Trial Information

Prognostic Relevance of Perioperative Cancer Cell Dissemination and Systemic Immune Suppression in Resectable Ductal Pancreatic Adenocarcinoma

Prognostic relevance of circulating cancer cells and immunosuppression in resectable
pancreatic carcinoma

1. Introduction

Pancreatic adenocarcinoma is the fourth commonest cause of death from cancer in men and
women, with 5-year survival for all stages of disease of less than 5%. The
case-fatality rate for pancreatic cancer is extremely high and virtually identical to
the number of new cases (World Health Organization. World cancer report. Lyon: IACR
Press; 2003). Most patients with cancer of the pancreas are diagnosed at an advanced
stage and have a median survival of 4 to 10 months, depending on the extent of disease
and irrespective the type of treatment modality. Overall, fewer than 5% of the patients
have resection with curative intent, though over 80% will develop cancer recurrence
(Jemal A, Thomas A, Murray T, Thun M. Cancer Statistics. CA Cancer J Clin 2002; 52:
23-47). The primary site of metastatic disease in patients with pancreatic cancer is
the liver and/or the peritoneal cavity.

Cancer recurrence is multifactorial and determined by patient-, tumour-, and
surgery-related factors. Any combination of several mechanisms have been proposed by
which cancer may recur following potentially curative resection (Coffey JC, Wang JH,
Smith MJF, Bouchier-Hayes D, Cotter TG,and Redmond HP. Excisional surgery for cancer
cure: therapy at a cost. Lancet Oncol 2003;4:760-68). Surgery may contribute to the
overall residual tumor burden by adding disseminated cancer cells during tumor
manipulation. Due to post-operative immunosuppression the patient may become more
susceptible to tumorigenesis and/or disseminated cancer cells may escape the immune
response more easily. Finally, surgical resection may alter biological properties of
cancer cells and lead to increased cellular proliferation and reduced cell death.

2. Cancer cell dissemination

Solid markers are lacking to predict cancer recurrence or cancer progression before
being measurable with conventional or modern diagnostic imaging. Molecular techniques
such as the reverse transcriptase polymerase chain reaction (RT-PCR) technique have
proven to be a highly sensitive method and a powerful tool in the study of cancer
metastasis (Raj GV, Moreno JG, Gomella LG. Utilization of Polymerase Chain Reaction
Technology in the Detection of Solid Tumors. Cancer 1998; 82: 1419-42). The prognostic
relevance of circulating cancer cells in patients with pancreatic cancer is hardly
studied. In patients with breast cancer the number of circulating cancer cells
expressing EpCAM (epithelial cell adhesion molecule) has proven to be an independent
predictor of progression-free and overall survival (Cristofanilli M, Budd GT, Ellis MJ,
Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, Hayes
DF. Circulating tumor cells, disease progression, and survival in metastatic breast
cancer. N Engl J Med 2004; 351: 781-91). EpCAM is a marker present on most epithelial
cell tumours and frequently overexpressed in primary and metastatic adenocarcinoma
including pancreatic cancer (Rao CG, Chianese D, Doyle GV, Miller MC, Russell T,
Sanders RA Jr, Terstappen LW. Expression of epithelial cell adhesion molecule in
carcinoma cells present in blood and primary and metastatic tumors. Int J Oncol 2005;
27: 49-57). Also, other markers such as CEA, CK20, K-ras have been used to detect
disseminated pancreatic cancer cells. In patients with colorectal carcinoma circulating
cancer cells in peripheral or in mesenteric venous blood as detected by RT-PCR, using
CEA and CK20 mRNA transcripts, seem to represent a prognostic factor (Yamaguchi K,
Takagi Y, Aoki S, Futamura M, Saji S. Significant detection of circulating cancer cells
in the blood by reverse transcriptase-polymerase chain reaction during colorectal
cancer resection. Ann Surg 2000; 232: 58-65. Guller U, Zajac P, Schnider A, Bösch B,
Vorburger S, Zuber M, Spagnoli GC, Oertli D, Maurer R, Metzger U, Harder F, Heberer M,
Marti WR. Disseminated single tumor cells as detected by real-time quantitative
polymerase chain reaction represent a prognostic factor in patients undergoing surgery
for colorectal cancer. Ann Surg 2002; 236: 768-76).

