efficiently kills tumor cells and in particular Panc02 mouse pancreatic carcinoma cells. To a minor part, these effects could be attributed to the induction of caspase-dependent apoptosis. In a first series of *in vivo* experiments, the SLS-deficient mutant *S. pyogenes* strain ΔsagA showed only impaired antitumoral activity. This strain has been described to exhibit strongly reduced epithelial cell killing compared with SLS-producing wildtype strains (Datta et al., 2005; Lin et al. 2009). Moreover, minimal evidence of necrosis and tissue injury is seen post infection with SLS-negative bacteria in murine models of skin lesions (Datta et al., 2005). Similarly, local infection with the ΔsagA strain exhibited antitumoral potential only at later timepoints post infection in our syngeneic murine Panc02 tumor model. This therapeutic success was significantly weaker than what we observed in our earlier studies using vital as well as lysed preparations of *S. pyogenes*. The hypothesis that SLS is a major antitumoral-acting molecule could be further substantiated in a second series of *in vivo* experiments. The application of streptococci depleted of active SLS by heating also resulted only in reduced antitumoral activity even after repetitive local treatments.

However, despite incomplete eradication of tumors, we want to strengthen the fact, that both preparations led to therapeutic responses as detected by significant reduction in tumor volumes. Thus, other factors are likely to contribute to the antitumoral effects of living and lysed *S. pyogenes* (e.g. M-Protein, superantigens, lipoteichoic acid) (Chau et al., 2009). Moreover, these findings hint towards a significant contribution of the immune system in partial control of tumor growth.

Bacterial Immunotherapy-Antitumoral Potential of the Streptococcal Toxin Streptolysin S- 175

Craft N, Bruhn KW, Nguyen BD, Prins R, Lin JW, Liau LM, Miller JF. 2005. The TLR7

Datta V, Myskowski SM, Kwinn LA, Chiem DN, Varki N, Kansal RG, Kotb M, Nizet V.

Fraser JD, Proft T. The bacterial superantigen and superantigen-like proteins. *Immunol Rev*.

Ginsburg I. 1999. Is streptolysin S of group A streptococci a virulence factor? *APMIS.* 107:

Hobohm U, Stanford JL, Grange JM. 2008.Pathogen-associated molecular pattern in cancer

Jacobs C, Duewell P, Heckelsmiller K, Wei J, Bauernfeind F, Ellermeier J, Kisser U, Bauer

Krieg AM. 2007.Development of TLR9 agonists for cancer therapy. *J Clin Invest.* 117: 1184-94.

Lin A, Loughman JA, Zinselmeyer BH, Miller MJ, Caparon MG. 2009.Streptolysin S inhibits

Linnebacher M, Maletzki C, Emmrich J, Kreikemeyer B. 2008.Lysates of S. pyogenes

Maletzki C, Linnebacher M, Kreikemeyer B, Emmrich J. 2008. Pancreatic cancer regression

Nizet V, Beall B, Bast DJ, Datta V, Kilburn L, Low DE, De Azavedo JC. 2008. Genetic

Prins RM, Craft N, Bruhn KW, Khan-Farooqi H, Koya RC, Stripecke R Miller JF, Liau LM.

Rechtsteiner G, Warger T, Osterloh P, Schild H, Radsak MP. 2005. Cutting edge: priming of

Taketo Y, Taketo A. 1966.Cytolytic effect of streptolysin S complex on Ehrlich ascites tumor

antitumor immune responses. *J Immunother*. 31: 704-13.

CA, Dauer M, Eigler A, Maraskovsky E, Endres S, Schnurr M. 2010. An ISCOM vaccine combined with a TLR9 agonist breaks immune evasion mediated by regulatory T cells in an orthotopic model of pancreatic carcinoma. Int J Cancer. 19.

neutrophil recruitment during the early stages of Streptococcus pyogenes infection.

serotype M49 induce pancreatic tumor growth delay by specific and unspecific

by intratumoural injection of live Streptococcus pyogenes in a syngeneic mouse

locus for streptolysin S production by group A streptococcus. *Infect Immun.* 68:

2006. TheTLR-7 agonist, imiquimod, enhances dendritic cell survival and promotes tumor antigen-specific T cell priming: relation to central nervous system antitumor

CTL by transcutaneous peptide immunization with imiquimod. *J Immunol*. 174:

activation and prevent toxic shock syndrome. *Nat Med*. 15: 641-8.

monocytogenes vaccine. *J Immunol*. 175: 1983–1990.

immunotherapy. *Crit Rev Immunol.* 28: 95-107.

2008 Oct; 225: 226-43. Review.

[Epub ahead of print]

*Infect Immun*. 77: 5190-201.

model. *Gut.* 57: 483-91.

immunity. *J Immunol.* 176: 157–164.

cells. *J Biochem*. 60: 357-362.

95.

1051-9.

Review.

4245-54.

2476–2480.

ligands on the staphylococcal cell wall downregulate superantigen-induced T cell

agonist imiquimod enhances the antimelanoma effects of a recombinant Listeria

2005. Mutational analysis of the group A streptococcal operon encoding streptolysin S and its virulence role in invasive infection. *Mol Microbiol.* 56: 681-

To understand the underlying immunological effects evoked by an inflammatory reaction in the ΔsagA treated animals, leading to reduced but not completely abolished antitumoral potential, *ex vivo* analyses were performed. Our observations imply an ongoing inflammatory response including systemic production of Th1 cytokines such as G-CSF and GM-CSF as well as raised levels of circulating NK cells and granulocytes. Functional analyses revealed recognition of syngeneic tumor cells as detected by IFN-γ release from lymphocytes obtained from infected animals. However, these effects were rather supposed to be mediated by activated NK cells than by tumor antigen specific T cells. This finding is additionally supported by the lymphocytes' killing activity especially towards the syngeneic tumor cell lines EL4 and CMT-93, but only marginally towards Panc02 cells.

To explain our findings, we propose that intratumoral injection of SLS-deficient streptococcal preparations leads to minor tumor cell damage driven by cytotoxic activity of other bacterial components. This triggers a comparably weak local inflammatory reaction followed by negligible systemic activation of the immune system. Thus, few specific antitumoral effector cells will be activated which can not totally control tumor growth whereas NK cells are the main effector type population mediating some degree of tumor growth control. The question, whether SLS may be an interesting molecule for tumor therapy will be addressed in ongoing trials.

#### **5. Conclusion**

Based on our previous findings on *Streptococcus pyogenes*-mediated eradication of established pancreatic murine tumors, we here elucidated the impact of the cytolytic toxin Streptolysin S on tumors *in vitro* and *in vivo*. We were able to show that direct exposure of the toxin to tumor cells results in a dose-dependent increase in cell damage. Contrary, the SLS-deficient ΔsagA strain showed only minor cytolytic potential. *In vivo*, a single i.t. injection of the ΔsagA strain affected pancreatic carcinomas only at later time points. This hints towards –at least partial- growth control of tumors by SLS, since comparable effects were observed following repetitive local applications of SLS-inactivated (by heating) bacteria. This finding is further supported by the histologic observation of ΔsagA-infected tumors. Those tumors showed slight increases in infiltrating granulocytes. Moreover, we found that recognition and killing of tumor cells was not restricted to Panc02 cells, but also detectable towards other syngeneic tumor entities. Taken together, we here provide clear evidence of strong antitumoral effects of SLS. However, in terms of the delayed, but significant impact on tumor growth *in vivo*, other factors are likely to contribute to the strong antitumoral effects of wildtype *S. pyogenes.*

#### **6. Acknowledgment**

We kindly thank Prof. Victor Nizet for providing the *S. pyogenes* strain ΔsagA.

#### **7. References**

Chau TA, McCully ML, Brintnell W, An G, Kasper KJ, Vinés ED, Kubes P, Haeryfar SM, McCormick JK, Cairns E, Heinrichs DE, Madrenas J. (2009). Toll-like receptor 2

To understand the underlying immunological effects evoked by an inflammatory reaction in the ΔsagA treated animals, leading to reduced but not completely abolished antitumoral potential, *ex vivo* analyses were performed. Our observations imply an ongoing inflammatory response including systemic production of Th1 cytokines such as G-CSF and GM-CSF as well as raised levels of circulating NK cells and granulocytes. Functional analyses revealed recognition of syngeneic tumor cells as detected by IFN-γ release from lymphocytes obtained from infected animals. However, these effects were rather supposed to be mediated by activated NK cells than by tumor antigen specific T cells. This finding is additionally supported by the lymphocytes' killing activity especially towards the syngeneic

To explain our findings, we propose that intratumoral injection of SLS-deficient streptococcal preparations leads to minor tumor cell damage driven by cytotoxic activity of other bacterial components. This triggers a comparably weak local inflammatory reaction followed by negligible systemic activation of the immune system. Thus, few specific antitumoral effector cells will be activated which can not totally control tumor growth whereas NK cells are the main effector type population mediating some degree of tumor growth control. The question, whether SLS may be an interesting molecule for tumor

Based on our previous findings on *Streptococcus pyogenes*-mediated eradication of established pancreatic murine tumors, we here elucidated the impact of the cytolytic toxin Streptolysin S on tumors *in vitro* and *in vivo*. We were able to show that direct exposure of the toxin to tumor cells results in a dose-dependent increase in cell damage. Contrary, the SLS-deficient ΔsagA strain showed only minor cytolytic potential. *In vivo*, a single i.t. injection of the ΔsagA strain affected pancreatic carcinomas only at later time points. This hints towards –at least partial- growth control of tumors by SLS, since comparable effects were observed following repetitive local applications of SLS-inactivated (by heating) bacteria. This finding is further supported by the histologic observation of ΔsagA-infected tumors. Those tumors showed slight increases in infiltrating granulocytes. Moreover, we found that recognition and killing of tumor cells was not restricted to Panc02 cells, but also detectable towards other syngeneic tumor entities. Taken together, we here provide clear evidence of strong antitumoral effects of SLS. However, in terms of the delayed, but significant impact on tumor growth *in vivo*, other factors are likely to contribute to the

tumor cell lines EL4 and CMT-93, but only marginally towards Panc02 cells.

therapy will be addressed in ongoing trials.

strong antitumoral effects of wildtype *S. pyogenes.*

We kindly thank Prof. Victor Nizet for providing the *S. pyogenes* strain ΔsagA.

Chau TA, McCully ML, Brintnell W, An G, Kasper KJ, Vinés ED, Kubes P, Haeryfar SM,

McCormick JK, Cairns E, Heinrichs DE, Madrenas J. (2009). Toll-like receptor 2

**5. Conclusion** 

**6. Acknowledgment** 

**7. References** 

ligands on the staphylococcal cell wall downregulate superantigen-induced T cell activation and prevent toxic shock syndrome. *Nat Med*. 15: 641-8.


**10**

*Italy* 

**Anesthesia and Pain Management:** 

Surgery for pancreatic cancer (PC) is widely viewed as a complex procedure associated with considerable perioperative morbidity and mortality. Many aspects of surgery for pancreatic cancer, such as the extent of resection, the value of vascular resection, the use of laparoscopy and the importance of treatment at high-volume centers are currently under debate. PC is the fourth leading cause of cancer related mortality in the United States with an estimated 42500 new cases and 35000 deaths from the disease each year (Jemal, 2009). Analysis of overall survival shows that the prognosis of PC is still quite poor despite the fact that 1-year survival has increased from 15.2% to 21.6% and 5-year survival has increased from 3% to 5% (ShaibYH et al., 2006). Surgery is the only chance of cure and the presence of negative resection margins of the primary tumor represent the strongest prognostic factor. Preoperative staging modalities include the combination of several imaging techniques such as computed tomography (CT scan), magnetic resonance imaging (MRI), endoscopic ultrasounds (EUS), staging laparoscopy and laparoscopic ultrasound which aim to identify patients with resectable disease. There is consensus that patients with distant metastases (liver, lung, peritoneum) or local invasion of the surrounding organs (stomach, colon, small bowel) are usually not surgical candidates. A decision analysis demonstrated that the best strategy to assess tumor resectability was based on CT as an initial test and the use of EUS to confirm the results of resectability by CT (Delbecke et al., 1999). Laparoscopic ultrasonography (LUS) has been introduced as an additional procedure to increase the detection of intrahepatic metastases, identify enlarged and suspicious lymph nodes and to evaluate local growth in the vascular structures (Tilleman et al., 2004). The routine use of staging laparoscopy and LUS in patients with radiographically resectable PC remains controversial as imaging modalities has significantly improved, thus reducing the risk of discovering non resectable disease at the time of surgery. Surgery for the PC can be considered an high-risk surgery. This term is rarely explicitly defined in scientific articles. There seems to be a common understanding among surgeons and anesthesiologists of what major surgery means. It can be defined as a surgical procedure that is extensive, involves removal of whole or parts of organs and/or is lifethreatening. It has also been defined as a surgical procedure with >1 mortality (Ghaferi et al., 2009). One possibility of evaluating the perioperative risk is the use of 1 of several risk scores. The American Society of Anesthesiologists score is widely used and easy to apply, but excludes age from its risk analysis (Kullavanijaya et al., 2001). Age is securely one of the most important, if not the single most predictive, risk factors for morbidity and mortality after major

surgery, including major pancreatic surgery (Riall et al., 2008).

**1. Introduction** 

**Techniques and Practice** 

Maurizio Marandola and Alida Albante *"Sapienza" University – Policlinico Umberto I, Rome,* 


### **Anesthesia and Pain Management: Techniques and Practice**

Maurizio Marandola and Alida Albante *"Sapienza" University – Policlinico Umberto I, Rome, Italy* 

#### **1. Introduction**

176 Pancreatic Cancer – Clinical Management

Xiong Z, Ohlfest JR. (2011) Topical imiquimod has therapeutic and immunomodulatory

Klier U, Maletzki C, Göttmann N, Kreikemeyer B, Linnebacher M. (2011) Avitalized bacteria

mediate tumor growth control via activation of innate immunity. *Cell Immunol*. 269:

effects against intracranial tumors. *J Immunother.* 34: 264-269.

120-127.

