Category: Pancreatic Cancer

Introduction

• Cancer epidemiology
• Cancer biology
• Carcinogenesis
• Molecular basis of cancer
• Cancer metastasis
• Pancreatic cancer

Epidemiology

In the current age of the cancer research, pancreatic ductal adenocarcinoma (PDA) is one of the most hostile and deadly cancer worldwide (Zhuo-Xin Cheng et al., 2011). Pancreatic cancer is a sixth leading cause of death due to cancer in Europe and in United States it is the sixth due to because of various reasons like poor diagnosis as well as prognosis, lack of screening tests, poor lifestyle, no effective treatment and various risk factors (Lakatos G et al., 2010). The risk factors for pancreatic cancer involve smoking, advancing age, gender, obesity, diabetes mellitus, high-fat diet, overweight, workplace exposure to particular chemicals, family history, chronic pancreatitis, inherited genetic syndrome. Cigarette smoking is a main cause of pancreatic cancer. Almost 20% of pancreatic tumours are due to the cigarette smoking because this smoking causes more genetic mutations in smokers (AudreyVincent et al., 2011).

Annually 168,000 deaths are caused by pancreatic cancer and it is also a ninth most common cause of cancer death. The rate of incidence is very high for cancer of the pancreas, it is even higher than the rate of mortality. The ratio of incidence rate and the mortality rate is very high and close to 98%. The mortality rate is extreme in developed countries whereas, the rate of mortality is not high as compared to developed countries in developing counties except central and temperate South America (Paola Pisani et al., 1999).

Ductal adenocarcinoma is a most common type of pancreatic cancer which has incidence of about 10 in 100,000 population annually. Men are more likely to have this cancer of pancreas than women, the ratio of male and females is 1.5:1. There is no such increase in the incidence since last few decades (Karger AG et al., 2010). The incidence of pancreatic cancer is maximum in ages between 60-80 and pancreatic cancer at below 40 years of age is very rare, but the incidence is approximately 200 in 100,000 annually in 80 years old age groups. In men, the pancreatic carcinoma is a 4th most prevalent cause od death by cancer and in women, it is fifth most frequent cause of death because of cancer (Krejs GJ, 2010).

The incidence rate of pancreatic carcinoma was highest in 2012 in Northern America i.e. 7.4 in 100,000 population and in western Europe 7.3 in 100,000 population followed by other regions like Australia, New Zealand and in other parts of Europe which was about 6.5 per 100,000 population. In South-Central Asia and Middle Africa, the rate of incidence was estimated was very low like 1 in 100,000 population. The differences between populations with high rates of incidence and low rates of incidence were twenty-fold (Milena Ilic and Irena Ilic, 2016). Worldwide, 458,918 new cases of incidence were estimated in 2018. The highest rate of incidence in 2018 was observed in Europe which was about 7.7 per 100,000 population and the lowest rate of incidence was estimated in Africa which was 2.2 in 100,000 population. The differences between the highest incidence rates and lowest incidence rates were 30-fold (Prashanth Rawla, et al., 2019).

In 2018, the mortality rate of pancreatic carcinoma was highest in western parts of Europe which estimated around 7.6 in 100,000 population and the incidence rate was also high in Eastern, Central and Northern Europe which recorded about 7.3 as well as in Northern America was about 6.5. the lowest rate of mortality was found in Eastern Africa (1.4), Western Africa and South-Eastern Asia (Evelina Mocci and Alison P. Klein, 2018). In men, the highest death rate was reported in Republic of Moldova which was 12.3 per 100,000 population and Uruguay which was 12.1 in 2018. Although, among women the death rate was highest in United Arab Emirates which was estimated about 10 per 100,000 population. On the other hand, the lowest death rate was observed in Guinea which was 2.0 and in Pakistan (Tagore Sunkara, et al., 2019).

Survival rates of pancreatic carcinoma is very low in both developed and developing countries and the rate of survival for five-year is around 6 %. Pancreatic carcinoma in both men and women and in all races were 9.4% which was diagnosed although, 29.3% was five-year survival for localized disease during 2006-2012 (Nation Cancer Institute, United States). The five-year survival rate was lower than 3% in men as well as women in England and Whales, it was 3.8% in Denmark and Sweden and in Italy it was 1.2%. the highest five-year survival rate in male was 7% in Estonia and in female, it was 7.5% in Czech Republic. Although there was almost zero survival rate in Malta and in females it was 1.3% in Slovenia (Lei Huang, et al., 2018).

History of pancreatic cancer

In 4th century BC, the pancreas was first described by a Greek physician who is one of the founders of the school of medicine in Alexandria known as Herophilos. The name ‘pancreas’ is a Greek terminology for ‘all flesh’ and this passed to another physician of Greek Ruphos in the 2nd century AD. During 138-201 AD, Galen a Rome physician and also a Rome Emperor illustrated that the function of pancreas was to provide cushion and pad to protect large blood vessels which are directly trailing. As he was very famous physician and Galen’s word was law, his illustration was not challenged for over thousand years. In 1663, the pancreas was first demonstrated as an exocrine gland by Regnier de Graaf in Leiden. After this demonstration 10 years later the first experiment of pancreatectomies on animals was carried out by Johann Brunner in Paris (David Ljungman, 2013).

The studies on pancreas was commenced on March 2, 1642 and pancreatic duct in pancreas was discovered by a German émigré Johann Georg Wirsüng in the San Francisco monastery in Padua, Italy. However, the role of pancreatic duct was still unknown but then it was named as “Duct of Wirsüng”. During 1673-1683, Anthony Van Leeuwenhoek’s microscope models were being gradually improved. Then in 1852, the histology of pancreas was first described in Paris. In 1869, the islets of the pancreas, as well as endocrine system in pancreas, were elucidated by Paul Langerhans and then the islets of pancreas was named as “islets of Langerhans”. In 1889, total pancreatectomy in the dogs resulted in diabetes was proved by Joseph F. Von Mering. Then the Insulin was discovered by a medical student Charles Herbert Best in 1921. The resections of the cancers of the ampulla of Vater and head of the pancreas was undertaken by many surgeons in 1898 and Allen O. Whipple one of these surgeons was known as “Father of Pancreatic Surgery”. In 1974, the biochemical steps in synthesis of protein, transport, ultrastructure unit, secretion, storage and segregation in exocrine pancreatic cell was described by Romanian-American Georg Palade (John M. Howard, Pancreas club 2019).

The Pancreas

The pancreas is an upper abdominal organ which lies behind the stomach and surrounded by other organs like liver, small intestine and spleen. the pancreas is around 15.24 centimetres long, slender in shape. Although pancreas is mainly an exocrine gland which secretes variety of digestive enzymes, the pancreas also consists endocrine function. The pancreas is a part of gastrointestinal system in which it mainly plays role in digestion system by secreting important digestive enzymes and it also plays a role as endocrine gland secreting various hormones into the blood to regulate metabolism and storage throughout the body (Jessie Szalay, 2018).

There are mainly two parts of pancreas based on their function and nature,

Exocrine pancreas

Exocrine pancreas is one component which secrets digestive enzymes into the duodenum and this pancreas consists of acinar and duct cells associated with connective tissue, blood vessels and nerves. These exocrine portions of pancreas comprise more than 95% of pancreas mass.

Endocrine pancreas

It is a small portion of pancreas which produce the various hormones like insulin, glucagon, somatostatin and pancreatic polypeptide in islets and secretes directly into the blood. These islets comprise 1-2% of pancreatic mass.