3. Molecular characteristics of metastatic cancer cells

The development of metastasis is a highly selective process favoring the survival of a
minor subpopulation of metastatic cells. To produce metastases, tumor cells from this
subpopulation must complete a sequence of interrelated steps. To produce clinically
relevant distant metastases, the successful metastatic cell must therefore exhibit a
complex phenotype that is regulated by transient or permanent changes at the gene
level. Many malignant tumors, including pancreatic cancer, contain heterogeneous
subpopulations of cells. This heterogeneity is exhibited in a wide range of genetic,
biochemical, immunological, and biological characteristics such as growth rate,
antigenic and immunogenic status, cell surface receptors and products, enzymes,
karyotypes, cell morphologies, invasiveness, drug resistance, and metastatic potential.
It is likely that specific tumor cells or colonies within the larger heterogeneous
tumor specimen are the forerunners of distant metastases. The overall efficiency of the
whole metastatic process itself is low. Although the absolute number of cancer cells
required for metastasis in humans is unknown, a positive relationship between the
number of circulating cancer cells and the subsequent development of metastases has
been the subject of several experimental studies (Fisher, E.R. and Fisher, B.
Experimental study of factors influencing development of hepatic metastases from
circulating tumor cells. Acta Cytol. 9: 146-159, 1965. Fidler, I.J. The relationship of
embolic homogeneity, number, size and viability to the incidence of experimental
metastasis. Europ. J. Cancer 9: 223-227, 1973. Liotta, L.A., Kleinerman, J., Saidel, G.
Quantitative relationships of intravascular tumor cells, tumor vessels and pulmonary
metastasis. Cancer Res. 34: 977-1004, 1974. Weiss, L., Mayhew, E., Glaves, R.P. and
Holmes, J.C. Metastatic inefficiency in mice bearing B16 melanomas. Br. J. Cancer 45:
44-53, 1982. Topal B, Aerts JL, Roskams T, Fieuws S, Van Pelt J, Vandekerckhove P,
Penninckx F. Cancer cell dissemination during curative surgery for colorectal liver
metastases. Eur J Surg Oncol 2005; 31: 506-511).

The poor prognosis and lack of effective treatments for pancreatic cancer arise from
several causes. There are currently no effective biomarkers useful for early detection
of pancreatic cancer that tends to rapidly invade surrounding structures and undergo
early metastatic spreading. Moreover, pancreatic cancer is highly resistant to both
chemotherapy and radiation therapy. Currently, the molecular basis for these
characteristics is unknown. Gene expression profiles provide important information
about the molecular characteristics of the cancers and can be used to distinguish
closely related cancer subtypes. Gene profiling can also be used to develop candidate
biomarkers and to identify groups of genes involved in specific functional aspects of
tumor biology. Gene expression profiles that could distinguish between pancreatic
tumors, chronic pancreatitis and normal pancreatic tissue have been studied extensively
using different microarrays and microarray platforms. The differentially expressed
genes in pancreatic tumor tissues were found to be quite specific and highly
reproducible among platforms and studies (Crnogorac-Jurcevic T, Efthimiou E, Nielsen T,
Loader J, Terris B, Stamp G, Baron A, Scarpa A and Lemoine NR. Expression profiling of
microdissected pancreatic adenocarcinomas. Oncogene 2002; 21: 4587-4594. Han H, Bearss
DJ, Browne LW, Calaluce R, Naagle RB, Von Hoff DD. Identification of differentially
expressed genes in pancreatic cancer cells using cDNA microarray. Cancer Res 2002; 62:
2890-96. Iacobuzio-Donahue CA, Ashfaq R, Maitra A, Adsay NV, Shen-Ong GL, Berg K,
Hollingsworth MA, Cameron JL, Yeo CJ, Kern SE, Goggins M, Hruban RH. Cancer Res 2003;
63: 8614-22. Logsdon CD, Simeone DM, Binkley C, Arumugam T, Greenson JK, Giordano TJ,
Misek DE, Hanash S. Molecular profiling of pancreatic adenocarcinoma and chronic
pancreatitis identifies multiple genes differentially regulated in pancreatic cancer.
Cancer Res 2003; 63: 2649-57).