Surgery for pancreatic cancer (PC) is widely viewed as a complex procedure associated with considerable perioperative morbidity and mortality. Many aspects of surgery for pancreatic cancer, such as the extent of resection, the value of vascular resection, the use of laparoscopy and the importance of treatment at high-volume centers are currently under debate. PC is the fourth leading cause of cancer related mortality in the United States with an estimated 42500 new cases and 35000 deaths from the disease each year (Jemal, 2009). Analysis of overall survival shows that the prognosis of PC is still quite poor despite the fact that 1-year survival has increased from 15.2% to 21.6% and 5-year survival has increased from 3% to 5% (ShaibYH et al., 2006). Surgery is the only chance of cure and the presence of negative resection margins of the primary tumor represent the strongest prognostic factor. Preoperative staging modalities include the combination of several imaging techniques such as computed tomography (CT scan), magnetic resonance imaging (MRI), endoscopic ultrasounds (EUS), staging laparoscopy and laparoscopic ultrasound which aim to identify patients with resectable disease. There is consensus that patients with distant metastases (liver, lung, peritoneum) or local invasion of the surrounding organs (stomach, colon, small bowel) are usually not surgical candidates. A decision analysis demonstrated that the best strategy to assess tumor resectability was based on CT as an initial test and the use of EUS to confirm the results of resectability by CT (Delbecke et al., 1999). Laparoscopic ultrasonography (LUS) has been introduced as an additional procedure to increase the detection of intrahepatic metastases, identify enlarged and suspicious lymph nodes and to evaluate local growth in the vascular structures (Tilleman et al., 2004). The routine use of staging laparoscopy and LUS in patients with radiographically resectable PC remains controversial as imaging modalities has significantly improved, thus reducing the risk of discovering non resectable disease at the time of surgery. Surgery for the PC can be considered an high-risk surgery. This term is rarely explicitly defined in scientific articles. There seems to be a common understanding among surgeons and anesthesiologists of what major surgery means. It can be defined as a surgical procedure that is extensive, involves removal of whole or parts of organs and/or is lifethreatening. It has also been defined as a surgical procedure with >1 mortality (Ghaferi et al., 2009). One possibility of evaluating the perioperative risk is the use of 1 of several risk scores. The American Society of Anesthesiologists score is widely used and easy to apply, but excludes age from its risk analysis (Kullavanijaya et al., 2001). Age is securely one of the most important, if not the single most predictive, risk factors for morbidity and mortality after major surgery, including major pancreatic surgery (Riall et al., 2008).

Anesthesia and Pain Management: Techniques and Practice 179

decreased breath sounds, dullness to percussion, wheezes, rhonchi and a prolonged expiratory phase that can predict an increase in the risk of pulmonary complications (Lawrence et al., 1996). The value of routine preoperative pulmonary testing remains controversial. There is consensus that such testing should be performed selectively in patients undergoing no-lung resection. It has been suggested that an increased risk of pulmonary complications is associated with a forced expiratory volume in one second (FEV1) or forced vital capacity (FVC) of less than 70 percent of the predicted value or a ratio of FEV1 to FVC of less than 65 percent (Gass & Olsen, 1986). A partial pressure of arterial carbon dioxide (PaCO2) greater than 45 mmHg can't be considered as a risk factor for pulmonary complications. Several strategies can be adopted in the perioperative period

Nearly 80% of PC patients have either frank diabetes or impaired glucose tolerance. Diabetes is usually diagnosed either concomitantly or during the two years preceding the diagnosis (Gullo et al. 1994; Permet et al. 1993). The link between abnormal glucose and PC exists only for type II diabetes. Better glycaemic control in diabetic patients undergoing major surgery has been shown to improve perioperative mortality and morbidity. Diabetics are at increased risk of myocardial ischaemia, cerebrovascular infarction and renal ischaemia because of their increased incidence of coronary artery disease, arterial atheroma and renal parenchymal disease. Increased mortality is found in all diabetics undergoing surgery and type I diabetics are particularly at risk of post-operative complications. Increased wound complications are associated with diabetes and anastomotic healing is severely impaired when glycaemic control is poor (Treiman, 1994; Verhofstad & Hendriks, 1996; Zacharias & Habib, 1996). Type 2 diabetics not receiving insulin and undergoing minor surgery usually can be managed satisfactory without insulin. However, diabetic patients scheduled for major surgery, who are receiving hypoglicaemic medication or who have poor glycaemic control, should be established on insulin therapy preoperatively. Continuous i.v. infusion of insulin is a better option than intermittent s.c. bolus regimens and may be associated with improved outcome. The immediate perioperative problems facing the diabetic patient are: a) surgical induction of the stress response with catabolic hormone secretion; b) interruption of food intake, which will be prolonged in PC surgery; c) circulatory disturbances associated with anesthesia and surgery, which may alter the absorption of subcutaneous insulin. Surgery evokes the "stress response", that is the secretion of catecholamines, cortisol, growth hormone and, in some cases, glucagone. These hormones oppose glucose homeostasis, as they have anti-insulin and hyperglicaemic effects. Although diabetics need increased insulin during the perioperative period, requirements for glucose and insulin in this period are unpredictable and close monitoring is essential, especially in the unconscious or sedated patients. The main concern for the anesthetist in the perioperative management of diabetic patients has been the avoidance of harmful hypoglicaemia; mild hyperglicaemia has tended to be seen as acceptable. High-dose opiate anesthetic techniques produce not only haemodinamic, but also hormonal and metabolic stability. Abolition of the catabolic hormonal response to surgery will abolish the hyperglicaemia seen in normal patients and may be of benefit in the diabetic patients. Tight metabolic control in the perioperative period is imperative and is a goal which is attainable in most patients. IV infusion of insulin is the standard therapy for the perioperative management of diabetes, especially in type 1 diabetic patients and patients with type 2

reducing the risks of complications (Table 1).

**2.2 Diabetes** 

#### **2. Preanesthetic considerations**

Patients undergoing pancreatic surgey require a complete history and physical examination. Coexisting medical illnesses may complicate the surgical and anesthetic course. The objectives of the preanesthetic evaluation include establishing a doctor-patient relationship, becoming familiar with the surgical illness and coexisting medical conditions, developing a management strategy for perioperative anesthetic care and obtaining informed consent for the anesthetic plan.

#### **2.1 History of smoking**

The risk of PC in smokers ranks second to lung cancer and is proportionate to the frequency, duration and cumulative smoking dose (Lynch et al., 2009; Neugut et el., 1995). The patients who smoke have an increased risk of intra- and postoperative complications, particularly of a pulmonary or cardiovascular nature, compared with nonsmoking patients (Bluman et al., 1998; Myles et al., 2002). As carbon monoxide (CO) preferentially binds to hemoglobin in place of oxygen, the short-term effects of cigarette smoking include elevated blood CO levels that result in a 3% to 12% reduction of oxygen availability in the periphery (Pearce & Jones, 1984). Moreover, nicotine stimulates a surgical stress response with increase in heart rate, arterial blood pressure and peripheral vascular resistance. Postoperative pulmonary complications are an important part of the risk of surgery and prolong the hospital stay by an average of one to two weeks. A careful history taking and physical examination are the most important parts of preoperative pulmonary risk assessment. One should seek a history of exercise intolerance, chronic cough or dyspnea. The physical examination may identify

PREOPERATIVE

Encourage cessation of cigarette smoking for at least 8 wk

Treat airflow obstruction in patients with chronic obstructive pulmonary disease or asthma

Administer antibiotics and delay surgery if respiratory infection is present Begin patient education regarding lung-expansion maneuvers

INTRAOPERATIVE

Limit duration of surgery to less than 3 hr Use spinal or epidural anesthesia Use laparoscopic procedures when possible Substitute less ambitious procedure for upper abdominal or thoracic surgery when possible

POSTOPERATIVE

Use deep-breathing exercises or incentive spirometry Use continuous positive airway pressure Use epidural analgesia Use intercostal nerve blocks

Table 1. Risk-Reduction strategies

decreased breath sounds, dullness to percussion, wheezes, rhonchi and a prolonged expiratory phase that can predict an increase in the risk of pulmonary complications (Lawrence et al., 1996). The value of routine preoperative pulmonary testing remains controversial. There is consensus that such testing should be performed selectively in patients undergoing no-lung resection. It has been suggested that an increased risk of pulmonary complications is associated with a forced expiratory volume in one second (FEV1) or forced vital capacity (FVC) of less than 70 percent of the predicted value or a ratio of FEV1 to FVC of less than 65 percent (Gass & Olsen, 1986). A partial pressure of arterial carbon dioxide (PaCO2) greater than 45 mmHg can't be considered as a risk factor for pulmonary complications. Several strategies can be adopted in the perioperative period reducing the risks of complications (Table 1).

#### **2.2 Diabetes**

178 Pancreatic Cancer – Clinical Management

Patients undergoing pancreatic surgey require a complete history and physical examination. Coexisting medical illnesses may complicate the surgical and anesthetic course. The objectives of the preanesthetic evaluation include establishing a doctor-patient relationship, becoming familiar with the surgical illness and coexisting medical conditions, developing a management strategy for perioperative anesthetic care and obtaining informed consent for

The risk of PC in smokers ranks second to lung cancer and is proportionate to the frequency, duration and cumulative smoking dose (Lynch et al., 2009; Neugut et el., 1995). The patients who smoke have an increased risk of intra- and postoperative complications, particularly of a pulmonary or cardiovascular nature, compared with nonsmoking patients (Bluman et al., 1998; Myles et al., 2002). As carbon monoxide (CO) preferentially binds to hemoglobin in place of oxygen, the short-term effects of cigarette smoking include elevated blood CO levels that result in a 3% to 12% reduction of oxygen availability in the periphery (Pearce & Jones, 1984). Moreover, nicotine stimulates a surgical stress response with increase in heart rate, arterial blood pressure and peripheral vascular resistance. Postoperative pulmonary complications are an important part of the risk of surgery and prolong the hospital stay by an average of one to two weeks. A careful history taking and physical examination are the most important parts of preoperative pulmonary risk assessment. One should seek a history of exercise intolerance, chronic cough or dyspnea. The physical examination may identify

Treat airflow obstruction in patients with chronic obstructive pulmonary disease or

Substitute less ambitious procedure for upper abdominal or thoracic surgery when

Administer antibiotics and delay surgery if respiratory infection is present

**2. Preanesthetic considerations** 

the anesthetic plan.

PREOPERATIVE

INTRAOPERATIVE

POSTOPERATIVE

Use epidural analgesia Use intercostal nerve blocks Table 1. Risk-Reduction strategies

Limit duration of surgery to less than 3 hr

Use laparoscopic procedures when possible

Use continuous positive airway pressure

Use deep-breathing exercises or incentive spirometry

Use spinal or epidural anesthesia

asthma

possible

Encourage cessation of cigarette smoking for at least 8 wk

Begin patient education regarding lung-expansion maneuvers

**2.1 History of smoking** 

Nearly 80% of PC patients have either frank diabetes or impaired glucose tolerance. Diabetes is usually diagnosed either concomitantly or during the two years preceding the diagnosis (Gullo et al. 1994; Permet et al. 1993). The link between abnormal glucose and PC exists only for type II diabetes. Better glycaemic control in diabetic patients undergoing major surgery has been shown to improve perioperative mortality and morbidity. Diabetics are at increased risk of myocardial ischaemia, cerebrovascular infarction and renal ischaemia because of their increased incidence of coronary artery disease, arterial atheroma and renal parenchymal disease. Increased mortality is found in all diabetics undergoing surgery and type I diabetics are particularly at risk of post-operative complications. Increased wound complications are associated with diabetes and anastomotic healing is severely impaired when glycaemic control is poor (Treiman, 1994; Verhofstad & Hendriks, 1996; Zacharias & Habib, 1996). Type 2 diabetics not receiving insulin and undergoing minor surgery usually can be managed satisfactory without insulin. However, diabetic patients scheduled for major surgery, who are receiving hypoglicaemic medication or who have poor glycaemic control, should be established on insulin therapy preoperatively. Continuous i.v. infusion of insulin is a better option than intermittent s.c. bolus regimens and may be associated with improved outcome. The immediate perioperative problems facing the diabetic patient are: a) surgical induction of the stress response with catabolic hormone secretion; b) interruption of food intake, which will be prolonged in PC surgery; c) circulatory disturbances associated with anesthesia and surgery, which may alter the absorption of subcutaneous insulin. Surgery evokes the "stress response", that is the secretion of catecholamines, cortisol, growth hormone and, in some cases, glucagone. These hormones oppose glucose homeostasis, as they have anti-insulin and hyperglicaemic effects. Although diabetics need increased insulin during the perioperative period, requirements for glucose and insulin in this period are unpredictable and close monitoring is essential, especially in the unconscious or sedated patients. The main concern for the anesthetist in the perioperative management of diabetic patients has been the avoidance of harmful hypoglicaemia; mild hyperglicaemia has tended to be seen as acceptable. High-dose opiate anesthetic techniques produce not only haemodinamic, but also hormonal and metabolic stability. Abolition of the catabolic hormonal response to surgery will abolish the hyperglicaemia seen in normal patients and may be of benefit in the diabetic patients. Tight metabolic control in the perioperative period is imperative and is a goal which is attainable in most patients. IV infusion of insulin is the standard therapy for the perioperative management of diabetes, especially in type 1 diabetic patients and patients with type 2

Anesthesia and Pain Management: Techniques and Practice 181

Malnurished patients who require major operations are predisposed to infectious complications and poor outcome. A low preoperative body mass index (BMI, kg/m2) may be regarded as an overall indicator of the size of the patient's reserves; a BMI<20 kg/m2 is an accepted indicator of malnutrition. However, it has been recognized that acutely malnourished patients may still have a normal or even elevated BMI. Serum protein markers such as albumin (for evaluating long-term nutritional status) and prealbumin (for evaluating acute responses to nutritional support) have been shown to be useful additional measurements for assessing nutritional status. Low albumin levels have been identified as an independent risk factor for postoperative morbidity and mortality (Gibbs et al., 1999). It should be emphasized that, although preoperative enteral or parenteral nutritional support clearly benefits surgical cancer patients, a systematic review showed that "preventive" administration of parenteral support in non-malnourished patients did not positively influence outcome and may even be potentially harmful for certain patient subgroups (Koretz et al., 2001). More recently, the concept of immunonutrition has evolved, in which enteral formulas are supplemented with arginine and glutamine, nucleotides or omega-3 fatty acids in an attempt to positively modulate the immune system, but the benefits of immunonutrition remain debatable. Where as perioperative nutrition in the malnourished patient can improve postoperative outcome, immunonutrition seems to attenuate the inflammatory response and

Jaundice results from an abnormally high bilirubin in the blood whose origin may be difficulty in eliminating; it's then an obstructive jaundice. This is the most symptom in patients with periampullary cancer (located near the Vater's ampulla) or cancer of the pancreatic head. It can be considered a risk factor for postoperative complications. [8, 9] Many studies demonstrate that it could be associated with a higher incidence of insufficient postoperative renal function, but also of sepsis, haemorrhage, of liver failure and risk of mortality from about 16% (Jiang & Puntis, 1997). Jaundice causes a retention of acids and bile salts. In the long term, may cause ascending cholangitis and secondary hepatocellular damage. In case of interruption of bile flow, bile acids and salts can't inhibit the phenomenon of translocation and endotoxemia caused by gram-negative from the digestive tract. These bacteria will then multiply and, for a phenomenon of translocation, can contribute to the dissemination of endotoxins into the systemic circulation then creating a pro-inflammatory state with production of cytokines by activated macrophages and a subsequent risk of multiple organ failure, including the appearance of coagulation disorders. Since surgery in patients with jaundice is thought to increase the risk of postoperative complications, preoperative biliary drainage was introduced to improve the postoperative outcome. In several experimental studies preoperative biliary drainage reduced morbidity and mortality after surgery (Van der Gaag et al., 2009). In a multicentre, randomized trial, Van der Gaag et al compared preoperative biliary drainage with surgery alone for patients with cancer of the pancreatic head and they found that endoscopic preoperative drainage with placement of a plastic stent did not have a beneficial effect on the surgical outcome and early surgery without preoperative drainage did not increase the risk of complications (Van der Gaag, 2010). The preoperative oral administration of bile salts

interferes with certain immune functions in selected patient groups.