Gross Anatomy

the long slender-shaped pancreas mass or body consists of three parts i.e. Head, body and tail. The head situated near the duodenum and tail extends to the hilum of the spleen. The head of the pancreas lies in between the loop of the duodenum which exits from the stomach. The body of pancreas is situated posterior to the distal region of stomach. The parts of pancreas which lie anterior to the aorta is bit thinner than adjacent portion of head and body and the common bile duct passes through the head and joins to the main duct of pancreas which is near to the duodenum. The minor papilla is region where accessory pancreatic duct empties into the duodenum and the place where the main pancreatic duct introduce into duodenum known as major papilla. The major papilla is also known as ampulla of Vater (Dan S. Longnecker, Anatomy and histology of pancreas, 2014)

Histology and Ultrastructure

There are very thin connective tissue capsules which surround the pancreas which invades into the glands which results in formation of septae between the glands which play as a stage for the large blood vessels. These septae divides the pancreas and ultimately forms distinctive lobules.

The Acinus

The exocrine pancreas is also known as a compound tubuloacinous gland. The cells in exocrine pancreas which are arranged in grape-like clusters, synthesising and secreting various digestive enzymes known as acini. This acinus is very similar to the salivary gland.

Pancreatic ducts

These digestive enzymes which are synthesized and secreted into the duodenum from acinar cells. These secretions of the acini pass through a tree like series of ducts. These duct cells secret watery fluid which is rich in bicarbonate that flush the enzymes through the ducts and play very important role in neutralizing acids. The pancreatic ducts are classified into four types;

Intercalated ducts

These ducts made up of flattened cuboidal epithelium cells which stretched up into lumen of the acinus which results in formation of centroacinar cells. It is a duct that receives secretion from acini.

Intralobular ducts

It is made up of classical cuboidal epithelium cells. This duct consists within the lobules which receive the secretion from intercalated ducts.

Interlobular ducts

These ducts are situated between the lobules and within the septae of connective tissue. These ducts differ in size. Smaller ducts are made up of cuboidal epithelium, whereas larger one is made up of columnar epithelium. These interlobular ducts carry the secretion from intralobular ducts forward to the major pancreatic duct.

The main pancreatic duct

This main pancreatic duct receives the secretion from interlobular ducts and penetrates through the wall of duodenum. In few species, including human, the pancreatic duct joins the bile duct before entering into the intestine (‘Pancreatic Histology: Exocrine Tissue’).

Function of pancreas

The pancreas has two primary functions, one is to synthesis the various enzymes to digest the proteins, fats, and carbohydrates in intestine and other is to produce the hormones such as insulin, glucagon (Szalay, Jessie, et al., 2018).

Exocrine function:

The pancreas consists of exocrine gland which produces different enzymes which are very essential for the digestion. These enzymes from exocrine gland are mainly trypsin, chymotrypsin to digest the variety proteins and it also includes amylase enzyme which digests the carbohydrates and lipase enzyme to breakdown the fats. These enzymes are produced when food reaches into the stomach and these enzymes travel through a series of ducts and arrive to the main pancreatic duct, then pancreatic duct meet the bile duct which arises from gall bladder and liver towards the duodenum. This meeting point is known as ampulla of Vater (‘The Pancreas and Its Functions | Columbia University Department of Surgery).

Endocrine function:

The endocrine portion of the pancreas includes islets which is also called as islets of Langerhans. This endocrine component produces essential hormones secret directly into the bloodstream. It produces two important hormones known as insulin and glucagon which regulates the blood sugar. Insulin lowers the blood sugar whereas, glucagon increases the blood sugar. Maintaining optimum blood sugar in bloodstream is a very crucial function of the endocrine pancreas (Héctor Del Zotto, et al., 1999).

Pancreas conditions

There are two main conditions of pancreas that includes diabetes type 1, diabetes type 2, cystic fibrosis, pancreatitis, pancreatic cancer, islet cell tumour, enlarged pancreas.

1. Diabetes type 1: It is an autoimmune disorder in which body’s immune system attacks and destroy the insulin-producing cells.
2. Diabetes type 2: In this condition, body becomes resistant to the insulin and no uptake of insulin by cells and blood sugar rises.

Cystic fibrosis:

It is a genetic disorder which affects multiple body systems that includes the pancreas.

Pancreatitis:

It is inflammation of pancreas in which pancreas produces digestive enzymes in excess so that it starts to digest the organ and it causes acute painful inflammation.

Pancreatic cancer:

Pancreatic adenocarcinoma is a most common type of pancreatic cancer in which tumour builds up from the cell lining of pancreatic duct. An endocrine tumour is a rare form of pancreatic cancer.

Islet cell tumour:

It is an endocrine tumour of pancreatic cancer. This tumour produces hormones in very large amounts. It divides and multiplies rapidly from a benign or malignant tumour.

Enlarged pancreas:

It is an anatomical abnormality in which pancreas is larger in size than the normal one.

• Types of pancreatic cancer
• The pancreatic cancer genome
• Ductal adenocarcinoma

1. Panc-1

• Risk factors
• Biological and morphological features
• Etiology of panc-1 cell line
• Treatments for pancreatic cancers
• Surgical resection
• Radiotherapy
• Chemotherapy
• Chemoresistance in panc-1
• Pancreatic cancer stem cells
• Cancer stem cell markers
• Therapeutic resistance
• Origin
• Hypoxia
• Molecular biology of HIF
• Hypoxia induced EMT and CSCs
• NF-kB
• Activation pathway
• NF-kB & chemoresistance
• Role in hypoxia induced EMT
• Drug development
• New drug application
• Repurposing the drug
• Disulfiram
• Pharmacology of disulfiram
• Treatments – anti-alcoholisum , anti-cancer
• Mechanism of action
• Targeting CSCs with Disulfiram
• Proteasome/ NFkB Inhibition
• ALDH inhibition
• Cyclodextrin and its solubility with disulfiram

Abstract

Introduction

Predicting the clinical outcome in pancreatic cancer is often challenging due to the lack of reliable and cost-effective prognostic parameters. Red Cell Distribution Width (RDW), an index of the variability in the size of the circulating RBCs has been reported to have prognostic significance in some malignancies. However, there is a scarcity of literature supporting its relevance in pancreatic cancer.

Objective

1. To study the association between RDW and tumour stage in patients with pancreatic cancer attending a tertiary care hospital in South India.
2. To correlate study the association between RDW and duration of survival among them after surgery for pancreatic cancer.

Methods

Retrospective data of 254 pancreatic cancer patients, who had undergone surgery at a tertiary care centre between 2002 and 2015, was obtained from digital medical records. This was supplemented with data obtained from telephone conversations with the patients and/or their next of kin. Data analysis was performed using SPSS version 20.

Results

Higher RDW values were associated with advanced tumour stages- 84.2% of patients with stage 3 cancer and 92.3% with stage 4 cancer had high RDW values. The mean RDW value for stage 1 patients was 14.93, while that for patients with tumour stages 2, 3, and 4 was 16.14, 17.51, and 18.68 respectively. High RDW values were also significantly associated with lower survival. The mean duration of survival for people with normal RDW values was 83 months while that for patients with higher values was significantly lower at 72 months. This difference was proven to be statistically significant by a log-rank test, which showed a p-value of 0.015. RDW values were found to be independent of age and gender.

Conclusion

There is appears to be a significant association between RDW and tumour stage in pancreatic cancer. RDW also correlates with the duration of survival in pancreatic cancer patients. Thus it may be of considerable useful in predicting the clinical outcome in pancreatic cancer.