4. Peri-operative immunological changes

Suppression of the immune system is a feature of the post-operative stress response, in
which natural killer (NK) and lymphocyte activated killer (LAK) cells form an integral
component in immune anti-tumor surveillance. However, a tumor type-specific immune
reaction in pancreatic cancer or in patients undergoing surgery is lacking.
Anti-tumoral activities of NK and LAK cells are reduced immediately after surgery so
the additive effects of surgery may be sufficient to improve the ability of
disseminated cancer cells to evade the host immune response long enough for metastases
to develop (Da Costa ML, Finnegan N, Flynn M, Bouchier-Hayes DJ. Laparotomy and
laparoscopy differentially accelerate experimental flank tumourgrowth. Br J Surg 1998;
85: 1439-42.) (Da Costa ML, Redmond HP, Bouchier-Hayes DJ. The effect of laparotomy and
laparoscopy on the establishment of spontaneous tumour metastases. Surgery 1998; 124:
516-26).

The suppressive immunological changes that follow surgical stress occur along a
temporal pattern. Depression of cellular immunity to tumor-associated antigen and
depression of delayed-type hypersensitivity are present for up to 3 weeks after
surgery. In patients with pancreatic cancer the prevalence of regulatory T cells (Treg)
is increased in the peripheral blood as well as in the tumour microenvironment. These
Treg cells (CD4+25+) are phenotypically similar to Treg cells from normal donors by
coexpression of CD45R0 and CTLA-4 molecules as well as cytokine profile. Regulatory
CD4+25+ lymphocytes infiltrate tumor and regional lymph nodes, and suppress activation
of CD8+ lymphocytes and helper CD4+25- lymphocytes (Liyanage, U. K., T. T. Moore, H. G.
Joo, Y. Tanaka, V. Herrmann, G. Doherty, J. A. Drebin, S. M. Strasberg, T. J. Eberlein,
P. S. Goedegebuure, and D. C. Linehan. 2002. Prevalence of regulatory T cells is
increased in peripheral blood and tumor microenvironment of patients with pancreas or
breast adenocarcinoma. J Immunol 169:2756).

However, the oncological significance of changes in activities and numbers of NK, LAK,
Treg and dendritic cells still has to be confirmed.

5. Aims / Endpoints

- Is the number of circulating cancer cells a determinant for cancer recurrence
and/or survival following potentially curative resection of pancreatic cancer?

- Is the genetic profile of circulating cancer cells a determinant for cancer
recurrence and/or survival following potentially curative resection of pancreatic
cancer?

- Is peri-operative immunosuppression correlated with cancer recurrence and/or
survival following potentially curative resection of pancreatic cancer?

6. Patient selection and inclusion criteria: Potentially curable/ resectable pancreatic
cancer

7. Study interventions

- Quantitative analysis of circulating cancer cells

In the present study peripheral venous blood samples will be obtained before the start
of surgery, at the end of surgery, on the first day after and on day 7 after surgery
through a central venous catheter. Tumour tissue samples and random liver tru-cut
biopsies will be obtained intra-operatively. Real-time quantitative RT-PCR technique
(TaqManR), based on 3 different target markers (CEA, CK20 and EpCAM mRNA transcripts)
and beta-glucuronidase (GUS) as control gene, will be used to detect and quantify
circulating cancer cells in the blood stream. Normalized copy numbers (NCN) will be
determined in each sample (analyzed in duplicate). RNA extraction from all samples will
be performed using RNeasy mini kit(Qiagen, BD Company). Sample preparation, cDNA
synthesis and PCR will be performed in separated rooms in order to prevent
cross-contamination.

- Genetic profiling of disseminated cancer cells

Oligonucleotide microarray analysis of mRNA extracted from the primary pancreatic tumor
and from disseminated cancer cells before, during and the day after surgery. The focus
of this study is not to identify specific markers for diagnosis or for drug targets but
to find indications of metastasis through the study of gene expression in cancer cells
circulating in the bloodstream. Our main objective is to analyze expression profiles of
circulating pancreatic cancer cells. Changes will be analyzed in the expression of
genes representative of several different pathways frequently altered during the
progression of cancer such as tumor suppressors, oncogenes, signal transduction
molecules, growth factors and angiogenesis factors. Gene expression profile data will
be obtained on apoptosis, cell cycle, cell growth and differentiation, cell migration
and motility, signal transduction and other cancer-related genes. Latest developments
in microarray technology comprise the manufacturing of the complete genome and in
designing new amplification strategies to be able to analyze very small samples less
than 1 ng total RNA (Xiang CC, Chen M, Kozhich OA, Phan QN, Inman JM, Chen Y,
Brownstein MJ. Probe generation directly from small number of cells for DNA microarray
studies. Biotechniques 2003; 34: 386-8, 390, 392-3. Schindler H, Wiese A, Auer J,
Burtscher H. cRNA target preparation for microarrays: comparison of gene expression
profiles generated with different amplification procedures. Anal Biochem 2005; 344:
92-101. Puskás LG, Zvara Á, Hackler L Jr, Van Hummelen P. RNA amplification results in
reproducible microarray data with slight ratio bias. Biotechniques 2002; 32:
1330-1341). In the present study the number of cancer cells circulating in the blood
stream is expected to be extremely small. Therefore, linear RNA amplification (T7
RNA-polymerase) is used in order to generate enough material for labeling and
hybridization on the arrays. Two rounds of amplification are performed routinely. This
has been performed successfully and reproducibly starting from as little as 700 cells
isolated via Laser Capture Microdissection (data unpublished).