**2.3 Nutritional status** 

**2.4 Patient with jaundice** 

diabetes undergoing major procedure (Clement et al., 2004). Institutions around the world use a variety of insulin infusion algorithms that can be implemented by nursing staff. Recently, several insulin infusion protocols have been reported in the literature. Two main methods of insulin delivery have been used either combining insulin with glucose and potassium in the same bag (GIK regimen) or giving insulin separately with an infusion pump. The GIK is initiated at a rate of 100 mL/h in a solution of 500 mL of 10% dextrose, 10 mmol of potassium, and 15 U of insulin. Adjustments in the insulin dose are made in 5 U increments according to blood glucose measurements performed at least every 2 hours. The combined GIK infusion is efficient, safe and effective but does not permit selective adjustment of insulin delivery without changing the bag. Separate continuous glucose and insulin infusions are used more frequently than the glucose-potassium-insulin infusion (Coursin et al., 2004; Furnary et al., 2003; Goldberg et al., 2004; Rehman & Mohammed, 2003). A proposed regimen for separate IV insulin infusion for perioperative diabetes management is shown in Table 2.

I) Initiating continuous insulin infusion (CII):

Prepare solution: 1 unit (U) per 1 mL of 0.9% normal saline.

Start continuous insulin infusion (CII) when blood glucose level ≥140 mg/dL (x 2).

Patients with known diabetes treated with insulin can start CII when blood glucose ≥70 mg/dL.

Initial rate: divide blood glucose level (mg/dL) by 100, then round to nearest 0.5 U

II) Insulin infusion rate change:

BloodGlucose (mg/dL) instructions:


III) Patient monitoring:

Check capillary blood glucose every hour until it is within goal range for 2 hours, and then decrease to every 2 hours.

Hourly monitoring may be indicated for critically ill patients even if they have stable blood glucose.

If a patient is eating, hourly blood glucose monitoring is necessary for at least 3 hours after eating.

Decrease insulin infusion rate by 50% if nutritional therapy (e.g. total parenteral nutrition or tube feeds) are discontinued or significantly reduced.

#### **2.3 Nutritional status**

180 Pancreatic Cancer – Clinical Management

diabetes undergoing major procedure (Clement et al., 2004). Institutions around the world use a variety of insulin infusion algorithms that can be implemented by nursing staff. Recently, several insulin infusion protocols have been reported in the literature. Two main methods of insulin delivery have been used either combining insulin with glucose and potassium in the same bag (GIK regimen) or giving insulin separately with an infusion pump. The GIK is initiated at a rate of 100 mL/h in a solution of 500 mL of 10% dextrose, 10 mmol of potassium, and 15 U of insulin. Adjustments in the insulin dose are made in 5 U increments according to blood glucose measurements performed at least every 2 hours. The combined GIK infusion is efficient, safe and effective but does not permit selective adjustment of insulin delivery without changing the bag. Separate continuous glucose and insulin infusions are used more frequently than the glucose-potassium-insulin infusion (Coursin et al., 2004; Furnary et al., 2003; Goldberg et al., 2004; Rehman & Mohammed, 2003). A proposed regimen for separate IV insulin infusion for perioperative diabetes

management is shown in Table 2.

II) Insulin infusion rate change: BloodGlucose (mg/dL) instructions: >200 ↑rate by 2 U/h >160–200 ↑rate by 1.0 U/h >120–160 ↑rate by 0.5 U/h 80–120 No change in rate

III) Patient monitoring:

blood glucose.

after eating.

then decrease to every 2 hours.

mg/dL.

I) Initiating continuous insulin infusion (CII):

Prepare solution: 1 unit (U) per 1 mL of 0.9% normal saline.

60–80 If <10% lower blood glucose, rate by 1 U/h,

If >10% lower blood glucose, 2 rate by 50%,

restart infusion at 50% of previous rate

or tube feeds) are discontinued or significantly reduced.

Table 2. Continuous insulin infusion (CII) protocol

< 60 Stop infusion (give IV dextrose 12.5 g IV bolus),

Check BG within 30 min

Check BG within 30 min

Start continuous insulin infusion (CII) when blood glucose level ≥140 mg/dL (x 2). Patients with known diabetes treated with insulin can start CII when blood glucose ≥70

Initial rate: divide blood glucose level (mg/dL) by 100, then round to nearest 0.5 U

Check blood glucose within 30 min. When blood glucose>100 mg/dL,

Check capillary blood glucose every hour until it is within goal range for 2 hours, and

Hourly monitoring may be indicated for critically ill patients even if they have stable

If a patient is eating, hourly blood glucose monitoring is necessary for at least 3 hours

Decrease insulin infusion rate by 50% if nutritional therapy (e.g. total parenteral nutrition

Malnurished patients who require major operations are predisposed to infectious complications and poor outcome. A low preoperative body mass index (BMI, kg/m2) may be regarded as an overall indicator of the size of the patient's reserves; a BMI<20 kg/m2 is an accepted indicator of malnutrition. However, it has been recognized that acutely malnourished patients may still have a normal or even elevated BMI. Serum protein markers such as albumin (for evaluating long-term nutritional status) and prealbumin (for evaluating acute responses to nutritional support) have been shown to be useful additional measurements for assessing nutritional status. Low albumin levels have been identified as an independent risk factor for postoperative morbidity and mortality (Gibbs et al., 1999). It should be emphasized that, although preoperative enteral or parenteral nutritional support clearly benefits surgical cancer patients, a systematic review showed that "preventive" administration of parenteral support in non-malnourished patients did not positively influence outcome and may even be potentially harmful for certain patient subgroups (Koretz et al., 2001). More recently, the concept of immunonutrition has evolved, in which enteral formulas are supplemented with arginine and glutamine, nucleotides or omega-3 fatty acids in an attempt to positively modulate the immune system, but the benefits of immunonutrition remain debatable. Where as perioperative nutrition in the malnourished patient can improve postoperative outcome, immunonutrition seems to attenuate the inflammatory response and interferes with certain immune functions in selected patient groups.

#### **2.4 Patient with jaundice**

Jaundice results from an abnormally high bilirubin in the blood whose origin may be difficulty in eliminating; it's then an obstructive jaundice. This is the most symptom in patients with periampullary cancer (located near the Vater's ampulla) or cancer of the pancreatic head. It can be considered a risk factor for postoperative complications. [8, 9] Many studies demonstrate that it could be associated with a higher incidence of insufficient postoperative renal function, but also of sepsis, haemorrhage, of liver failure and risk of mortality from about 16% (Jiang & Puntis, 1997). Jaundice causes a retention of acids and bile salts. In the long term, may cause ascending cholangitis and secondary hepatocellular damage. In case of interruption of bile flow, bile acids and salts can't inhibit the phenomenon of translocation and endotoxemia caused by gram-negative from the digestive tract. These bacteria will then multiply and, for a phenomenon of translocation, can contribute to the dissemination of endotoxins into the systemic circulation then creating a pro-inflammatory state with production of cytokines by activated macrophages and a subsequent risk of multiple organ failure, including the appearance of coagulation disorders. Since surgery in patients with jaundice is thought to increase the risk of postoperative complications, preoperative biliary drainage was introduced to improve the postoperative outcome. In several experimental studies preoperative biliary drainage reduced morbidity and mortality after surgery (Van der Gaag et al., 2009). In a multicentre, randomized trial, Van der Gaag et al compared preoperative biliary drainage with surgery alone for patients with cancer of the pancreatic head and they found that endoscopic preoperative drainage with placement of a plastic stent did not have a beneficial effect on the surgical outcome and early surgery without preoperative drainage did not increase the risk of complications (Van der Gaag, 2010). The preoperative oral administration of bile salts

Anesthesia and Pain Management: Techniques and Practice 183

epidural space. The choice of anesthetics must consider the interference pharmacokinetic: benzodiazepines should be avoided for premedication; propofol are the preferred induction agent; morphine should be used with caution in patients with hepatic or renal function (accumulation); muscle relaxants not metabolized by hepatobiliary system (atracurium, *cis*atracurium) are to be used in the first intent with adequate monitoring. The antibiotic prophilaxis (Enterobacteriaceae and Staphylococcus) is essential in this surgery. Fluid and volume therapy is an important cornerstone of treating critically ill patients in the operating room. New findings concerning the vascular barrier, its physiological functions and its role regarding vascular leakage have lead to a new view of fluid and volume administration. Avoiding hypervolemia, as well as hypovolemia, plays a pivotal role when treating patients both perioperatively and in the intensive care unit. The postoperative phase may be studded with complications: sepsis, hepatic dysfunction, coagulation and metabolic disorders, renal and pulmonary failure and, in addition to the typical risks associated with abdominal surgery, some specific to the Whipple procedure, the two most common are pancreatic fistula and delayed gastric emptying (Buchler et al., 2003). Therefore the recovery in the

Pre-, intra-and postoperative use of benzodiazepines (BZP) is widely not recommended because of their hepatic metabolism that exposed to an increased half-life, an extension the duration of action and delayed recovery. In premedication for anxiolysis, with the exception of jaundiced patients, midazolam 0.1-0.4 mg/Kg is indicated; after i.v. administration, the onset of central nervous system effects occurs in 2 to 3 minutes. BZP enhance inhibitory neurotransmission by increasing the affinity of GABAA receptors for GABA . Effects are terminated by redistribution, the metabolism is tipically hepatic and renal the elimination. Administration of a BZP to a patient receveing the anticolvulsivant valproate may

Thiopental has no longer the place it has had for very many years. In addition, its use was largely dissuaded in the presence of hepatobiliary disease because of its hepatic metabolism (cytochrome P450). Thiopental is metabolized to pentobarbital, an active metabolite with a longer half- life. Its use therefore exposed to delayed awakening. Similar to propofol, barbiturates facilitate inhibitory neurotransmission by enhancing GABAA receptor function. They also inhibit exicitatory neurotransmission via glutamate and nicotinic acetylcholine receptors. Absolutely contraindicated in patient with acute intermittent porphyria, variegate porphyria and hereditary coproporphyria (barbiturates induce porphyrin synthetic enzymes such as δ-aminolevulinic acid synthetase). Ketamine for its variable pharmacokinetics in the presence of extrahepatic biliary obstruction and postoperative hallucinatory effects has a limited use in clinical practice. Propofol is the agent of choice, not only for the induction, but also for sedation in patients requiring postoperative ventilatory support. It has a short action effect and the rapid metabolism is not influenced in the presence of liver failure. It is prepared as a 1% isotonic oil-in water emulsion, which contains egg lecithin, glycerol and

postanesthesia care unit (PACU) is necessary for these fragile patients.

**3.1 Pharmacology of anesthetics** 

precipitate a psychotic episode.

**3.1.2 Induction agents** 

**3.1.1 Benzodiazepines** 

or lactulose has been proposed in order to reduce the risk of endotoxemia by blocking bacterial translocation phenomenon from the gut. The effectiveness of this practice has not been validated. Anti-inflammatory and antibiotic prophylaxis should be avoided. In severe cases, a preoperative hemodiafiltration session can address the surgery with more serenity.

#### **2.5 The general physical examination**

The physical examination should be thorough but focused. Special attention is directed toward evaluation of the airway, heart, lungs and neurologic status.

#### **2.5.1 Vital signs and head and neck**

Height and wheight are useful in estimating drug dosages and determining volume requirements and the adequacy of perioperative urine output. Ideal body weight should be calculated in obese patients to help determine proper drug dosages and ventilator settings (e.g. tidal volume). Blood pressure should be recorded in both arms and any disparity noted (significant differences may imply disease of the thoracic aorta or its major branches). At same time should be observed and noted the respiration rate and oxygen saturation. One should evaluate maximal mouth opening, the size of the tongue, the ability to visualize the posterior pharyngeal structures and Mallampati classification. A thyromental distance shorter or longer than three fingerbreadth may be a sign of a difficult intubation.

#### **2.6 Laboratory studies**

A routine laboratory screening tests are necessary to evaluate a recent hematocrit/ hemoglobin level, the platelet activity and the coagulation status before surgery. An ECG should be obtained in any patient with risk factors for coronary artery disease (CAD). It can also detect new dysrhythmias and be useful to evaluate the stability of known abnormal rhythms. A chest radiography should be obtained in all patients to evaluate the cardiovascular image and to document any tracheal deviation or cervical masses.

#### **3. Anaesthetic management**

General anesthesia with mechanical ventilation is the rule. Spinal anesthesia is impractical owing to the length of the operation. However, epidural analgesia could, in theory, be used as the sole anesthetic technique. It's our belief that the length of surgery, insertion of central lines and the high likelihood of conversion to general anesthesia make epidural alone unsatisfactory. Epidural analgesia may be beneficial post-operatively in reducing venous thromboembolic events, the incidence of respiratory failure and in providing superior analgesia in comparison with opioids. However, there may be clotting abnormalities perioperatively leading to an increased risk of neurological complications. Epidural can make assessment of the patient's volume status more difficult and, with large fluid shifts occurring in this group, a period of hypovolemia could be worsened by concomitant vasodilatation secondary to the epidural analgesia. A balance of these risks needs to be addressed before embarking on an epidural anesthesia technique. It's our practice to routinely use epidural analgesia as a part of combined general and regional technique in these patients. Postoperative analgesia is then provided by a catheter left in place in epidural space. The choice of anesthetics must consider the interference pharmacokinetic: benzodiazepines should be avoided for premedication; propofol are the preferred induction agent; morphine should be used with caution in patients with hepatic or renal function (accumulation); muscle relaxants not metabolized by hepatobiliary system (atracurium, *cis*atracurium) are to be used in the first intent with adequate monitoring. The antibiotic prophilaxis (Enterobacteriaceae and Staphylococcus) is essential in this surgery. Fluid and volume therapy is an important cornerstone of treating critically ill patients in the operating room. New findings concerning the vascular barrier, its physiological functions and its role regarding vascular leakage have lead to a new view of fluid and volume administration. Avoiding hypervolemia, as well as hypovolemia, plays a pivotal role when treating patients both perioperatively and in the intensive care unit. The postoperative phase may be studded with complications: sepsis, hepatic dysfunction, coagulation and metabolic disorders, renal and pulmonary failure and, in addition to the typical risks associated with abdominal surgery, some specific to the Whipple procedure, the two most common are pancreatic fistula and delayed gastric emptying (Buchler et al., 2003). Therefore the recovery in the postanesthesia care unit (PACU) is necessary for these fragile patients.