Introduction

Pancreatic cancer is a malignant neoplasm of the pancreatic tissue. Although infrequent it is one of the most rapidly lethal cancers with a 5-year survival rate of less than 7 percent [1], causing more than 331,000 deaths per year[2]. Predicting the clinical outcome of pancreatic cancer is often very challenging. Although CA 19-9 levels have increasingly been used as a diagnostic as well as prognostic tool for pancreatic cancer, several some studies have emerged proving its inadequacy show no correlation [3][4]. The American Society of Clinical Oncology does not recommend using CS CA19-9 as a standalone diagnostic and prognostic tool for pancreatic cancer[5].In such a scenario, identification of additional parameters that are reliable, reproducible, and cost-effective could prove to be of substantial help in predicting disease outcomes in pancreatic cancer patients.

Red Cell Distribution Width (RDW) is an index of the variability in the size of the circulating Red Blood Corpuscles. RDW values are widely available to clinicians as a part of routine haemograms, but it is rarely used except to distinguish different types of anemia. Recent research, however, has furnished mounting evidence that a high RDW value is linked to several cardiovascular diseases, sepsis, COPD, and hepatitis [6][7]. Reports indicating that an increase in RDW values correlates with tumour stage and prognosis in lung cancers (Koma et al) [8] and tumour stage and grade in renal cell carcinoma (Wang et al)[9]have garnered considerable attention. There is, however, a deficiency paucity of literature focusing on RDW in the setting of pancreatic cancer. ????[10].The present study attempts to gain insight into the prognostic value of RDW in pancreatic cancer by looking into theat its association between RDW and with tumour stage as well as the impact of RDW values on survival.

Methodology

In this study, we retrospectively analyzed the data of 254 consecutive patients (where Follow up was possible) who had undergone potentially curative resectional surgery for pancreatic cancer at a tertiary referral centre between 2002 and 2015. Data regarding the patient’s demographics, tumour stage, RDW value, and treatment details were obtained from the digital records. Patients(or their next of kin) were then contacted by telephone and the nature of the study was explained to them. A total of eg 280 pts were operated on – 26 patients either had not followed up and could not be contacted telephonically either. They were also informed that their participation in the study was solely on a voluntary basis and not mandatory, however, no one declined. All data (including life status) was entered into a Microsoft Excel spreadsheet. Based on the standard lab reference values for RDW at our centre (11.6 to 14.8) a cutoff value of 14.8 was taken and the patients were divided into two categories –those with high RDW values (more than 14.8) and those with low or normal RDW values(less than or equal to 14.8). Patients were also grouped based on their AJCC tumour stage. The data was then analyzed using SPSS version 20 to look for any association between RDW and tumour stage, variation in RDW values with age and sex, and the impact of RDW on survival using Kaplan Meir survival analysis

Results

The mean age of study participants was 63.69 years. The majority of the patients were in the age group of 60 to 69years (31.88%), followed by 50 to 59 years (28.74%). 64.96% of the study participants were males and 35.04% were females. Out of the 254 cases, the majority (60.2%) were diagnosed with stage 2 cancer.[Table 1]

Table 1

Sociodemographics

Tumour stage number percentagenumber percentage

1. Stage 1 56 22%
2. Stage 2 153 60.2%
3. Stage 3 19 7.5%
4. Stage 4 26 10.2%

• Total 254 100%

Out of 254 patients, 177 (69.8%) had high RDW values (more than 14.8) while remaining 77 (30.31%) had RDW values less than or equal to 14.8.[Table 2]

As the tumour stage advanced, the number of individuals in the high RDW category showed a progressive increase from 48.2% of stage one individuals to 99.3% of stage 4 individuals. (Table 2).

Table 2

Distribution of individuals based on tumour stage and RDW category

Tumour stage low RDW HIGH RDW

1. Stage 1 (56) 29(51.8%) 27(48.2%)
2. Stage 2 (153) 43(28.2%) 110(71.8%)
3. Stage 3 (19) 3(15.8%) 16(84.2%)
4. Stage 4 (26) 2 (0.7%) 24(99.3%)

• Total 77 177

The mean RDW value was found to progressively increase from stage 1 to stage 4. Stage 1 had a mean of 14.9, Stage 2 had a mean of 16.1, stage 3 had a mean of 17.5, and stage 4 had a mean of 18.7. RDW values were found to be independent of age and gender.

The mean duration of survival for people with low RDW values(less than or equal to 14.8) was 83 months while those with RDW values above 14.8 had lower mean duration of survival, 72 months. This is depicted in the Kaplan Meir survival analysis curve in Figure 1. This difference was proven to be statistically significant by a log-rank test, which obtained a p-value of 0.015. The median duration of survival could not be calculated as close to 15% of the individuals who were followed up were still alive.

FIGURE 1

Kaplan Meir survival analysis curve for pancreatic cancer patients with respect to their RDW values

Discussion

RDW definition

Start a short para on RDW

In the current study, RDW values were positively correlated positively with the stage of pancreatic cancer, with a higher RDW value correlating with a more advanced tumour stage. This finding concurs with the results of a previously Studies correlating RDW to tumour stage in other cancers conducted as well as the only study found in English indexed literature by Yilmaz et al in pancreatic cancer [10] which reported a strong association between tumour stage and RDW value among pancreatic cancer patients. Studies correlating RDW to tumour stage in other cancers also showed similar results [8] [9]. The precise mechanism of the association between RDW and tumour stage is unknown. Several factors affecting RDW values such as RBC circulation half-life and membrane deformability can be affected by inflammation [11] which is recognized as a hallmark of tumour development. Research studies point toward a possible correlation between RDW and C-reactive protein (CRP) values [12]. Neote et al [13]detected several receptors for inflammatory factors on the RBC surface and hypothesized that RBCs are involved in the inflammatory process. Forhecz et al[14] found that inflammatory factors potentially affect iron metabolism, and RBC life span, and cause the release of immature red blood cells into circulation causing an increase in the RDW. Inflammatory factors have also been hypothesized to suppress the action of erythropoietin on erythroid stem cells and prevent the antiapoptotic effect of erythropoietin on maturating RBCs. [14]

The current study also showed that the mean duration of survival for patients with low RDW values(less than or equal to 14.8) was significantly higher than those with high RDW values (above 14.8). This result is in agreement with several previously published studies. Podhorecka et al showed that elevated RDW was associated with shorter survival time in patients with chronic lymphocytic leukemia [15]. A metanalysis by Ai et al indicated that increased pretreatment RDW predicted poorer overall survival, progress-free survival, and event-free survival in patients suffering from hematological malignancies [16]. The reason for this association too remains unknown. A possible explanation is that RDW has correlated with several circulating cytokines such as Il-6, tumor necrosis factor-alpha, and hepcidin that can affect the biological behavior of tumor cells [17, 18].

While the finding of the current study that RDW was found to be independent of sex gender is in agreement with studies such as that by Hoffman et al[19], our finding that RDWhowever unlike them we was also found to be independent of no correlation with age differs from the conclusions arrived at by Hoffman et al[19] who found that RDW increases with age. While the possible as the aforementioned study was conducted among presumably healthy individuals. It is likely that the present study was conducted in individuals with malignancies, a condition associated with there could be increased signaling along the IGFs/mTOR pathway. Persistent IGF-1/mTOR signaling enhances erythropoiesis by activating erythropoietin [20] [21] resulting in heterogeneity of RBC size due to a high turnover rates. So the RDW becomes affected by the metabolically driven (growth factor – IGF-1/mTOR) signaling rather than chronological aging.