- Preparation of cell suspension microarrays to isolate circulating cancer cells.

Although most of the microarray challenges are known and under control, the present
study has an important specific challenge that is the isolation and enrichment of the
circulating cancer cells. Currently, the proportion of circulating cancer cells to
other nucleated blood cells (leucocytes) is unknown in patients suffering from
pancreatic cancer. Therefore, the first step will be to determine the number of cancer
cells per ml blood in pancreatic cancer patients and to determine the variability among
different patients. A second step is to develop an enrichment method to concentrate the
cancer cells without selecting specific types of cancer cells or loss of information on
the initial proportion of cancer cells in each patient's blood stream. The fastest way
to isolate the circulating cancer cells is using flow cytometry after density-gradient
pre-enrichment in combination with negative selection strategies to discard leukocytes
(CD45+ and CD34+) from the sorted 'circulating' cancer cells. Isolating cells with LCM
has been used many times successfully for microarray studies. It has been reported that
LCM can easily dissect several thousands of cells from pancreatic tissue with a
RNA-yield of around 1ng/1000 cells (Datson NA, Meijer L, Steenbergen PJ. Morsiak MC,
van der Laan S, Meijer OC, de Kloet ER. Expression profiling in laser-microdissected
hippocampal subregions in rat brain reveals large subregion specific differences in
expression. Eur J Neurosci 2004; 20: 2541-54. Player A, Barrett JC, Kawasaki ES. Laser
capture microdissection, microarrays and the precise definition of a cancer cell.
Expert Rev Mol Diagn 2004; 4: 831-40. Espina V, Geho D, Mehta AI, Petricoin EF 3rd,
Liotta LA, Rosenblatt KP. Pathology of the future: molecular profiling for targeted
therapy. Cancer Invest 2005; 23: 36-46. Crnogorac-Jurcevic T, Efthimiou E, Nielsen T,
Loader J, Terris B, Stamp G, Baron A, Scarpa A and Lemoine NR. Expression profiling of
microdissected pancreatic adenocarcinomas. Oncogene 2002; 21: 4587-4594).

- Immunological changes

Since a tumor type-specific immune reaction in pancreatic cancer or in patients
undergoing surgery is lacking, the present study will assess the systemic immunological
changes including cytokine levels before and at different time intervals following
potentially curative surgery in pancreatic cancer patients.

PBMC will be extracted from peripheral venous blood and used for further analysis under
FACS (fluorescence-activated cell sorter) to determine the prevalence of:

- CD3-19+ (B-lymphocytes)

- CD3+56+ (Killer cells)

- CD3-56+CD16+ (NK natural killer cells)

- CD8+ (Cytotoxic T-lymphocytes)

- CD4+25- (Helper T-lymphocytes)

- CD4+25+127- (Regulatory T-lymphocytes)

- Inflammatory cytokines in peripheral blood (IL-1b, IL-6, IL-8, IL-10, IL-12 and
TNF) will be analysed using a commercially available 'BD Cytometric Bead Array
(CBA) Human Inflammatory Kit'.

- PBMC (peripheral blood mononuclear cells) and tumour tissue will be stored in
liquid nitrogen for analysis in future studies.

8. Statistical analysis

Univariate analysis as well as corrected, multivariate analysis will be performed in
order to identify the optimally combined prognostic group of factors: patient-,
tumour-, and treatment-associated factors as well as the amounts of disseminated cancer
cells, their genetic expression profile and immunological changes following surgery.
The events to study will be cancer recurrence (disease free survival DFS) and death
(overall survival OS).

9. Expected patient accrual "Single center study": UZ.Gasthuisberg - KU.Leuven, Belgium
Expected annual patient registration: 30 pts / year Duration of patient accrual: 2
years