#### **3.1 Pharmacology of anesthetics**

#### **3.1.1 Benzodiazepines**

182 Pancreatic Cancer – Clinical Management

or lactulose has been proposed in order to reduce the risk of endotoxemia by blocking bacterial translocation phenomenon from the gut. The effectiveness of this practice has not been validated. Anti-inflammatory and antibiotic prophylaxis should be avoided. In severe cases, a preoperative hemodiafiltration session can address the surgery with more serenity.

The physical examination should be thorough but focused. Special attention is directed

Height and wheight are useful in estimating drug dosages and determining volume requirements and the adequacy of perioperative urine output. Ideal body weight should be calculated in obese patients to help determine proper drug dosages and ventilator settings (e.g. tidal volume). Blood pressure should be recorded in both arms and any disparity noted (significant differences may imply disease of the thoracic aorta or its major branches). At same time should be observed and noted the respiration rate and oxygen saturation. One should evaluate maximal mouth opening, the size of the tongue, the ability to visualize the posterior pharyngeal structures and Mallampati classification. A thyromental distance

A routine laboratory screening tests are necessary to evaluate a recent hematocrit/ hemoglobin level, the platelet activity and the coagulation status before surgery. An ECG should be obtained in any patient with risk factors for coronary artery disease (CAD). It can also detect new dysrhythmias and be useful to evaluate the stability of known abnormal rhythms. A chest radiography should be obtained in all patients to evaluate the

General anesthesia with mechanical ventilation is the rule. Spinal anesthesia is impractical owing to the length of the operation. However, epidural analgesia could, in theory, be used as the sole anesthetic technique. It's our belief that the length of surgery, insertion of central lines and the high likelihood of conversion to general anesthesia make epidural alone unsatisfactory. Epidural analgesia may be beneficial post-operatively in reducing venous thromboembolic events, the incidence of respiratory failure and in providing superior analgesia in comparison with opioids. However, there may be clotting abnormalities perioperatively leading to an increased risk of neurological complications. Epidural can make assessment of the patient's volume status more difficult and, with large fluid shifts occurring in this group, a period of hypovolemia could be worsened by concomitant vasodilatation secondary to the epidural analgesia. A balance of these risks needs to be addressed before embarking on an epidural anesthesia technique. It's our practice to routinely use epidural analgesia as a part of combined general and regional technique in these patients. Postoperative analgesia is then provided by a catheter left in place in

shorter or longer than three fingerbreadth may be a sign of a difficult intubation.

cardiovascular image and to document any tracheal deviation or cervical masses.

toward evaluation of the airway, heart, lungs and neurologic status.

**2.5 The general physical examination** 

**2.5.1 Vital signs and head and neck** 

**2.6 Laboratory studies** 

**3. Anaesthetic management** 

Pre-, intra-and postoperative use of benzodiazepines (BZP) is widely not recommended because of their hepatic metabolism that exposed to an increased half-life, an extension the duration of action and delayed recovery. In premedication for anxiolysis, with the exception of jaundiced patients, midazolam 0.1-0.4 mg/Kg is indicated; after i.v. administration, the onset of central nervous system effects occurs in 2 to 3 minutes. BZP enhance inhibitory neurotransmission by increasing the affinity of GABAA receptors for GABA . Effects are terminated by redistribution, the metabolism is tipically hepatic and renal the elimination. Administration of a BZP to a patient receveing the anticolvulsivant valproate may precipitate a psychotic episode.

#### **3.1.2 Induction agents**

Thiopental has no longer the place it has had for very many years. In addition, its use was largely dissuaded in the presence of hepatobiliary disease because of its hepatic metabolism (cytochrome P450). Thiopental is metabolized to pentobarbital, an active metabolite with a longer half- life. Its use therefore exposed to delayed awakening. Similar to propofol, barbiturates facilitate inhibitory neurotransmission by enhancing GABAA receptor function. They also inhibit exicitatory neurotransmission via glutamate and nicotinic acetylcholine receptors. Absolutely contraindicated in patient with acute intermittent porphyria, variegate porphyria and hereditary coproporphyria (barbiturates induce porphyrin synthetic enzymes such as δ-aminolevulinic acid synthetase). Ketamine for its variable pharmacokinetics in the presence of extrahepatic biliary obstruction and postoperative hallucinatory effects has a limited use in clinical practice. Propofol is the agent of choice, not only for the induction, but also for sedation in patients requiring postoperative ventilatory support. It has a short action effect and the rapid metabolism is not influenced in the presence of liver failure. It is prepared as a 1% isotonic oil-in water emulsion, which contains egg lecithin, glycerol and

Anesthesia and Pain Management: Techniques and Practice 185

absorbed carbon dioxide. After several years of clinical application, no renal failure was observed and appropriate studies on compound A did not show any renal effects in human. Desflurane is largely appreciated for its high stability. Less than 0.02% of desflurane is metabolized, thus, plasma fluorine levels are very low. The very low solubility of desflurane allows for a surprisingly rapid emergence from anesthesia. Nitrous oxide has a controversial role in the modern anesthesia. For one and a half centuries it has played a relevant role in general anesthesia. Many of the side effects of nitrous oxide correlate with its physical properties. Its ability to diffuse into air filled cavities increases the likelihood of pneumothorax, air emboli and pressure in the cuff of the endotracheal tube. Nitrous oxide diffusion causes an increase in the middle ear pressure and distension of the bowel, possibly resulting in increases in postoperative nausea and vomiting. The results of a questionnaire proposed by the Association of Anesthesist of Great Britain and Ireland indicate that 49% of anesthesist had reduced their use of nitrous oxide (Henderson et al., 2002). According to Baum, nitrous oxide should not be used routinely as a carrier gas and the safer mixture of oxygen/medical air is able to replace this old anesthetic with some economical advantages (Baum, 2004). The combination of halogenated agents with short acting opioids results in the possibility of limiting the clinical application of nitrous oxide. Attempts to replace nitrous oxide with other gases has led to an increase in studies on xenon. This inert gas does not undergo metabolic biotransformation and has no direct negative environmental effects. Xenon has a very low solubility in the blood and its potency is higher when compared to nitrous oxide solubility (Hecker et al., 2004). Xenon cannot be synthesized and the available amount is very low. Consequently, at present, the cost of compound may be a limiting factor for the clinical use. The pharmacokinetic advantages of inhalation anesthetics are unique. By increasing or decreasing their inspired concentration, it is possible to increase or decrease their concentration in the blood and tissues, allowing for rapid changes in anesthesia depth and providing a simple method for inducing, maintaining and reversing general anesthesia. The flexibility of inhalation anesthesia cannot be reproduced with modern intravenous hypnotics or opioids. Furthermore, it is important to underline the protective effects of

Non depolarizing blockade is produced by reversible competitive antagonism of Ach at the α subunits of the AChRs. The principal pharmacologic effect is to interrupt transmission of synaptic signaling at the neuromuscular junction. The neuromuscular blocking agents in biliary excretion (e.g. vecuronium) should be avoided in favor of those metabolized by way of Hoffman (atracurium, *cis*-atracurium). In all cases, the use of a monitoring of neuromuscular blockade is obviously essential (Chiu & White, 2000; Murphy &

Standard monitoring for general anaesthesia involves oxygenation (analyzer and pulse oximetry), ventilation (capnography and minute ventilation), circulation (ECG with STsegment analysis, blood pressure and perfusion assessment) and temperature if necessary. Additional monitoring may be added such as invasive arterial and venous pressure

inhalation agents on several different organs.

**3.1.5 Neuromuscolar blocking drugs** 

Szokol,2004).

**3.2 Monitoring** 

soybean oil. Bacterial growth is inhibited by ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), sulfite, or benzyl alcohol depending on the manufacturer (don't use opened propofol after 6 hours to prevent inadvertent bacterial contamination). Mode of action: facilitation inhibitory neurotransmission by enhancing the function of GABAA receptors in the central nervous system; the modulation of glycine receptors, N-etyl-D aspartate receptors, cannabinoid receptors and voltage-gated ion channels may also contribute to propofol's actions. After the infusion it can be observed dose–dependent decreases in preload, afterload and contractility that lead to decrease in blood pressure and cardiac output. Hypotension may be marked in hipovolemic, elderly, or hemodynamically compromised patients. Heart rate is minimally affected and baroreceptor reflex is blunted. Adverse effects are: venous irritation, lipid disorders, myoclonus and hiccups, "propofol infusion syndrome".

#### **3.1.3 Opioids**

Morphine and its derivatives are essential for the perioperative period (commonly used in general anesthesia) and are frequently used to ensure postoperative analgesia. Opioids, including morphine and fentanyl, have been accused to increase the bile ducts tone and to determine a spasm of Oddi's sphincter. However, the consequences in clinical practice are limited: the pressure is most often in the bile duct within normal limits and the delay of the bile's drainage in the duodenum is not significant. Opioids differ in their potencies, pharmacokinetics and site effects. The mode of action is due to the interaction with specific receptors in the brain, spinal cord and peripheral neurons (Kumamoto et al., 2011). After i.v. administration, the onset of action is within minutes for the fentanyl derivatives; due to their lower lipid solubility hydromorphone and morphine may take from 20 to 30 minutes for their peak effect. Elimination is primarily by the liver and depends on hepatic blood flow. In patients with renal failure, the accumulation of morphine -6- glucuronide, the active metabolite, may cause prolonged narcosis and respiratory depression. Fentanyl is metabolized by hydrolysis and N-dealkylation and its metabolites are excreted in the urine. Function liver in the normal range is necessary to plasma clearance in case of repeated injections. The pharmakocinets of alfentanil is also changed, with a longer duration of action and an initial effect over pronounced. The sufentanil phamacokinetics is not altered even in cases of moderate hepatic insufficiency. The short duration of action of remifentanil (context-insensitive half-time) and especially its extrahepatic metabolism (by non specific esterases in tissues, primarily skeletal muscle) are purely an advantage (Dershwitz et al., 1996). Opioids exert emetogenic effects and represent a significant cause of patient discomfort. Nausea and vomiting can occur because of the direct stimulation of the chemoreceptor trigger zone, of the vestibular apparatus, inhibition of gut motility (Porreca & Ossipov, 2009).

#### **3.1.4 Halogenated**

Inhalation agents represent a basic drug used in modern balanced anesthesia. Actually the most important halogenated in the clinical use are sevoflurane and desflurane. They were developed in the late 1960s and tested in clinical practice much later. Sevoflurane was not immediately introduced to the USA because of its fluorine release and its reaction with

soybean oil. Bacterial growth is inhibited by ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), sulfite, or benzyl alcohol depending on the manufacturer (don't use opened propofol after 6 hours to prevent inadvertent bacterial contamination). Mode of action: facilitation inhibitory neurotransmission by enhancing the function of GABAA receptors in the central nervous system; the modulation of glycine receptors, N-etyl-D aspartate receptors, cannabinoid receptors and voltage-gated ion channels may also contribute to propofol's actions. After the infusion it can be observed dose–dependent decreases in preload, afterload and contractility that lead to decrease in blood pressure and cardiac output. Hypotension may be marked in hipovolemic, elderly, or hemodynamically compromised patients. Heart rate is minimally affected and baroreceptor reflex is blunted. Adverse effects are: venous irritation, lipid disorders, myoclonus and

Morphine and its derivatives are essential for the perioperative period (commonly used in general anesthesia) and are frequently used to ensure postoperative analgesia. Opioids, including morphine and fentanyl, have been accused to increase the bile ducts tone and to determine a spasm of Oddi's sphincter. However, the consequences in clinical practice are limited: the pressure is most often in the bile duct within normal limits and the delay of the bile's drainage in the duodenum is not significant. Opioids differ in their potencies, pharmacokinetics and site effects. The mode of action is due to the interaction with specific receptors in the brain, spinal cord and peripheral neurons (Kumamoto et al., 2011). After i.v. administration, the onset of action is within minutes for the fentanyl derivatives; due to their lower lipid solubility hydromorphone and morphine may take from 20 to 30 minutes for their peak effect. Elimination is primarily by the liver and depends on hepatic blood flow. In patients with renal failure, the accumulation of morphine -6- glucuronide, the active metabolite, may cause prolonged narcosis and respiratory depression. Fentanyl is metabolized by hydrolysis and N-dealkylation and its metabolites are excreted in the urine. Function liver in the normal range is necessary to plasma clearance in case of repeated injections. The pharmakocinets of alfentanil is also changed, with a longer duration of action and an initial effect over pronounced. The sufentanil phamacokinetics is not altered even in cases of moderate hepatic insufficiency. The short duration of action of remifentanil (context-insensitive half-time) and especially its extrahepatic metabolism (by non specific esterases in tissues, primarily skeletal muscle) are purely an advantage (Dershwitz et al., 1996). Opioids exert emetogenic effects and represent a significant cause of patient discomfort. Nausea and vomiting can occur because of the direct stimulation of the chemoreceptor trigger zone, of the vestibular apparatus, inhibition of gut motility (Porreca

Inhalation agents represent a basic drug used in modern balanced anesthesia. Actually the most important halogenated in the clinical use are sevoflurane and desflurane. They were developed in the late 1960s and tested in clinical practice much later. Sevoflurane was not immediately introduced to the USA because of its fluorine release and its reaction with

hiccups, "propofol infusion syndrome".

**3.1.3 Opioids** 

& Ossipov, 2009).