While the present study shows that RDW has a strong association with tumour stage and survival rate, many of our study subjects(53% )were lost to follow up and this may have led to attrition bias.

In conclusion, there is appears to be a significant association between RDW and tumour stage in pancreatic cancer patients.RDW also correlates with duration of survival in these patients. It is a simple and easily available parameter, obtained at no additional cost incurred to the patient. Therefore RDW may be of considerable use in predicting the clinical outcome in pancreatic cancer patients.

However, in view of the limitations of the present study, larger-scale prospective studies be would be required to confirm these results.

References

1. Michaud D.S. (2017) Epidemiology of Pancreatic Cancer. In: Loda M., Mucci L., Mittelstadt M., Van Hemelrijck M., Cotter M. (eds) Pathology and Epidemiology of Cancer. Springer, Cham DOI https://doi.org/10.1007/978-3-319-35153-7_25Publisher Name Springer, Cham Print ISBN 978-3-319-35151-3 Online ISBN978-3-319-35153-7
2. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. GLOBOCAN 2012 v1.0, Cancer Incidence and 2.Mortality Worldwide: IARC CancerBase No. 11. Lyon, France: International Agency for Research on Cancer; 2013. Accessed 2016-03-04.
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4. Howaizi M, Abboura M, Krespine C, Sbai-Idrissi MS, Marty O, Djabbari-Sobhani M. A new cause for CA19.9 elevation: heavy tea consumption. Gut. 2003;52:913–914.
5. Locker GY, Hamilton S, Harris J, et al. ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer. J ClinOncol. 2006;24:5313–5327.
6. Ye Z, Smith C, Kullo IJ. Usefulness of red cell distribution width to predict mortality in patients with peripheral artery disease. Am J Cardiol. 2011;107:1241–1245.
7. Patel KV, Semba RD, Ferrucci L, Newman AB, Fried LP, Wallace RB, Bandinelli S, Phillips CS, Yu B, Connelly S, Shlipak MG, Chaves PH, Launer LJ, Ershler WB, Harris TB, Longo DL, Guralnik JM. Red cell distribution width and mortality in older adults: a meta-analysis. J Gerontol A BiolSci Med Sci. 2010;65:258–265
8. Increased red blood cell distribution width is associated with cancer stage and prognosis in patients with lung cancer. Koma Y, Onishi A, Matsuoka H, Oda N, Yokota N, Matsumoto Y, et al. (2013)
9. Red Cell Distribution Width Is Associated with Presence, Stage, and Grade in Patients with Renal Cell Carcinoma Fang-Ming Wang, 1 Gongjun Xu, 2 Yan Zhang, 3 and Lu-Lin Ma
10. Effect of pre-operative red blood cell distribution on cancer stage and morbidity rate in patients with pancreatic cancer. A Yilmaz,1 FU Malya,2 G Ozturk,1 B Citgez,2 Y Ozdenkaya,1 C Ersavas,1 AF Agan,3 H Senturk,4 and O Karatepe1
11. Weiss G, Goodnough LT. Anemia of Chronic Disease. N Engl J Med. 2005;35210:1011–23.
12. Lippi G, Plebani M. Red blood cell distribution width (RDW) and human pathology. One size fits all. ClinChem Lab Med 2014;52:1247–9.
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Pancreatic Cancer

Pancreatic cancer is a silent killer and “is the fourth leading cause of death in the United States” (Reynolds and Folloder, 2014, p. 356). “Pancreatic cancer will often develop without clear early signs or symptoms, and the eventual manifestations will depend on the tumor location within the gland” (Reynolds and Folloder, 2014, p. 357). Pancreatic cancer is of significant concern for nursing because the signs and symptoms are very broad and can overlap with other disease processes. Pancreaticoduodenectomy is a treatment option for patients who can have the tumor removed. If the tumor is unable to be removed then their treatment options would either be systemic chemotherapy or chemoradiation. Educating the patient and caregivers about side effects of the treatment or therapy can help them to take immediate action for these adverse events.

The purpose of this paper is to educate on the anatomy and physiology or pancreatic cancer, describe the treatment of the patient, how a pancreaticoduodenectomy is effective but still has some consequences that the patient should be aware of. The subject, M.V., and the medical treatment received by this patient will be compared to the textbook management. M.V.’s health deviations that were present during the clinical setting and the nursing care provided will be discussed. The article Clinical Management of Pancreatic Cancer written by Reynolds and Folloder (2014) will be used to discuss the nursing practice and clinical management of patients who are diagnosed with pancreatic cancer.

Basic Conditioning Factors

The patient M.V. was admitted on February 3rd, 2019 to Long Beach Memorial Medical Center (LBMMC). M.V.’s medical diagnosis was pancreatic cancer. Her medical doctor was Dr. Arguita, the responsible nurse was Andrew, and her coassigned nurse assistant was Belinda. M.V.’s surgery on February 6th, 2019, was a Whipple procedure which is also known as a pancreaticoduodenectomy, to treat pancreatic cancer. M.V. is a 61-year-old Hispanic female who is a retired certified nursing assistant (CNA). Her family roles are a wife, mother, grandmother, and daughter. She has no allergies and her previous medical history are hypertension and diabetes mellitus. M.V.’s code status was full while a patient at LBMMC.

According to Potter, Perry, Stockert, and Hall (2017), M.V.’s developmental level is Generativity versus Self-Absorption and Stagnation. This is the middle age adult developmental level where a person contributes in a positive way to their family and/or community. Stagnation is the opposite of this because this person is concerned only for them self and does not want to help the next generation. One could achieve generativity by contributing to the growth and development of the next generation by teaching, volunteering, or any other way to help in the community. If a person does not want to contribute to the development of the next generation they fall under stagnation. Stagnation is when a person is only worried about themselves and how they benefit from what they are doing at work, with friends and family, or will they benefit from helping others.

During clinical rotation, M.V. was asked what she enjoyed doing with her family, what she looked forward to once discharged, and any hobbies she had. M.V. shared that she enjoyed just being in the company of her mother, children, grandchildren, and husband. She goes to church and volunteers when asked. She enjoys taking her grandchildren to school and babysitting them while their parents are at work or school. She looked forward to being home and recovering with her family and also getting a new manicure and pedicure. She also shared that she just wanted to be home because that is where she knew she would get better quicker because some of her kids couldn’t make it to visit her in the hospital. M.V. has currently achieved generativity based on the definition given by Potter, Perry, Stockert, and Hall (2017).

Anatomy and Physiology of the Involved Organs

According to Hinkle and Cheever (2018), the pancreas, common bile duct, gallbladder, and duodenum are all the organs involved in the tumor of the head of the pancreas. The function of the pancreas is to produce enzymes to help with digestion in the exocrine system and produces hormones for blood glucose regulation in the endocrine system. The common bile duct carries bile from the gallbladder and empties the bile into the small intestine. The gallbladder stores bile, which is an enzyme that is produced by the liver, which then releases the bile through the common bile duct into the duodenum. The function of the duodenum is chemical digestion of the secretions from the pancreas, liver, and gallbladder which all mix with chyme from the stomach. The pancreas is located in the upper abdomen and is between the duodenum and spleen. The common bile duct is located at the end of the gallbladder and is attached to the head of the pancreas which then leads into the duodenum. The gallbladder is located below the liver. The duodenum is located anterior to the pancreas and is the beginning of the small intestine.

Pathophysiology

According to Hinkle and Cheever (2018), people that are at higher risk for pancreatic cancer are individuals that have a history of smoking cigarettes, diabetes, chronic pancreatitis, and any hereditary pancreatitis. “Pancreatic cancer is the fifth leading cause of cancer death in women” (Hinkle & Cheever, 2018, p. 1450). Hinkle and Cheever (2018), continue to explain that pancreatic cancer can occur at the head, body, or tail of the pancreas. Thy symptoms are not specific and patients do not seek treatment until it is late into the disease. Patients can present with pain in the abdomen, weight loss, and jaundice. The pain can sometimes move to the midback and can become severe over time. Metastasis can occur if the malignant cells from the pancreatic cancer go into the peritoneal cavity. Sometimes ascites can occur because of this metastasis. The onset of insulin deficiency, glucosuria, hyperglycemia, and an abnormal glucose tolerance is a very important sign that some patients will present with if the malignant cells have moved and caused metastasis. “There is no known biomarker specific to pancreatic cancer, but carbohydrate 19-9 (CA 19-9)…” (Reynolds and Folloder, 2014, p. 358).

Hinkle and Cheever (2018), describe the medical management of pancreatic cancer depends on if it can be resectable and how localized is the tumor. If the tumor is resectable then the patient will undergo a very comprehensive procedure which is called the Whipple procedure. To remove the tumor completely is not possible sometimes due to two factors, one, how large the tumor has grown, and secondly, if the malignant cells have metastasized into the liver, lungs, or bones. If these factors occur then palliative measures will more than likely be the treatment option. Pancreatic tumors are resistant to basic radiation therapy, so the patient may need to be treated with chemotherapy and radiation.

Hinkle and Cheever (2018), advise that the nursing management for pancreatic cancer is pain management and assessing the nutritional requirements to help with improving the patient’s comfort level. Due to the pain that the patient may have because of the pancreatic cancer, opioids are used to help alleviate the pain. Patients who have severe and excruciating pain may need to be considered to have patient-controlled analgesia (PCA). Patients who are at the end of life may need to be referred to hospice care.

M.V. went to her primary care physician with a chief complaint of abdominal pain for two weeks. She also presented with nausea, vomiting, and diarrhea. M.V. then came to the emergency department Dr. Arguita admitted her with a diagnosis for abdomen pain. The emergency department ran a CBC that showed elevated liver function tests (LFTs) and a CT scan with findings of gallstones. Admitting physician, Dr. Argita, and the surgeon, Dr. Do, preoperatively diagnosed M.V. with gallstones and elevated LFTs (Long Beach Memorial Medical Center, 2019). The signs and symptoms that M.V. presented with correlate with the signs and symptoms Hinkle and Cheever (2018) described which was abdominal pain and her history of diabetes mellitus also put her at risk for pancreatic cancer.

M.V.’s preoperative diagnosis of gallstones was what lead to getting a biopsy done prior to continuing with removal of the gallstones. Intraoperatively, the biopsy findings were that of pancreatic cancer. Dr. Do proceeded with removing the gallstones and then the extensive Whipple procedure. (Long Beach Memorial Medical Center, 2019). The surgery resulted in the removal of the head of the pancreas and the rest of the pancreas was attached to the side of the jejunum. The common bile duct is attached to the side of the jejunum and the end of the stomach is sutured to the side of the jejunum as well (Hinkle & Cheever, 2018, p. 1452). The estimated blood loss was 100 milliliters. The intraoperative findings resulted in the postoperative diagnosis of pancreatic cancer.

Medical Orders

• Diet
• Activity

Nurse Responsibilities

NPO due to radiology procedure to insert a central venous catheter with placement of port.

Bedside commode with assistance

Notify MD if WBCs were still high to make the decision of proceeding with port placement

Saline lock

• Jackson Pratt drains – note color, amount, and characteristics of drainage
• G-tube enterostomy tube – note any infection, color, amount, and characteristics of drainage.

Note: (Long Beach Memorial Medical Center, 2019)

Medications

• Drug
• Dose
• Route
• Frequency
• Therapeutic effect
• Nursing responsibilities
• Relation to medical diagnosis
• amlodipine
• (Norvasc
• 5 mg
• PO
• Once daily
• Lower blood pressure.

Monitor blood pressure and pulse, intake and output, and daily weight. Assess for signs of angina.

M.V. has a history of hypertension. This does not relate to the medical diagnosis.

Antihypertensive to lower blood pressure by causing vasodilation.

cefoxitin (Mefoxin) 2 g in NS 100 mL

IVPB

Once daily

To prevent infection.

Assess for signs and symptoms of infection: surgical site. Assess WBC lab results. No labs to be drawn within 2 hours od administering this medication due to false increase of serum & urine creatinine.

M.V.’s Whipple procedure and her pancreatic cancer increase the risk of infection. Postoperative infection risks can be reduced when administering antibiotics.

• hydrocodone/ acetaminophen
• (Norco)
• 7.5-325 mg
• PO
• q 4 hours PRN
• 1 tab for pain (4-6)
• 2 tabs for pain (7-10)

For pain management

Do not exceed 4 g/day. Assess blood pressure, pulse, respiratory rate, and bowel function. If the respiratory rate is 60

• 65-99
• 136-145
• 3.5-5.1
• 8.5-10.1
• 15-37 u/L
• 13-56 u/L
• 45-117 u/L
• 0.2-1.0 mg/dL
• 73-393
• 2-37
• 6.0
• 14.3
• 43.1
• 223
• 6
• 0.76
• >60
• 126
• 139
• 3.9
• 9.3
• 445
• 811
• 232
• 2.1
• 315
• 32 (02/07/19)
• 11.6
• 9.5
• 29.4
• 332
• 8
• 0.67
• >60
• 167
• 137
• 3.6
• 7.9
• 37
• 59
• 152
• 0.4
• UTZ
• CT
• Abdomen

MRCP without contrast

Note: (Long Beach Memorial Medical Center, 2019), (Pagana, Pagana, & Pagana, 2015)

When diagnosing M.V. with gallstones as stated before the main lab values and tests to determine this diagnosis were the CT scan and the CBC. On her admission date February 3rd, 2019, her LFTs which consist of the enzymes that are found in the liver, that include alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase, and bilirubin. M.V.’s ALT levels were elevated at 811, AST levels were elevated at 445, alkaline phosphatase levels were elevated at 232, and bilirubin levels were elevated at 2.1 (Pagana, Pagana, & Pagana, 2017). The LFTs check to see how well the liver is working. The CT scan also solidified the diagnosis with its findings of gallstones obstructing the common bile duct (Long Beach Memorial Medical Center, 2019).

Health Deviations

Balance Between Solitude and Social Interaction

Health deviations brought on by pancreatic cancer. The patient may not be able to interact in a social environment due to pain and would be in solitude. The pain from pancreatic cancer can inhibit a patient from meeting these universal needs. The psychosocial concerns for these patients would be about relieving the pain.

Maintenance of Sufficient Intake of Air

Health deviation brought on by pancreatic cancer. The patient may hyperventilate as a response to pain. Hyperventilation causes a release of carbon dioxide quickly which stimulates an increase in respiratory rate which leads to respiratory alkalosis (Potter, Perry, Stockert, & Hall, 2017). This can be a consequence of insufficient intake of air because the brain won’t have enough air to profuse which then the patient may have a lack of consciousness. Pancreatic cancer is effected more because the heart rate and blood pressure will increase as a response to pain.

Maintenance of Sufficient Intake of Food and Water

Health deviation brought on by pancreatic cancer. The malabsorption of nutrients and fat-soluble vitamins can obstruct the common bile duct can decrease the secretions of bile which aids in the breakdown of chyme from the stomach. Pancreatic cancer can lead to anorexia because of discomfort and pain in the abdominal area (Hinkle & Cheever, 2018).