**3.1.4 Halogenated** 

absorbed carbon dioxide. After several years of clinical application, no renal failure was observed and appropriate studies on compound A did not show any renal effects in human. Desflurane is largely appreciated for its high stability. Less than 0.02% of desflurane is metabolized, thus, plasma fluorine levels are very low. The very low solubility of desflurane allows for a surprisingly rapid emergence from anesthesia. Nitrous oxide has a controversial role in the modern anesthesia. For one and a half centuries it has played a relevant role in general anesthesia. Many of the side effects of nitrous oxide correlate with its physical properties. Its ability to diffuse into air filled cavities increases the likelihood of pneumothorax, air emboli and pressure in the cuff of the endotracheal tube. Nitrous oxide diffusion causes an increase in the middle ear pressure and distension of the bowel, possibly resulting in increases in postoperative nausea and vomiting. The results of a questionnaire proposed by the Association of Anesthesist of Great Britain and Ireland indicate that 49% of anesthesist had reduced their use of nitrous oxide (Henderson et al., 2002). According to Baum, nitrous oxide should not be used routinely as a carrier gas and the safer mixture of oxygen/medical air is able to replace this old anesthetic with some economical advantages (Baum, 2004). The combination of halogenated agents with short acting opioids results in the possibility of limiting the clinical application of nitrous oxide. Attempts to replace nitrous oxide with other gases has led to an increase in studies on xenon. This inert gas does not undergo metabolic biotransformation and has no direct negative environmental effects. Xenon has a very low solubility in the blood and its potency is higher when compared to nitrous oxide solubility (Hecker et al., 2004). Xenon cannot be synthesized and the available amount is very low. Consequently, at present, the cost of compound may be a limiting factor for the clinical use. The pharmacokinetic advantages of inhalation anesthetics are unique. By increasing or decreasing their inspired concentration, it is possible to increase or decrease their concentration in the blood and tissues, allowing for rapid changes in anesthesia depth and providing a simple method for inducing, maintaining and reversing general anesthesia. The flexibility of inhalation anesthesia cannot be reproduced with modern intravenous hypnotics or opioids. Furthermore, it is important to underline the protective effects of inhalation agents on several different organs.

#### **3.1.5 Neuromuscolar blocking drugs**

Non depolarizing blockade is produced by reversible competitive antagonism of Ach at the α subunits of the AChRs. The principal pharmacologic effect is to interrupt transmission of synaptic signaling at the neuromuscular junction. The neuromuscular blocking agents in biliary excretion (e.g. vecuronium) should be avoided in favor of those metabolized by way of Hoffman (atracurium, *cis*-atracurium). In all cases, the use of a monitoring of neuromuscular blockade is obviously essential (Chiu & White, 2000; Murphy & Szokol,2004).

#### **3.2 Monitoring**

Standard monitoring for general anaesthesia involves oxygenation (analyzer and pulse oximetry), ventilation (capnography and minute ventilation), circulation (ECG with STsegment analysis, blood pressure and perfusion assessment) and temperature if necessary. Additional monitoring may be added such as invasive arterial and venous pressure

Anesthesia and Pain Management: Techniques and Practice 187

the rule in all patient ones, necessary for fluid management and the control of the renal

The primary goals of general anesthesia are to maintain the health of the patient while providing amnesia, hypnosis (lack of awareness), analgesia and immobility. Secondary goals may vary depending on the patient's medical condition and the surgical procedure. Perioperative planning involves the integration of preoperative, intraoperative and postoperative care. Flexibility, the ability to anticipate problems before they occur and the ability to execute contingency plans are skills that define the expert anesthetist. An anesthetic plan developed prior to entering the operating room helps the anesthetist marshal appropriate resources and anticipate potential difficulties. Important elements to consider in the anesthetic plain include: risk assessment (ASA classification), specific homeostatic challenges, intravenous access, monitoring, airway management, medications, perioperative analgesia, postoperative transport and disposition. Preoperative medications is realized with midazolam 0.1-0.4 mg/Kg (except cases of jaundice) for anxiety control. It is also important to consider aspiration prophylaxis; drugs to neutralize gastric acid and decrease gastric volume are used: metoclopramide 10 mg and ranitidine 50 mg usually. Induction of anesthesia produces an unconscious patient with depressed reflexes who is dependent on the anesthetist for maintenance of homeostatic mechanisms and safety. The patient's position for induction is usually supine, with extremities resting comfortably on padded surface in a neutral anatomic position. The head should rest comfortably on a firm support, which is raised in a "sniff" position. Routine pre-induction administration of oxygen minimizes the risk of hypoxia developing during induction of anesthesia. High flow (8 to 10 L/minute) oxygen should be delivered via a face mask placed gently on the patient's face. Commonly, for the induction of anesthesia, we use propofol 4-6 mg/Kg, a non- depolarizing neuromuscular blocking agent (cis–atracurium 0,15 mg/Kg is the usual choice) and sufentanil 0.1-0.5 mcg/Kg. Hypertensive patients may have an exaggerated pressor response to laryngoscopy. To obtund this response, opioids or β-blockers can be used. Tracheal intubation is performed with laryngoscopy usually. An appropriate ETT size depends on the patient's age, body habitus. Proper placement of the ETT needs to be verified by the detection of carbon dioxide in end-tidal or mixed expiratory gas as well as inspection and auscultation of the stomach and both lung fields during positive-pressure ventilation. Tidal volumes of 8-10 ml/ Kg and a respiratory rate of 10 to 12 breaths/minute are set and low level PEEP is beneficial. For the maintenance of anesthesia we use normally a mixture of oxygen and air (40%/60%) and an halogenated (sevoflurane or desflurane) with a continuous infusion of sufentanil until the end of operation. The infusion of sufentanil generally is continued in the PACU to better adapt the patient to the mechanical ventilation. If we decide for a blended anesthesia, before the induction of anesthesia, we perform a

thoracic epidural anesthesia (T8-T10) with the patient in sitting position.

The epidural space is surrounded by the outer surface of the dura mater and the bony and ligamentous walls of the spinal canal and extends from the foramen magnum to the sacral hiatus. The cross-sectional area of the epidural space becomes smaller cranially, as the theca

**3.4 Epidural anaesthesia / analgesia** 

functionality.

**3.3 Conduct of anaesthesia** 

monitoring, trans esophageal echocardiography (TEE), neuromuscular blockade and central nervous system monitoring. Automated noninvasive blood pressure is the most common noninvasive method of measuring blood pressure in the operating room for minor surgery. Invasive blood pressure (IBP) monitoring is imperative in the pancreatic surgery; there is potential for rapid swings in blood pressure and acid-base balance often needs managing (acidosis is common). IBP uses an indwelling arterial catheter coupled through fluid-filled tubing to a pressure transducer. The transducer converts pressure into a electrical signal to be displayed. Generally the catheter size is 18 to 20 gauge for adults. The radial artery is the most common site. Other locations include ulnar, brachial, axillary, femoral and dorsal pedis arteries. The procedure should be perfomed aseptically. Local anesthetic may be used to raise a skin wheal if the patient is awake. For catheter insertion it can be used the Seldinger technique. The modified Allen test has been recommended to assess the relative patency and contribution of the radial and ulnar arteries to the blood supply to the hand, but the results are unreliable. Central venous catheter (CVC) is essential; ultrasound guidance can be useful in the patients that have had multiple previous cannulation. The central venous pressure (CVP) and cardiac output (CO) are monitored by CVC. CVP is measured by coupling the intravascular space to a pressure transducer using a fluid-filled tubing. Pressure is monitored at the level of the vena cava or the right atrium. The normal CVP is 2 to 6 mmHg. Positive- pressure ventilation affects both cardiac output and venous return. According to the Starling rule, the transmural pressure, which is the difference between the atrial pressure and extracardiac pressure, correlates with the cardiac output. At low level of PEEP, the CVP increases with increased PEEP, at high levels of PEEP (over 15 cmH2O), CVP increases as the cardiac output is depressed because of impaired right ventricular output. Common locations include internal jugular and subclavian vein. Multiple lumen catheters are directly inserted and are available with one to four lumens to provide access for multiple drugs, pressure monitoring and blood sampling. Temperature may be measured continuously; the limitation of more external methods of temperature determination is that they may not reflect changes in the core body temperature, especially in the presence of vasoconstriction. Oropharyngeal temperature monitoring is preferred in any lengthy laparotomy, which has potential for blood loss and perioperative clotting abnormalities. Ventilation is assessed by end- tidal carbon dioxide measurements and spirometry. Capnometry and capnography are often used as synonyms, as both analyze and record carbon dioxide, with the latter including a waveform. Capnography not only evaluates respiration but also confirms of endotracheal intubation and its diagnostic of pathologic conditions. Neuromuscular blockade is utilized, above all for patients with coexisting renal failure. The adductor pollicis response to ulnar nerve stimulation at the wrist is most often used, because it is easily accessible, and the results are not confused with direct muscle activation. Cutaneous electrodes are placed at the wrist over the ulnar nerve and attached to a battery-driven pulse generator, which delivers a graded impulse of electrical current at a specified frequency. For maximal twitch response, the negative pole (active) should be placed distally over the ulnar nerve at the wrist. Evoked muscle tension can be estimated by feeling for thumb adduction or measured by using a force transducer attached to the thumb. After administration of a neuromuscular blocking drug (NMBD), the developed tension and twitch height decrease with the onset of blockade. Foley catheter is the rule in all patient ones, necessary for fluid management and the control of the renal functionality.

#### **3.3 Conduct of anaesthesia**

186 Pancreatic Cancer – Clinical Management

monitoring, trans esophageal echocardiography (TEE), neuromuscular blockade and central nervous system monitoring. Automated noninvasive blood pressure is the most common noninvasive method of measuring blood pressure in the operating room for minor surgery. Invasive blood pressure (IBP) monitoring is imperative in the pancreatic surgery; there is potential for rapid swings in blood pressure and acid-base balance often needs managing (acidosis is common). IBP uses an indwelling arterial catheter coupled through fluid-filled tubing to a pressure transducer. The transducer converts pressure into a electrical signal to be displayed. Generally the catheter size is 18 to 20 gauge for adults. The radial artery is the most common site. Other locations include ulnar, brachial, axillary, femoral and dorsal pedis arteries. The procedure should be perfomed aseptically. Local anesthetic may be used to raise a skin wheal if the patient is awake. For catheter insertion it can be used the Seldinger technique. The modified Allen test has been recommended to assess the relative patency and contribution of the radial and ulnar arteries to the blood supply to the hand, but the results are unreliable. Central venous catheter (CVC) is essential; ultrasound guidance can be useful in the patients that have had multiple previous cannulation. The central venous pressure (CVP) and cardiac output (CO) are monitored by CVC. CVP is measured by coupling the intravascular space to a pressure transducer using a fluid-filled tubing. Pressure is monitored at the level of the vena cava or the right atrium. The normal CVP is 2 to 6 mmHg. Positive- pressure ventilation affects both cardiac output and venous return. According to the Starling rule, the transmural pressure, which is the difference between the atrial pressure and extracardiac pressure, correlates with the cardiac output. At low level of PEEP, the CVP increases with increased PEEP, at high levels of PEEP (over 15 cmH2O), CVP increases as the cardiac output is depressed because of impaired right ventricular output. Common locations include internal jugular and subclavian vein. Multiple lumen catheters are directly inserted and are available with one to four lumens to provide access for multiple drugs, pressure monitoring and blood sampling. Temperature may be measured continuously; the limitation of more external methods of temperature determination is that they may not reflect changes in the core body temperature, especially in the presence of vasoconstriction. Oropharyngeal temperature monitoring is preferred in any lengthy laparotomy, which has potential for blood loss and perioperative clotting abnormalities. Ventilation is assessed by end- tidal carbon dioxide measurements and spirometry. Capnometry and capnography are often used as synonyms, as both analyze and record carbon dioxide, with the latter including a waveform. Capnography not only evaluates respiration but also confirms of endotracheal intubation and its diagnostic of pathologic conditions. Neuromuscular blockade is utilized, above all for patients with coexisting renal failure. The adductor pollicis response to ulnar nerve stimulation at the wrist is most often used, because it is easily accessible, and the results are not confused with direct muscle activation. Cutaneous electrodes are placed at the wrist over the ulnar nerve and attached to a battery-driven pulse generator, which delivers a graded impulse of electrical current at a specified frequency. For maximal twitch response, the negative pole (active) should be placed distally over the ulnar nerve at the wrist. Evoked muscle tension can be estimated by feeling for thumb adduction or measured by using a force transducer attached to the thumb. After administration of a neuromuscular blocking drug (NMBD), the developed tension and twitch height decrease with the onset of blockade. Foley catheter is

The primary goals of general anesthesia are to maintain the health of the patient while providing amnesia, hypnosis (lack of awareness), analgesia and immobility. Secondary goals may vary depending on the patient's medical condition and the surgical procedure. Perioperative planning involves the integration of preoperative, intraoperative and postoperative care. Flexibility, the ability to anticipate problems before they occur and the ability to execute contingency plans are skills that define the expert anesthetist. An anesthetic plan developed prior to entering the operating room helps the anesthetist marshal appropriate resources and anticipate potential difficulties. Important elements to consider in the anesthetic plain include: risk assessment (ASA classification), specific homeostatic challenges, intravenous access, monitoring, airway management, medications, perioperative analgesia, postoperative transport and disposition. Preoperative medications is realized with midazolam 0.1-0.4 mg/Kg (except cases of jaundice) for anxiety control. It is also important to consider aspiration prophylaxis; drugs to neutralize gastric acid and decrease gastric volume are used: metoclopramide 10 mg and ranitidine 50 mg usually. Induction of anesthesia produces an unconscious patient with depressed reflexes who is dependent on the anesthetist for maintenance of homeostatic mechanisms and safety. The patient's position for induction is usually supine, with extremities resting comfortably on padded surface in a neutral anatomic position. The head should rest comfortably on a firm support, which is raised in a "sniff" position. Routine pre-induction administration of oxygen minimizes the risk of hypoxia developing during induction of anesthesia. High flow (8 to 10 L/minute) oxygen should be delivered via a face mask placed gently on the patient's face. Commonly, for the induction of anesthesia, we use propofol 4-6 mg/Kg, a non- depolarizing neuromuscular blocking agent (cis–atracurium 0,15 mg/Kg is the usual choice) and sufentanil 0.1-0.5 mcg/Kg. Hypertensive patients may have an exaggerated pressor response to laryngoscopy. To obtund this response, opioids or β-blockers can be used. Tracheal intubation is performed with laryngoscopy usually. An appropriate ETT size depends on the patient's age, body habitus. Proper placement of the ETT needs to be verified by the detection of carbon dioxide in end-tidal or mixed expiratory gas as well as inspection and auscultation of the stomach and both lung fields during positive-pressure ventilation. Tidal volumes of 8-10 ml/ Kg and a respiratory rate of 10 to 12 breaths/minute are set and low level PEEP is beneficial. For the maintenance of anesthesia we use normally a mixture of oxygen and air (40%/60%) and an halogenated (sevoflurane or desflurane) with a continuous infusion of sufentanil until the end of operation. The infusion of sufentanil generally is continued in the PACU to better adapt the patient to the mechanical ventilation. If we decide for a blended anesthesia, before the induction of anesthesia, we perform a thoracic epidural anesthesia (T8-T10) with the patient in sitting position.