Maintenance of Sufficient Elimination of Water.

Health deviation brought on by pancreatic cancer. Ascites is common in these patients. There for monitoring for any abdominal cavity swelling and distention is very important. These signs and symptoms can further look into any metastasized cells of pancreatic cancer (Hinkle & Cheever, 2018).

Maintenance of Balance Between Activity and Rest

Health deviation brought on by pancreatic cancer. Immobility due to the presence of pain that is caused by the pancreatic cancer. Acute pain can affect the patient’s ability to walk or rest comfortably. When patients are uncomfortable and in pain, sleeping patterns are disrupted.

Promotion of Normalcy

Health deviation brought on by pancreatic cancer. Normalcy is affected by the pain from pancreatic cancer. Routine lifestyle activities are limited. Patients are not comfortable with body movements and become malaise when getting up and using the toilet. Weight loss can occur due to the abdominal pain from pancreatic cancer as well. Resting would be affected if the pain is not able to be controlled by opioid drug therapy.

Prevention of Hazards

Health deviation brought on by pancreatic cancer. The pain brought on by pancreatic cancer causes the patient to be very cautious of how one moves. Patients guard and move to a comfortable position to maintain a level of relief.

Nursing Diagnosis

The priority nursing diagnosis for M.V. is acute pain related to surgical incision, secondary to the Whipple procedure, as evidenced by numeric pain scale of 6/6/4 and verbal report of pain that she felt in the lower abdominal region. M.V.’s goal on February 14th, 2019 for this nursing diagnosis was that she will state a pain less than six within one hour of administering Norco. The nursing interventions to help M.V. to reach this goal was to reassess her pain level in one hour, to administer the Norco prescription as needed for the pain every four hours according to the parameters, advise deep breathing exercise (Ladwing, Ackley, & Makic, 2017).

The potential complication for M.V. in regards to the pancreatic cancer is paralytic ileus (Potter, Perry, Stockert, & Hall, 2017). The nurse will monitor, manage, minimize paralytic ileus and collaboratively intervene to reach this goal. The nurse will monitor the signs and symptoms which are nausea, vomiting, constipation, loss of appetite, inability to pass gas, and the distention of the abdomen (Mestrovic, 2018). The nurse will manage paralytic ileus by a drug treatment with alvimopan or stimulate the bowel movement with prescribed lactulose or neostigmine (Mestrovic, 2018). The nurse will minimize paralytic ileus by getting M.V. to ambulate at least three times per shift, encourage patient to report any changes in her pain level, and encourage fluid intake.

Introduction

Pancreatic cancer (PC) is highly lethal malignancy and consider the fourth most common cause of cancer-related deaths worldwide. The majority of patients, approximately 80%, are diagnosed at an advanced and incurable stage, advanced local (III) or metastatic (IV), of disease, and only around 20% of cancers are suitable for surgical resection. The prognosis for PC is extremely poor, with only 7% five-year survival rate. Few chemotherapy treatments have been shown to slightly improve survival for advanced pancreatic cancer patients, but even with the best treatments currently available, the median survival for these patients is less than one year. For over 40 years, survival rates in pancreatic cancer have not shown any improvement, and there is a clear need for further research of this disease to improve patient outcomes.

Palliative indiscriminate chemotherapy still is the standard of care for patients with advanced PC with no biomarkers currently available to guide the likelihood of an individual’s response to treatment. In recent decades, the treatment of many cancers has been evolved, in which screening for specific molecular changes is applied to patients first, and then targeted therapy is applied accordingly. Examples of this approach include the use of EGFR and ALK inhibitors in non-small cell lung cancers harbouring EGFR mutations or ALK gene rearrangements; BRAF and MEK inhibitors in melanoma; and trastuzumab and pertuzumab in HER2 amplified breast cancer, all of which have led to clinically meaningful improvements in patient outcomes. Given the limitations of current treatment regimens, the prospect of applying precision medicine to the treatment of PDAC holds great appeal.

Pancreatic ductal adenocarcinoma (PDAC) accounts for about 90-95% of all pancreatic cancer cases

Genomic landscape of PDAC

Recent advances in next-generation sequencing technologies have allowed for significant progress in the characterization of the genomic landscape of pancreatic cancer, leading to better understanding of the pathogenesis of this disease and identification of a number of potential therapeutic targets.

An activating somatic mutation of the KRAS gene has long been implicated as a critical event occurring early in the development of the overwhelming majority of human PDAC. Mutation of this gene has been implicated in the progression of pre-malignant pancreatic intra-epithelial neoplasia (Pan-IN) into invasive malignancy, and has also been demonstrated to play a vital role in tumour maintenance. Early mutation in KRAS is typically followed by the loss of multiple tumour suppressor genes, most notably CDKN2A, TP53 and SMAD4. However, beyond these common mutations, recent studies have revealed significant tumour heterogeneity among PDAC patients, highlighting a major challenge to the application of precision medicine to this disease.

In 2014, a large genome wide association study of more than 7000 PDAC patients identified numerous susceptibility loci for PDAC lying in close proximity to a variety of genes, some of which have previously been implicated in oncogenesis (e.g. BCAR1, KLF14, PDX1, CHEK2, TERT). More recently, whole exome sequencing on a smaller cohort of 109 patients identified that 5% of tumours contained 24 significantly mutated genes with potential prognostic significance (e.g. KRAS) as well as potential for therapeutic targeting (e.g. BRAF, PIK3CA). Further, comprehensive analysis of 24 PDAC tumours identified an average of 63 genetic mutations in each tumour and described alterations in 12 core signaling pathways, some of which (e.g. DNA damage repair, alterations in cell cycle regulation) may also be amenable to targeted therapy. Indeed, a number of gene mutations with potential for targeted therapy can be identified using resources such as the COSMIC database (Catalogue of Somatic Mutations in Cancer), but most occur at a low overall frequency in PDAC. Despite this, a recent comprehensive analysis integrating genomic, transcriptomic and proteomic profiling of 150 PDAC specimens identified that 42% of patients harboured at least one alteration which could potentially inform enrolment in a genotype directed clinical trial.

EUS FNA as a source of tissue and genetic material

Overwhelmingly, genomic profiling of PDAC has relied on surgical resection specimens to obtain tumour material. EUS FNA is a well-established, minimally invasive biopsy technique which can be utilized in patients at any stage of disease. It is generally considered a safe procedure, as evidenced by a large systematic review of over 10,000 patients undergoing EUS FNA across multiple institutions which reported reassuringly low morbidity (0.98%) and mortality (0.02%) rates associated with the procedure.

EUS FNA biopsy specimens can be used as a source of tissue for genetic analysis of PDAC, although widespread clinical use has been limited due to concerns regarding small tissue quantities, suboptimal yield of genetic material, and potential contamination of samples with non-malignant cells such as blood, inflammatory cells and stomach or intestinal wall cells. However, despite some inherent challenges, the potential to use this technique to obtain tissue from patients at all disease stages with relative ease is a clear advantage.

There are several approaches which have been demonstrated to improve the sensitivity and yield of EUS FNA-derived tissue for diagnosis and genetic analysis, including using larger needles and increased number of passes, utilizing on-site cytology services, and optimizing sample processing including using techniques such as snap freezing biopsies in liquid nitrogen and using RNA-preserving agents such as RNAlater™. Multiple studies have reported improved diagnostic sensitivity of the procedure using EUS FNA-derived genomic DNA to detect KRAS mutations, showing that this technique can be reliably used as a source of genomic DNA. Several studies have also demonstrated that EUS FNA can be used to reliably extract and sequence RNA for gene-expression profiling and to derive diagnostic gene signatures.

EUS FNA biopsies are also poised to play a role in establishing valuable pre-clinical disease models for precision therapy, such as patient-derived xenografts and organoid cultures. Pre-clinical disease models which accurately reflect the histological and molecular make-up of a tumour allow for rapid in-vivo testing and drug screening, and can support in-vitro findings. PDX involves implanting patient-derived cancer cells into immunodeficient mice to grow tumours, which have been shown to reliably retain the original histological architecture, cellular characteristics and molecular profiles of the original patient tumour over multiple passages. However, the lack of human stromal elements and the absence of a functional immune system is a limitation of these PDX models, particularly when considering in-vivo testing of immunotherapeutic agents. These limitations can be addressed to a degree through “humanizing” PDX models in-vivo, by reconstituting with human immune cells or tissue.

While PDX models have historically been largely reliant on surgical specimens, our group is among those to recently show that EUS FNA biopsies can also be successfully used to create PDX models which maintain their original tumour characteristics. Using defined media and conditions, patient derived tumour cells can also be used to grow organoid cultures, which provide three-dimensional cell cultures that can be used to complement PDXs in molecular analysis of PDAC and for longitudinal therapeutic testing in “real-time”. These organoid cultures can be generated using EUS FNA derived tissue in a short period of time, serially passaged, and used to create PDX models.

Our group has recently optimized a novel protocol for the simultaneous extraction of genomic DNA and RNA from EUS FNA biopsies, and demonstrated that EUS-FNA derived PDAC biopsies can be used to establish patient derived xenografts (PDXs) which can then be utilized for in-vivo testing of targeted therapies on molecular-profiled tumours. This translational pipeline allows for molecular screening and testing of targeted therapy on tumours derived from all clinical stages of disease. The benefits of this approach and contribution to bench-to-bedside care are evidenced by the subsequent initiation of the phase II clinical trial “Panitumumab in KRAS wild-type pancreatic cancer” at the Monash Health Translation Precinct, which is described in detail in the methodology section below. This pipeline leaves us well poised to examine our immune targets of interest in PDAC.

Immune regulation in pancreatic cancer

Dysregulation of the innate immune system can lead to chronic inflammation in the pancreas, which is well established as a risk factor for development and progression of invasive malignancy. Pro-inflammatory factors in the tumour microenvironment including cytokines (e.g. TNF-α, IL-6), chemokines (e.g. CCL-2), reactive oxygen species, angiogenic factors and growth factors, can promote growth of the underlying pancreatic epithelium and facilitate oncogene activation, leading to promotion of tumour cell proliferation, invasion and metastasis.

The tumour microenvironment in PDAC is rich in immune cells, and antigen presenting cells are capable of evoking an anti-tumour response by activating natural killer (NK) and cytotoxic T cells to eradicate tumour cells. However, spontaneous regression of tumours is extremely rare; and it is evident that pancreatic cancer cells are capable of inducing local immune dysfunction, leading to an immunosuppressive tumour microenvironment and protecting the tumour cells from immune attack. Immune cell function has also been demonstrated to be modulated by chemotherapy agents, and via interaction with stromal elements such as pancreatic stellate cells, which contribute to the typical desmoplastic reaction seen in PDAC.

In recent years, we have seen the emergence of immunotherapy as a viable and effective treatment modality which has been demonstrated to improve overall survival rates in several malignancies while maintaining a favourable toxicity profile; the most notable clinical responses are seen in melanoma, renal cell carcinoma and non-small cell lung cancer. These successes, along with the above observations noting an immune cell rich tumour microenvironment capable of eliciting anti-tumour response in PDAC, support the targeting of innate and adaptive immune responses as an appealing therapeutic prospect despite the known heterogeneity of the tumour genetics and local environment. There are a number of broad approaches to immunotherapy, including passive approaches such as the use of antibodies or effector T cells to target tumour-specific antigens or molecules involved in tumour progression; and active approaches such as vaccination with tumour antigens or irradiated tumour cells, or targeting immune checkpoint inhibition. However, to date the application of immunotherapy in unselected PDAC patients has proven disappointing. Applying a precision medicine approach to immunotherapy in PDAC may improve responses, by identifying predictive biomarkers to select and stratify patients towards specific treatments.

Our group has performed RNA sequencing on a number of EUS FNA-derived PDAC biopsies, and identified toll-like receptor 2 (TLR2), bone-morphogenetic protein 4 (BMP4) and interleukin 6 (IL-6) as genes of interest which display dysregulated expression across all clinical stages of PDAC. All three genes regulate a wide range of oncogenic processes affecting both tumour and immune cells, have previously been associated with carcinogenesis, and also represent potentially “actionable” targets with available therapeutic inhibitors. They are therefore attractive candidates to study further for immune-based precision medicine in PDAC. My initial focus is on TLR2, which is discussed in further detail below.

Abstract:

Epigallocatechin-3-gallate (EGCG) is a polyphenol from green tea extract is known to suppress the human pancreatic cancer in-vitro. Its anti-proliferative action mediated by caspase-3-activation, nuclear condensation and poly-ADP ribose polymerase cleavage. Pancreatic cancer cell death by EGCG is mediated by arresting growth at an initial stage of cell cycle. EGCG involves depolarisation mitochondrial membrane to allow cytochrome-c release into cytosal. EGCG increased the production of intracellular release oxygen species (ROS), along with C-Jun-N-terminal kinase activation in pancreatic carcinoma cells. In brief EGCG induces stress signals by substracting mitochondria and ROS-mediated JNK activation in MIA PaCa-2 pancreatic cancer cells.

Keywords: Epigallocatechin-3-gallate (EGCG), caspase-3-activation, nuclear condensation, poly-ADP ribose polymerase cleavage, ROS and MIA PaCa-2 pancreatic cancer cell.

Introduction:

Cancer is a disease of cells characterized by progressive, persistent, purposeless and uncontrolled proliferation of tissues. It has the ability to spread to other parts of the body from its origin. Green tea, popularly consuming drink around the world is known has anti-cancer activity through its polyphenols such as EGCG. EGCG is a broad spectrum anticancer activity against pancreatic carcinoma. Green tea has non-toxic activity and it is preferably in treating cancer in different organ mainly pancreatic cancer.

Materials And Methods

Materials:

• Human pancreatic carcinoma
• MIA PaCa-2
• PANC-1
• AsPc-1
• The above mentioned are growth in Petri plate, supplemented with 10% fetal bovine serum (FBS) and 50 µg/ml gentamicin at 37 degrees Celcius in 5% CO2 humidified atmosphere.

Methods:

• Soft agar assay

A total of ~20 000 cells was resuspended in 0.35% noble agar supplemented with 10% FBS and plated on 6-cm plates containing a solidified bottom layer (0.5% noble agar in growth medium). The plates were incubated in a humidified incubator at 37oC for 10–14 days. The plates were stained with 0.005% crystal violet. The colonies were counted by using an inverted microscope.

• Analysis of cell death

Chromatin condensation was determined by 4’, 6-diamidino-2-phenylindole (DAPI) fluorescence as described previously. A total of 5×106 cells (in triplicate plates) was cultured in the growth medium in the presence or absence of 0.1–0.2 µM EGCG for 24 h. After fixing and permeabilization, the cells were mounted in a fluid containing 2 µg/ml DAPI (Vector Laboratories, Burlingame, CA). A Nikon Eclipse E600 fluorescence microscope (Huntley, IL) was used to visualize the nuclear stain.