#### **3.4 Epidural anaesthesia / analgesia**

The epidural space is surrounded by the outer surface of the dura mater and the bony and ligamentous walls of the spinal canal and extends from the foramen magnum to the sacral hiatus. The cross-sectional area of the epidural space becomes smaller cranially, as the theca

Anesthesia and Pain Management: Techniques and Practice 189

block) during mobilization than bupivacaine (Macias et al., 2002). Panousis et al. evaluated the effect of different epidurally administered concentrations of ropivacaine on inhaled anesthetic, fluid and vasopressor requirement and hemodynamic changes. They concluded that ropivacaine 0.5% compared with a ropivacaine 0.2 % concentration led to a greater inhaled anesthetic-sparing effect at the same levels of IV fuid supply and vasopressor support (Panousis et al., 2009). In a critical appraisal published on 2008, Pratt WB et al. concluded that although it may provide more effective initial pain control, epidural analgesia does not necessarily improve the critical outcome as after pancreatoduodenectomy. The Authors explained it with the high propensity for rapid fluid shifts and excessive blood loss during this operation, which may negate the proposed benefits of administering analgesic medications by epidural infusion and they reinforced these results considering the frequent need to terminate epidural infusions because of hemodynamic compromise or inadequate analgesia. Spinal epidural hematoma (SHE) after epidural analgesia is a rare but serious complication. Most cases of SHE after epidural block are attributed to a bleeding tendency or anticoagulant therapy. Placement of an epidural catheter may cause SHE more often than expected, but most SEHs remain asymptomatic (Inoue, 2002). The incidence of significant spinal bleeding (paraplegia requiring laminectomy) has been estimated at 1:1,000,000 in patients without clinically apparent coagulation disorders. Vandermeulen et al. found spinal bleeding immediately after removal of the epidural catheter in 15 of the 32 cases that he reviewed. Spontaneous SHE has been reported in a few cases (Skilton, 1998; Vandermeulen, 1994). The maximum incidence of clinically important spinal bleeding after epidural catheter blocks without specific additional risk factors probably list between 1:190,000-200,000. Approximately 60- 80% of all clinically important spinal bleeding is associated with haemostatic disorders or a blood tap. Removal of an epidural catheter should be considered a significant risk factor for spinal bleeding because 30-60% of clinically important spinal hematomas occurs after catheter removal (Tryba, 1998). A practical approach to the patients with anticoagulant/antiaggregant therapy is reported in Table 3, according to the last

Where central neural block is contraindicated (e.g systemic sepsis, in anti-coagulated patients), or where epidural catheterization is technically impossible, bilateral paravertebral nerve blocks (PVB) is a suitable alternative. The paravertebral space is a potential space, which is turned into a temporary cavity by fluid. Anaesthesia occurs because of direct penetration of local anesthetic (LA) into the neurological structures contained within the PVB (anterior and posterior ramus of the intercostals nerve, sympathetic chain, rami comunicantes, sinu-vertebral nerve). The spinal nerve, lacking both an epineurvium and part of the perinervium and with only a thin membranous root sheath is easily penetrated by LA and hence easily and efficiently blocked (Karmaker, 2001). We recommend the use of levobupivacaine or ropivacaine for bilateral blocks. Good preservation of postoperative pulmonary function has been demonstrated, particularly in thoracotomy, which is a significant benefit over epidural analgesia (Davies et al., 2006). The incidence of complications such as pneumothorax and hypotension is low. For bilateral PVB a variety of techniques, including loss of resistance, nerve stimulators and ultrasound, have been used. Potential or relative contraindications to the use of PVB are: coagulation disordes, tumor in the PVB and empyema. The relationship of regional anaesthesia to wound healing, chronic postoperative pain, and cancer recurrence rates with this and other block is important.

guidelines of the European Society of Anaesthesiology.

and its contents tend to occupy a greater proportion of space. Hence, a given volume of drugs affects a greater number of segments the more cranially it is introduced. The epidural space contains nerve roots, fat, spinal arteries and lymphatics, as well as a valveless venous system that communicates directly with both the intracranial sinuses via the basovertebral veins and the general circulation via the azygos vein. Dorsal and ventral spinal nerve roots covered by dura mater pass across the epidural space and drugs within this space can act on any nerve that traverses it – whether it be motor, sensory or autonomic. Epidural analgesics may prevent the release of neurotransmitters from afferent pain fibres, block receptors to neurotransmitters released by primary afferent pain fibres or interrupt the transmission of pain-related information in the dorsal horn of the spinal cord. Drugs introduced into the epidural space also have the potential to pass into the brain and the general circulation depending on their pharmacokinetics. Epidural analgesia was originally achieved with local anaesthetic agents but, more recently, with opioids or a combination of local anaesthetics and opioids. This combination has a synergistic action that allows the concentration of each drug to be reduced, thereby limiting unwanted effect produced by higher concentrations. Ketamine, midazolam or clonidine has also been used in combination with local anaesthetics and opioids to obtain the best intra- and post-operative pain control. Local anaesthetics penetrate axonal membranes within the epidural space and bind to sodium channels in nerves. This inhibits sodium conductance and reduces action potential depolarization, thereby reducing nerve stimulus propagation. The drawback is that the effect is non selective, involving both autonomic and somatic nerves. Thinner nerve fibres are affected by lower local anaesthetic concentrations than thicker fibres, suggesting that neuronal block is a function of diameter. With increasing local anesthetic concentration, the thinner C fibres (pain and autonomic fibres) are blocked first, followed by B fibres (preganglionic sympathetic fibres) and finally the largest A fibres (touch, pressure sensation and motor fibres). Epidural analgesia aims to produce a differential nerve block, affecting predominantely nociceptive fibres with few motor effects. Opioids act on opioid receptors that are widespread throughout the nervous system, but more concentrated in the medullary dorsal horn of the spinal cord and the periaqueductal grey matter of the brain. Opioid receptors belong to the family of guanine nucleotide-binding protein receptors. They exist as three principle types (OP1, OP2 and OP3) and opioids acting at these receptors have the advantage of selectively blocking pain without affecting motor function or the sense of touch. Epidural opioids act mainly on presynaptic and postsynaptic receptors in the substantia gelatinosa of the dorsal horn of the spinal cord (Fotiadis et al., 2004). The combination of thoracic epidural analgesia (TEA) and general anesthesia has become a widespread anesthetic technique for the perioperative treatment of patients undergoing major abdominal surgery. The neuraxial application of local anesthetics and opioids provides superior pain relief, reduced hormonal and metabolic stress, enhanced normalization of gastrointestinal function and thus a shortened postoperative recovery time, facilitating mobilization and physiotherapy. TEA is currently thought to mitigate this effect by blocking nociceptive afferent nerves and thoracolumbar sympathetic efferent routes. In a very recent cohort study Van Lier F. et al. (Van Lier et al., 2011) demonstrated that epidural analgesia reduces postoperative pneumonia in patients with chronic obstructive pulmonary disease (COPD) undergoing major abdominal surgery. Among the long-acting local anesthetics, the S-enantiomer, ropivacaine, is gaining increasing preference for continuous epidural analgesia. Ropivacaine has lower central nervous system and cardiac toxicity and a less frequent incidence of motor block (differential

and its contents tend to occupy a greater proportion of space. Hence, a given volume of drugs affects a greater number of segments the more cranially it is introduced. The epidural space contains nerve roots, fat, spinal arteries and lymphatics, as well as a valveless venous system that communicates directly with both the intracranial sinuses via the basovertebral veins and the general circulation via the azygos vein. Dorsal and ventral spinal nerve roots covered by dura mater pass across the epidural space and drugs within this space can act on any nerve that traverses it – whether it be motor, sensory or autonomic. Epidural analgesics may prevent the release of neurotransmitters from afferent pain fibres, block receptors to neurotransmitters released by primary afferent pain fibres or interrupt the transmission of pain-related information in the dorsal horn of the spinal cord. Drugs introduced into the epidural space also have the potential to pass into the brain and the general circulation depending on their pharmacokinetics. Epidural analgesia was originally achieved with local anaesthetic agents but, more recently, with opioids or a combination of local anaesthetics and opioids. This combination has a synergistic action that allows the concentration of each drug to be reduced, thereby limiting unwanted effect produced by higher concentrations. Ketamine, midazolam or clonidine has also been used in combination with local anaesthetics and opioids to obtain the best intra- and post-operative pain control. Local anaesthetics penetrate axonal membranes within the epidural space and bind to sodium channels in nerves. This inhibits sodium conductance and reduces action potential depolarization, thereby reducing nerve stimulus propagation. The drawback is that the effect is non selective, involving both autonomic and somatic nerves. Thinner nerve fibres are affected by lower local anaesthetic concentrations than thicker fibres, suggesting that neuronal block is a function of diameter. With increasing local anesthetic concentration, the thinner C fibres (pain and autonomic fibres) are blocked first, followed by B fibres (preganglionic sympathetic fibres) and finally the largest A fibres (touch, pressure sensation and motor fibres). Epidural analgesia aims to produce a differential nerve block, affecting predominantely nociceptive fibres with few motor effects. Opioids act on opioid receptors that are widespread throughout the nervous system, but more concentrated in the medullary dorsal horn of the spinal cord and the periaqueductal grey matter of the brain. Opioid receptors belong to the family of guanine nucleotide-binding protein receptors. They exist as three principle types (OP1, OP2 and OP3) and opioids acting at these receptors have the advantage of selectively blocking pain without affecting motor function or the sense of touch. Epidural opioids act mainly on presynaptic and postsynaptic receptors in the substantia gelatinosa of the dorsal horn of the spinal cord (Fotiadis et al., 2004). The combination of thoracic epidural analgesia (TEA) and general anesthesia has become a widespread anesthetic technique for the perioperative treatment of patients undergoing major abdominal surgery. The neuraxial application of local anesthetics and opioids provides superior pain relief, reduced hormonal and metabolic stress, enhanced normalization of gastrointestinal function and thus a shortened postoperative recovery time, facilitating mobilization and physiotherapy. TEA is currently thought to mitigate this effect by blocking nociceptive afferent nerves and thoracolumbar sympathetic efferent routes. In a very recent cohort study Van Lier F. et al. (Van Lier et al., 2011) demonstrated that epidural analgesia reduces postoperative pneumonia in patients with chronic obstructive pulmonary disease (COPD) undergoing major abdominal surgery. Among the long-acting local anesthetics, the S-enantiomer, ropivacaine, is gaining increasing preference for continuous epidural analgesia. Ropivacaine has lower central nervous system and cardiac toxicity and a less frequent incidence of motor block (differential block) during mobilization than bupivacaine (Macias et al., 2002). Panousis et al. evaluated the effect of different epidurally administered concentrations of ropivacaine on inhaled anesthetic, fluid and vasopressor requirement and hemodynamic changes. They concluded that ropivacaine 0.5% compared with a ropivacaine 0.2 % concentration led to a greater inhaled anesthetic-sparing effect at the same levels of IV fuid supply and vasopressor support (Panousis et al., 2009). In a critical appraisal published on 2008, Pratt WB et al. concluded that although it may provide more effective initial pain control, epidural analgesia does not necessarily improve the critical outcome as after pancreatoduodenectomy. The Authors explained it with the high propensity for rapid fluid shifts and excessive blood loss during this operation, which may negate the proposed benefits of administering analgesic medications by epidural infusion and they reinforced these results considering the frequent need to terminate epidural infusions because of hemodynamic compromise or inadequate analgesia. Spinal epidural hematoma (SHE) after epidural analgesia is a rare but serious complication. Most cases of SHE after epidural block are attributed to a bleeding tendency or anticoagulant therapy. Placement of an epidural catheter may cause SHE more often than expected, but most SEHs remain asymptomatic (Inoue, 2002). The incidence of significant spinal bleeding (paraplegia requiring laminectomy) has been estimated at 1:1,000,000 in patients without clinically apparent coagulation disorders. Vandermeulen et al. found spinal bleeding immediately after removal of the epidural catheter in 15 of the 32 cases that he reviewed. Spontaneous SHE has been reported in a few cases (Skilton, 1998; Vandermeulen, 1994). The maximum incidence of clinically important spinal bleeding after epidural catheter blocks without specific additional risk factors probably list between 1:190,000-200,000. Approximately 60- 80% of all clinically important spinal bleeding is associated with haemostatic disorders or a blood tap. Removal of an epidural catheter should be considered a significant risk factor for spinal bleeding because 30-60% of clinically important spinal hematomas occurs after catheter removal (Tryba, 1998). A practical approach to the patients with anticoagulant/antiaggregant therapy is reported in Table 3, according to the last guidelines of the European Society of Anaesthesiology.

Where central neural block is contraindicated (e.g systemic sepsis, in anti-coagulated patients), or where epidural catheterization is technically impossible, bilateral paravertebral nerve blocks (PVB) is a suitable alternative. The paravertebral space is a potential space, which is turned into a temporary cavity by fluid. Anaesthesia occurs because of direct penetration of local anesthetic (LA) into the neurological structures contained within the PVB (anterior and posterior ramus of the intercostals nerve, sympathetic chain, rami comunicantes, sinu-vertebral nerve). The spinal nerve, lacking both an epineurvium and part of the perinervium and with only a thin membranous root sheath is easily penetrated by LA and hence easily and efficiently blocked (Karmaker, 2001). We recommend the use of levobupivacaine or ropivacaine for bilateral blocks. Good preservation of postoperative pulmonary function has been demonstrated, particularly in thoracotomy, which is a significant benefit over epidural analgesia (Davies et al., 2006). The incidence of complications such as pneumothorax and hypotension is low. For bilateral PVB a variety of techniques, including loss of resistance, nerve stimulators and ultrasound, have been used. Potential or relative contraindications to the use of PVB are: coagulation disordes, tumor in the PVB and empyema. The relationship of regional anaesthesia to wound healing, chronic postoperative pain, and cancer recurrence rates with this and other block is important.

Anesthesia and Pain Management: Techniques and Practice 191

In patients with epidural catheter the analgesia can be continued with a volumetric or elastomeric pump with a rate infusion of 5-8 ml/h, by using local anesthetics alone or in combination with opioids. Generally we use ropivacaine 2mg/ml and sufentanil 5 mcg/ml. In patients where was impossible the positioning of an epidural catheter the postoperative analgesia is performed with NSAIDs or opioids or mixture of them. Several protocols are reported in literature for IV analgesia, but generally morphine is the leader drug. The patient controlled analgesia (PCA) is the best route of administration with a primary dose of 2-10 mg and a rescue dose of 0.5-2 mg with a lock-out of 5-10 minutes (Miaskowski, 2005). A specific role have the COX-2 inhibitors. Parecoxib (40-80 mg) is disposable for intravenous

Pancreatic diseases such as cancer can cause clinically significant pain in the upper abdomen, which may radiate to the back. Pain management for pancreatic cancer patients is one of the most important aspects of their care, as it is one of the most weakening symptoms. The best therapy involves adequate therapy with constant assessment. The current management of pancreatic pain follows the WHO three-step ladder for pain control, starting with non-opioid analgesics such as nonsteroidal anti-inflammatory drugs (NSAIDs) and progressing to increasing doses of opioid analgesics (WHO, 2008). For pain that does not respond to drugs, or when oral or topical medication leads to unacceptable side effects such as nausea, constipation, somnolence, confusion, dependence and addiction, an alcohol nerve block can be indicated. This provides pain relief by acting directly on the nerves (celiac plexus) that carry painful stimuli from the diseased pancreas to the brain. Pancreatic cancer causes severe pain in 50% to 70% of patients. This kind of pain is multi-factorial (pancreatic duct obstruction and hypertension, neural invasion) and it is often difficult to treat (Staatas 2001). Different mechanisms perpetuate pancreatic pain: infiltration of nerve sheaths and neural ganglia, increased ductal and interstitial pressure and gland inflammation. Pancreatic pain is generally transmitted through the celiac plexus, a neural structure located in the upper abdomen, near the emergence of the celiac trunk from the aorta. Celiac plexus neurolysis was first described by Kappis (1919) and is done at the level of the L1 vertebral body, with the patient in the prone position. There are a number of variations on the technique (Giménez, 1993). It has been described in the literature since the 1950s but the first prospective study was published in 1990 and the first randomized in 1992. Celiac plexus neurolysis can be done surgically under fluoroscopic guidance or under computed tomography (CT) guidance. The target for celiac axis destruction are the splanchnic nerves and/or celiac ganglia. The splanchnic nerves cross the diaphragm, enter the abdominal cavity and form the celiac plexus. The celiac ganglia are located around the celiac artery anterior to the aorta, in varying positions, from T12 to L2. They can be reached percutaneously by different routes, with one needle through the anterior approach (under CT or ultrasound guidance) or with one or two needles through the posterior approach. During abdominal surgical procedures for pancreatic cancer chemical splanchnicectomy can be achieved by injecting the neurolytic solutions directly into the junction area of the splanchnic nerves with the celiac ganglia in the retroperitoneal area. With the advent of

**3.5 Postoperative care** 

**3.5.1 Postoperative I.V. analgesia** 

administration (Nussmeier et al, 2006).

**3.6 Pain and inoperable pancreatic cancer** 


*ACT, activated clotting time; aPTT, activated partial thromboplastin time; b.i.d., twice daily; ECT, ecarin clotting time; INR, international normalised ratio; IU, international unit;* 

*i.v., intravenously; NSAIDs, non-steroidal anti-inflammatory drugs; s.c., subcutaneously; q.d., daily. All time intervals refer to patients with normal renal function. Prolonged time interval in patients with hepatic insufficiency.* 

Table 3. Recommended time intervals before and after neuraxial puncture or catheter removal (Gogarten et al., 2010)

#### **3.5 Postoperative care**

190 Pancreatic Cancer – Clinical Management

4-6 h 1 h

36-42h 6-12h

prophylaxis, 10mg q.d.) 22-26 h 4–6 h (PT, standardised

2.5mg b.i.d.) 26-30 h 4–6 h ?

Coumarins INR ≤1.4 after catheter removal INR

(lepirudin, desirudin) 8-10 h 2-4 h aPTT, ECT Argatrobanc 4 h 2 h aPTT, ECT, ACT

*ACT, activated clotting time; aPTT, activated partial thromboplastin time; b.i.d., twice daily; ECT, ecarin clotting* 

*All time intervals refer to patients with normal renal function. Prolonged time interval in patients with hepatic* 

*i.v., intravenously; NSAIDs, non-steroidal anti-inflammatory drugs; s.c., subcutaneously; q.d., daily.* 

Table 3. Recommended time intervals before and after neuraxial puncture or catheter

Contraindicated according to the manufacturer

Time after puncture/catheter manipulation or removal

> 1 h 1 h

removal

removal

removal

Laboratory tests

Platelets during treatment for more than 5 days

> aPTT, ACT, platelets

Platelets during treatment for more than 5 days

Platelets during treatment for more than 5 days

(anti-Xa, standardised for specific agent)

for specific agent )

6 h ?

Time before puncture/catheter manipulation or removal

> i.v. 4–6 h s.c. 8–12 h

heparins (for prophylaxis) 12 h 4 h

heparins (for treatment) 24h 4h

Acetylsalicylic acid None None

Clopidogrel 7 days after catheter removal Ticlopidine 10 days after catheter removal Ticagrelor 5 days 6 h after catheter

Cilostazolc 42 h 5 h after catheter

Prasugrel 7-10 days 6 h after catheter

NSAIDs None None

*time; INR, international normalised ratio; IU, international unit;* 

Unfractionated heparins

Unfractionated heparins

Low-molecular-weight

Low-molecular-weight

(for prophylaxis, ≤ 15 000 IU per day)

(for treatment)

Fondaparinux (for prophylaxis, 2.5mg per day)

Rivaroxaban (for

Dabigatran (for

Hirudins

*insufficiency.* 

removal (Gogarten et al., 2010)

Apixaban (for prophylaxis,

prophylaxis, 150–220 mg)

#### **3.5.1 Postoperative I.V. analgesia**

In patients with epidural catheter the analgesia can be continued with a volumetric or elastomeric pump with a rate infusion of 5-8 ml/h, by using local anesthetics alone or in combination with opioids. Generally we use ropivacaine 2mg/ml and sufentanil 5 mcg/ml. In patients where was impossible the positioning of an epidural catheter the postoperative analgesia is performed with NSAIDs or opioids or mixture of them. Several protocols are reported in literature for IV analgesia, but generally morphine is the leader drug. The patient controlled analgesia (PCA) is the best route of administration with a primary dose of 2-10 mg and a rescue dose of 0.5-2 mg with a lock-out of 5-10 minutes (Miaskowski, 2005). A specific role have the COX-2 inhibitors. Parecoxib (40-80 mg) is disposable for intravenous administration (Nussmeier et al, 2006).

#### **3.6 Pain and inoperable pancreatic cancer**

Pancreatic diseases such as cancer can cause clinically significant pain in the upper abdomen, which may radiate to the back. Pain management for pancreatic cancer patients is one of the most important aspects of their care, as it is one of the most weakening symptoms. The best therapy involves adequate therapy with constant assessment. The current management of pancreatic pain follows the WHO three-step ladder for pain control, starting with non-opioid analgesics such as nonsteroidal anti-inflammatory drugs (NSAIDs) and progressing to increasing doses of opioid analgesics (WHO, 2008). For pain that does not respond to drugs, or when oral or topical medication leads to unacceptable side effects such as nausea, constipation, somnolence, confusion, dependence and addiction, an alcohol nerve block can be indicated. This provides pain relief by acting directly on the nerves (celiac plexus) that carry painful stimuli from the diseased pancreas to the brain. Pancreatic cancer causes severe pain in 50% to 70% of patients. This kind of pain is multi-factorial (pancreatic duct obstruction and hypertension, neural invasion) and it is often difficult to treat (Staatas 2001). Different mechanisms perpetuate pancreatic pain: infiltration of nerve sheaths and neural ganglia, increased ductal and interstitial pressure and gland inflammation. Pancreatic pain is generally transmitted through the celiac plexus, a neural structure located in the upper abdomen, near the emergence of the celiac trunk from the aorta. Celiac plexus neurolysis was first described by Kappis (1919) and is done at the level of the L1 vertebral body, with the patient in the prone position. There are a number of variations on the technique (Giménez, 1993). It has been described in the literature since the 1950s but the first prospective study was published in 1990 and the first randomized in 1992. Celiac plexus neurolysis can be done surgically under fluoroscopic guidance or under computed tomography (CT) guidance. The target for celiac axis destruction are the splanchnic nerves and/or celiac ganglia. The splanchnic nerves cross the diaphragm, enter the abdominal cavity and form the celiac plexus. The celiac ganglia are located around the celiac artery anterior to the aorta, in varying positions, from T12 to L2. They can be reached percutaneously by different routes, with one needle through the anterior approach (under CT or ultrasound guidance) or with one or two needles through the posterior approach. During abdominal surgical procedures for pancreatic cancer chemical splanchnicectomy can be achieved by injecting the neurolytic solutions directly into the junction area of the splanchnic nerves with the celiac ganglia in the retroperitoneal area. With the advent of

Anesthesia and Pain Management: Techniques and Practice 193

Delbeke D., Rose, D.M., Chapman, W.C., Pinson, C.W., Wright, J.K., Beauchamp, R.D., Shyr

Dershwitz, M., Hoke, J.F., Rosow, C.E., Michalowski, P., Connors, P.M., Muir, K.T et al.

Fotiadis, R.J., Badvie, S., Weston, M.D. & Allen-Mersh T.G. (2004). Epidural analgesia in

Furnary, A.P., Gao, G., Grunkemeier, G.L. et al.( 2003). Continuous insulin infusion reduces

Ghaferi, A.A., Birkmeyer, J.D. & Dimik, J.B. (2009). Variation in hospital mortality associated

Gibbs, J., Cull,W., Henderson,W., Daley, J., Hur, K. & Khuri, S.F.(1999). Preoperative serum

Giménez, A., Martínez-Noguera, A., Donoso, L., Catalá, E. & Serra, R.(1993). Percutaneous

Goldberg, P.A., Siegel, M.D., Sherwin, R.S., et al. (2004). Implementation of a safe and

Gullo, L., Pezzilli, R. & Morselli-Labate, A.M. (1994). Diabetes and the risk of pancreatic

Hecker K, Baumert, J.H., Horn, N. & Rossaint, R. (2004). Xenon, a modern anaesthesia gas.

Henderson**,** K.A., Raj, N. & Hall, J.E. (2002). The use of nitrous oxide in anaesthetic practice:

Inoue, K. et al. (2002). Spontaneous resolution of epidural hematoma after continuous

a questionnaire survey. Anaesthesia, Vol. 57, No. 12, pp. 1155-8, ISSN 0003-2409

epidural analgesia in a patient without bleeding tendency. *Anesthesiology,* Vol. 97,

cancer. *N Engl J Med,* Vol. 331, No. 2, pp. 81-84, ISSN 0028-4793

*Minerva Anestesiol*, Vol.70, No. 5, pp. 255-60, ISSN 0375-9393

guidance*. AJR Am J Roentgenol,* Vol. 161, No. 5,pp. 1061-3, ISSN 0361-803X Gogarten, W., Vandermeulen, E., Van Aken, H., Kozek, S., Van Llau, J. & Samama, C.M.

*Thorac Cardiovasc Surg,*Vol. 125, No. 5, pp. 1007–1021, ISSN 0022-5223 Gass, G.D. & Olsen, G.N. (1986). Preoperative pulmonary function testing to predict

418-26, ISSN 0007-0912

1784-1791, ISSN 0161-5505

0003-3022

0007-1323

0012-3692

0004-0010

1015, ISSN 0265-0215

Vol. 27, No. 2, pp. 461–467, ISSN 0149-5992

No. 3, pp. 735-7, ISSN 0003-3022

review and meta-analysis of randomized trials. *Br j Anaesth,* Vol. 96, No. 4, pp.

Y. & Learch, S.D. (1999). Optimal interpretation of FDG PET in the diagnosis, staging and management of pancreatic carcinoma. *J Nucl Med*, Vol. 40, No. 11, pp.

(1996). Pharmacokinetics and pharmacodynamics of remifentanil in volunteer subjects with severe liver disease. *Anesthesiology,* Vol. 84, No.4, pp. 812-20, ISSN

gastrointestinal surgery. *British Journal of Surgery,* Vol. 91, No.7, pp. 828-841, ISSN

mortality in patients with diabetes undergoing coronary artery bypass grafting. *J* 

postoperative morbidity and mortality. *Chest*, Vol. 89, No. 1, pp. 127-35, ISSN

with inpatient surgery. *N Engl J Med* Vol. 361, No. 14, pp. 1368-75, ISSN 1533-4406

albumin level as a predictor of operative mortality and morbidity : results from the National VA Surgical Risk Study. *Arch Surg,* Vol.134, No. 1, pp.36-42, ISSN

neurolysis of the celiac plexus via the anterior approach with sonographic

(2010). Regional anaesthesia and antithrombotic agents: recommendations of the European Society of Anaesthesiology. *Eur J Anaesthesiol*, Vol. 27, No. 12, pp. 999–

effective insulin infusion protocol in a medical intensive care unit. *Diabetes Care,* 

endoscopic ultrasonography (EUS) new therapeutic applications for endoscopy have been developed and a needle can now be guided safely in the celiac plexus (Puli, 2009). The celiac plexus is destroyed by alcohol injected under the guidance of real-time endosonography. First, using a linear array echo-endoscope, the region of the celiac ganglia is located from the lesser curve of the stomach, following the emergence of the celiac trunk from the aorta. The anterior approach avoids the retro-crural space and minimizes the risk of neurologic complications such as paraesthesia or paralysis. Anyway, although statistical evidence is minimal for the superiority of pain relief over analgesic therapy, the fact that CPB causes fewer adverse effects than opioids is important for patients.

#### **4. Conclusion**

Pancreatic ductal adenocarcinoma (90% of pancreatic cancers) remains a devastating disease. For a select group in which complete resection is possible, surgery prolongs survival. Pancreaticoduodenectomy, the "Cadillac" of abdominal operations, is a major surgery with significant morbidity and mortality. The pancreatico-enteric anastomosis has been the Achilles' heel of this operation. Adequate nutritional support, reduction of invasiveness, shorter operation times, combined regional/general anesthesia, and targetcontrolled fluid management are options for reducing postoperative morbidity. In recent decades, diagnostic modalities and the surgical and palliative treatments of PC have clearly progressed, although the overall prognosis has barely changed. The management of patient affected by PC is complex and requires exepertise in many fields. Multidisciplinary teams are necessary to optimize the overall care. The anesthesiologist plays a crucial role in the perioperative management of such patients and for patient with unresectable PC (anesthesia and analgesia). Careful patient selection, individualized preoperative evaluation and optimization go a long way in improving the short-term and long-term outcomes of these patients. In the future new protocols are necessary for pain control, adjuvant strategies, palliative measures in patients with pancreatic cancer.

#### **5. References**


endoscopic ultrasonography (EUS) new therapeutic applications for endoscopy have been developed and a needle can now be guided safely in the celiac plexus (Puli, 2009). The celiac plexus is destroyed by alcohol injected under the guidance of real-time endosonography. First, using a linear array echo-endoscope, the region of the celiac ganglia is located from the lesser curve of the stomach, following the emergence of the celiac trunk from the aorta. The anterior approach avoids the retro-crural space and minimizes the risk of neurologic complications such as paraesthesia or paralysis. Anyway, although statistical evidence is minimal for the superiority of pain relief over analgesic therapy, the fact that CPB causes

Pancreatic ductal adenocarcinoma (90% of pancreatic cancers) remains a devastating disease. For a select group in which complete resection is possible, surgery prolongs survival. Pancreaticoduodenectomy, the "Cadillac" of abdominal operations, is a major surgery with significant morbidity and mortality. The pancreatico-enteric anastomosis has been the Achilles' heel of this operation. Adequate nutritional support, reduction of invasiveness, shorter operation times, combined regional/general anesthesia, and targetcontrolled fluid management are options for reducing postoperative morbidity. In recent decades, diagnostic modalities and the surgical and palliative treatments of PC have clearly progressed, although the overall prognosis has barely changed. The management of patient affected by PC is complex and requires exepertise in many fields. Multidisciplinary teams are necessary to optimize the overall care. The anesthesiologist plays a crucial role in the perioperative management of such patients and for patient with unresectable PC (anesthesia and analgesia). Careful patient selection, individualized preoperative evaluation and optimization go a long way in improving the short-term and long-term outcomes of these patients. In the future new protocols are necessary for pain control, adjuvant strategies,

Baum, J.A. (2004). The carrier gas in anaesthesia: nitrous oxide/oxygen, medical air/oxygen

Bluman, L.G., Mosca, L., Newman, N. & Simon, D.G. (1998). Preoperative smoking habits

Buchler, M.W. et al. (2003). Changes in morbidity after pancreatic resection : toward the end

Chiu, J.W.& White, P.F. (2000). The pharmacoeconomics of neuromuscular blocking drugs. J

Clement, S., Braithwaite, S.S., Magee, M.F., et al. (2004). Management of diabetes and hyperglycemia in hospitals. *Diabetes Care*, Vol. 27, pp. 553–597, ISSN 0149-5992 Coursin, D.B., Connery, L.E. & Ketzler, J.T.(2004). Perioperative diabetic and hyperglycemic management issues. *Crit Care Med,* Vol. 32, pp. S116–S125, ISSN 0090-3493 Davies, R.G., Myles, P.S. & Graham, J.M. (2006). A comparison of the analgesic efficacy and

and pure oxygen. *Curr Opin Anaesthesiol* Vol. 17, No. 6, pp. 513-6, ISSN 0952-7907

and postoperative pulmonary complications. *Chest*, Vol. 113, pp. 883-9, ISSN

of completion pancreatectomy. Arch Surg, Vol. 138,No. 12, pp. 1310-1314, ISSN

site effects of paravertebral epidural blockade for thoracotomy- a systematic

fewer adverse effects than opioids is important for patients.

palliative measures in patients with pancreatic cancer.

Anaesth, Vol.90, pp. S19-S23, ISSN 0003-2999

**4. Conclusion** 

**5. References** 

0012-3692

0004-0010

review and meta-analysis of randomized trials. *Br j Anaesth,* Vol. 96, No. 4, pp. 418-26, ISSN 0007-0912


Anesthesia and Pain Management: Techniques and Practice 195

Pearce, A.C. & Jones, R.M. (1984). Smoking and anesthesia : preoperative abstinence and

Permet, J., Ihse, I., Jorfeldt, L., von Schenck H et al. (1993). Pancreatic cancer is associated

Puli, S.R. et al. (2009). US-guided celiac plexus neurolysis for pain due to chronic

Rehman, H.U. & Mohammed, K. (2003). Perioperative management of diabetic patients.

Shaib Y.H., Davila J.A. & El-Serag H.B. (2006). The epidemiology of pancreatic cancer in the

Skilton, R.W.H. & Justice, W. (1998). Epidural hematoma following anticoagulant treatment

Staatas, P.S. et al. (2001). The effects of alcohol celiac block, pain and mood on longevity in

controlled study. *Pain Medicine*, Vol. 2, No. 1, pp. 28-34, ISSN 1526-2375 Tilleman, E.H., Busch, O.R., Bemelman, W.A., van Gulik, T.M., Obertop, H. & Gouma, D.J.

Treiman, G.S., Treiman RL, Foran RF et al. (1994). The influence of diabetes mellitus on the

Tryba, M. (1998). European practice guidelines: Thromboembolism prophylaxis and

Van der Gaag, N.A., Kloek, J.J., de Castro, S.M., Busch, O.R., van Gulik, T.M. & Gouma, D.J.

Van der Gaag, N.A., Rauws, E.A.J., van Eijck, H.J., Bruno, M.J., van derd Harst, E., Kubben,

Van Lier, F., Van der Geest, P., Hoeks, S., Van Gestel, Y., Hol, J., Sin, D., Stolker, R.J. &

*Anesthesiology*, Vol. 115, No. 2, pp. 315-21, ISSN 0003-3022

*Digestive Diseases Science*, Vol. 54, No. 11, pp. 2330-7, ISSN 0163-2116 Riall, T.S., Reddy, D.M., Nelson, W.H. & Goodwin, J.S. (2008). The effect of age on short-

Vol.108, No. 6, (June 2009), pp. 1971-6, ISSN 0003-2999

Vol. 248, No. 3, pp. 459-67, ISSN 0003-4932

pp. 87-94, ISSN 0269-2813

691-701, ISSN 0003-2409

ISSN 0944-1166

1348

1098-7339

1091-255X

129-37, ISSN 0028-4793

*Curr Surg,* Vol.60, No. 6, pp. 607–611, ISSN 0149-7944

1102-4151

randomized, double-blinded, placebo-controlled study. *Anesthesia & analgesia*,

perioperative morbidity. *Anesthesiology*, Vol. 61, No. 5, pp. 576-84, ISSN 0003-3022

with impaired glucose metabolism. *Eur J Surg*, Vol. 159, No. 2, pp. 101-107, ISSN

pancreatitis or pancreatic cancer pain: A meta-analysis and systematic review.

term outcomes after pancreatic resection : a population-based study. *Ann Surg*,

United States: changes below the surface. *Aliment pharmacol Ther*, Vol. 24, No. 1,

in a patient with an indwelling epidural catheter. *Anesthesia,* Vol. 53, No. 7, pp.

patients with unresectable pancreatic pain: A double-blind, randomized, placebo-

(2004). Diagnostic laparoscopy in staging pancreatic carcinoma: Developments during the past decade. *J Hepatobiliary Pancreat Surg*, Vol. 11, No. 1, pp. 11-16,

risk of abdominal aortic surgery. *Am Surg*, Vol. 60, No. 6, pp. 436-40, ISSN 0003-

regional anesthesia. *Reg Anesth Pain Med*, Vol. 23 (Suppl. 2), pp. 178-82, ISSN

(2009). Preoperative biliary drainage in patients with obstructive jaundice : history and current status. *J Gastrointest Surg,* Vol. 13, No. 4, pp. 814-20, ISSN

F.J., Gerritsen, J., Greve, J.W., Gerhards, M.F. et al. (2010). Preoperative biliary drainage for cancer of the head of the pancreas. *N Engl J Med*, Vol. 362, No. 2, pp.

Poldermans, D. (2011). Epidural analgesia is associated with improved health outcomes of surgical patients with chronic obstructive pulmonary disease.


Jemal A. et al. (2009). Cancer statistics, 2009.*CA Cancer J Clin*, Vol. 59, No. 4, pp. 225-249,

Jiang, W.G .& Puntis MC. (1997). Immune dysfunction in patients with obstructive jaundice,

Karmaker, M.K. (2001). Thoracic paravertebral block. *Anesthesiology,* Vol. 95, No. 3, pp. 771-

Koretz, R.L., Lipman,T.O. & Klein, S. (2001). AGA technical review on parenteral nutrition.

Kullavanijaya, P., Treeprasertsuk, S., Thong-Nham, D., Kladcharoen, N., Mahachai, V. &

Kumamoto, e., Mizuta, K. & Fujita, T. (2011). Opiod actions in primary- afferent fibers-

Lawrence, V.A., Dhanda, R., Hilsenbeck, S.G. & Page, P.G. (1996). Risk of pulmonary

Lynch, S.M., Vrieling, A., Lubin, J.H., Kraft, P., Mendelson, J.B., Hartge, P., Canzian, F.,

Macias, A., Monedero, P., Adame, M., Torre, T., Fidalgo, I. & Hidalgo, F. (2002). A

Miaskowski, C. (2005). Patient-controlled modalities for acute postoperative pain management. *J Perianesth Nurs*,Vol. 20, No. 4,pp. 255-67, ISSN 1089-9472 Murphy, G.S. & Szokol, J.W. (2004). Monitoring neuromuscular blockade*. Int Anesthesiol* 

Myles, P.S., Iacono, G.A., Hunt, J.O., Fletcher, H., Morris, J., McIlory, D. & Fritschi, L. (2002).

Nussmeier, N.A., Whelton, A.A., Brown, M..T, Joshi, G.P., Langford, R.M., Singla, N.K.,

Neugut, A.I., Ahsan, H. & Robinson, E. (1995). Pancreas cancer as a second primary

Panousis,P., Heller, A.R., Koch, T. & Litz, R. (2009). Epidural ropivacaine concentrations for

*Anesth Analg,* Vol. 95, No. 5, pp. 1344-50, ISSN 0003-2999

*Clin*, Vol.42, No.2, pp.25-40, ISSN 0020-5907

pp. 842-7, ISSN 0003-3022

ISSN 0003-3022

0008-543X

*Gastroenterology*, Vol. 121, No. 4, pp. 970-1001, ISSN 0016-5085

mediators and implications for treatments. *HPB Surg,* Vol.10, No. 3, pp. 129-42,

Suwanagool, P. (2001). Adenocarcinoma of the pancreas: the clinical experience of 45 histopathologically proven patients, a 6 year study. *J Med Assoc Thai*, Vol. 84,

involvement in analgesia and anestesia. *Pharmaceuticals,* Vol. 4, ( January 2011),

omplications after elective abdominal surgery. *Chest*, Vol. 110, No.3, pp. 744-50,

Steplowski, E., Arslan, A.A., Gross,M.,Helzlsouer, K., Jacobs,E.J., La Croix,A., Petersen, G., Zheng, W., Albanes,D., Amundadottir, L., Bingham, S.A., Boffetta, P., Boutron-Ruault, M.C., Chanock, S.J., Clipp,S., Hoover, R.N., Jacobs, K. *et al.* (2009). Cigarette smoking and pancreatic cancer : a pooled analysis from the pancreatic cancer cohort consortium. *Am J Epidemiol* Vol*.* 170, No. 4,pp. 403-413,

randomized, double-blinded comparison of thoracic epidural ropivacaine, ropivacaine/ fentanyl, or bupivacaine / fentanyl for postthoracotomy analgesia.

Risk of respiratory complications and wound infection in patients undergoing ambulatory surgery : smokers versus nonsmokers. *Anesthesiology*, Vol. 97, No. 4,

Boye, M.E. & Verburg, K.M (2006). Anesthesiology,Vol. 104, No. 3, pp. 255-67,

malignancy. A population- based study. *Cancer*, Vol. 76, No. 4, pp. 589-592, ISSN

intraoperative analgesia during major upper abdominal surgery : A prospective,

ISSN 0007-9235

ISSN 0894-8569

80, ISSN 0003-3022

No 5,. pp. 640-647, ISSN 0125-2208

pp. 343-365, ISSN 1424-8247

ISSN 0012-3692

ISSN 0002-9262

randomized, double-blinded, placebo-controlled study. *Anesthesia & analgesia*, Vol.108, No. 6, (June 2009), pp. 1971-6, ISSN 0003-2999


**11**

*Lebanon* 

**Multi-Disciplinary Management**

**of Metastatic Pancreatic Cancer** 

Marwan Ghosn\*, Colette Hanna and Fadi El. Karak

Pancreatic cancer (PC) is a devastating disease with the worst mortality rate and an overall 5-year survival rate lower than 5% (2% in distant cases; 9% in regional cases and 22% in localized cases). Although accounting for only 3% of all cancers, this disease is the fourth leading cause of death and represents 6 – 7 % of all cancer related deaths. In males, the incidence ASR is 8.2 and 2.7 and the mortality ASR is 7.9 and 2.5 in more developed areas

In females, the incidence ASR is 5.4 and 2.1 and the mortality ASR is 5.1 and 2.0 in more

We noticed that the incidence and the mortality rates are very close (*Jemal et al. 2011).* Also, the death rate is increasing from 9.28 per 100,000 in 1991 to 9.48 in 2006 with an absolute

In the United States, the overall incidence is about 8–10 cases per 100,000 persons/year and

Pancreatic cancer remains one of the most difficult to treat due to late initial diagnosis and to intrinsic resistance to conventional treatments. About 50% of patients have distant disease at the time of diagnosis (locally advanced stage) and in 40% the tumor has spread

Risk factors have been identified, molecular pathogenesis has been elucidated, but advances in early detection and efficient treatments remain rather disappointing despite tremendous

Studies results show that long-term diabetes, even though risk diminishes over time, remains a risk factor for PC independent of obesity and smoking with a latency period of more than 5 years. Type 3 diabetes mellitus is an effect, and therefore a harbinger, of

pancreatic cancer in at least 30% of patients *(Magruder JT et al, 2011; Li D et al. 2011).*

**1. Introduction** 

(metastatic stage).

**2. Risk factors** 

Corresponding Author

efforts.

\*

and less developed areas, respectively.

change of 0.2 (2.1%). *(Jemal et al. 2010).* 

developed areas and less developed areas, respectively.

rises slowly over the years with 43 140 new cases in 2010.

*Faculty of Medicine, Saint-Joseph University, Beirut,* 