• Poly-ADP ribose polymerase (PARP) cleavage analysis

Cells were treated with specified concentrations of EGCG for designated time periods. After treatment, the total cellular proteins were extracted as described previously. After normalization for the total protein content, the resulting lysate was subjected to SDS–PAGE and immunoblotting with monoclonal against PARP (Pharmingen, San Diego, CA). Immunodetection was accomplished by enhanced chemiluminescence method.

• Cell cycle analysis by flow cytometry

After the designated treatment period with 0.2µMEGCG, cells were sorted by a fluorescence activated cell sorter at Ireland Cancer Center’s core facility as described previously. Mitochondrial membrane depolarization study by confocal microscopy MIA PaCa-2 cells was grown in a 6-well tissue culture dish. The cells were either treated with 0.2 µM EGCG or untreated for 14 h. After treatment, the medium was replaced with serum-free medium containing 10 mg/ml JC-1, a potential-dependent J-1 aggregate forming lipophilic cation (Molecular Probes, Eugene, OR). Cells were incubated at 37oC for 10 min followed by washing with PBS. Immediately, the cells were visualized by a confocal laser-scanning microscope (Leica SP2, Bannockburn, IL). The monomer and J-aggregate forms were simultaneously excited by 488-nm argon-ion laser sources. Polarized mitochondria were marked by punctate orange-red fluorescence staining.

• Determination of cytochrome c and oligomeric Bax by immunofluorescence microscopy

For cytochrome c, control and EGCG-exposed cells were fixed and permeabilized followed by immunostaining with mouse monoclonal cytochrome c antibody as primary and CY3 conjugated goat anti-mouse IgG as secondary. To detect oligomeric Bax, immunostaining was performed with mouse anti- Bax monoclonal antibody 6A7 as primary. Slides were visualized under a Leica SP2 confocal microscope.

• Immunocomplex kinase assay

Equal amount of protein from control and EGCG-treated MIA PaCa-2 cells were subjected to immunoprecipitation with anti-human c-Jun N-terminal kinase (JNK) antibody in lysis buffer containing 10 µM HEPES, pH 7.2, 142 µM KCl, 5 µM MgCl2, 1 µM EGTA, 0.2% NP-40 and protease inhibitors. After overnight incubation at 4oC, the immunocomplex was trapped in Protein A-sepharose CL4B beads (Amersham-Pharmacia Biotech) by incubating for 2 h at 4oC. After washing, the kinase reaction for JNK was performed by incubating the immunoprecipitated proteins in 25 µM HEPES, pH 7.5, 25 µM MgCl2, 25mMb-glycerophosphate, 1 µM DTT, 0.1 µM sodium orthovanadate, 10 µM ATP, 2 ml activated JNK (Calbiochem) and 10 µCi of [g-32P] ATP. Reactions were carried out at 30oC for 15 min. Kinase reactions were terminated by boiling with Lammeli’s sample buffer followed by SDS–PAGE and autoradiography.

• Measurement of reactive oxygen species (ROS)

Flow cytometric determination of ROS production was carried out as described previously. MIA PaCa-2 cells were collected by trypsinization and 200 µl of cell suspension (2×106 cells/ml) was mixed with 800 µl PBS. Cells were incubated with 10 µM 5-(and-6)-chloromethyl—2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester (molecular probes) for 15 min, followed by the addition of EGCG or hydrogen peroxide. The incubation was continued for 20 min at 370 C. The oxidative burst (hydrogen peroxide) was detected using a FACScan flow cytometer with excitation and emission settings of 488 and 530 nm, respectively.

‘Cancer’ – a word so small yet so destructive. In fact, in 2017, in the UK alone there was an astonishing 4 million cases and 9.56 million deaths worldwide. An inevitable disease that just continues to increase in size. As the philosopher Desiderius Erasmus once said, ‘prevention is better than cure’ – this is most certainly the case with cancer.

Cancer survival rates are the highest they have ever been. A way to improve survival rates even further is for cancer to be diagnosed even earlier. And the NHS’ plan to do such suggests that ‘by 2028, the proportion of cancers diagnosed at stages 1 and 2 will rise from around half now to three-quarters of cancer patients’. The plan includes to lower the threshold requirements for patients to be referred for a screening by their GP, and to maximize the variety of cancers that can be detected by screening [3]. Despite routine cancer screenings helping reduce the spread of cancer meaning it can be treated a lot easier and with a higher success rate, another vital factor to highlight is to emphasize the importance of such screenings, raising awareness to premature symptoms which will thus encourage people to go for a screening in the first place. Examples include the self-examination flow diagrams created by the NHS for women to check for abnormalities in their breasts. But cancer screenings need to be normalized- people should not be embarrassed, worried or ashamed to have an examination. I feel as if people see screening programmes as something to carry out only if symptoms are apparent, where this simply is not the case. Routine cancer screenings reduce the risks of developing later stages of cancer which are much more difficult to control and may potentially already be terminal at the time of screening.

More than one third of all humans are diagnosed with cancer, and this number could be massively reduced if people were to take advantage of the screening programmes prescribed by the NHS. In the UK, the NHS currently run screening programmes for breast, bowel and cervical cancer as are the three most common cancers, furthermore have the highest survival rates when diagnosed at an early stage. All women between the ages of 50 and 71 are entitled to a breast screening every 3 years. The process involves a mammogram of the tissue within both breasts. Screening is crucial as it maximizes the chances of finding cancerous tissue within the breast at an early stage, meaning it is much easier to conquer. A flaw however would be the fact that screens are not always entirely accurate; false negatives are unlikely but never impossible. Unclear mammograms may lead to a second scan being needed; this could cause the patient to feel distressed and lead to psychological complications such as anxiety or the mental despair caused by over-thinking about the reasons behind needing a second scan, which could lead to depression. Furthermore, going to one mammogram and receiving a negative result does not mean that the patient will never develop breast cancer, and this emphasizes the need for follow-up screenings. And as a result, through breast cancer screenings alone and hence early detection, 1,300 lives are annually saved in the UK. Controversy around radiation within breast cancer screening is apparent however this is outweighed by the potential positive impact of saving lives and preventing the need of a mastectomy or chemotherapy – not only does this positively increase the self-esteem of the patient but is also economically impactful for the NHS. Chemotherapy costs the NHS approximately £1.4 billion each year, equating to £30,000 for one round of treatment. This means that for every mammogram that catches the cancerous tissue at a stage prior to needing chemotherapy, the NHS are saving an incredible £30,000.

There are some cancers such as pancreatic cancer that have no symptoms and thus cannot always be picked up during a screening – the pancreas being so deeply embedded in the body hinders the ability of detecting pancreatic cancer via a CT scan also. This cancer only can be detected via the screening process when it has already begun to press onto nearby organs and at this point has grown to the extent that it has reached an advanced stage and is much more difficult to treat if not terminal. In this example, screening programmes are not very useful when detecting cancers with similar properties like pancreatic and thus is not offered commonly. Despite there being no symptoms, pancreatic cancer falls into the category where individuals might be at increased risk of developing the disease due to family history and therefore is an example where genetic testing is needed. Correspondingly, the plan devised by the NHS also outlines the necessity of ‘risk stratified screening’ and to test family members of patients with cancer where their risk of developing cancer is higher than average. Genetic testing delves into any gene mutations that cause the presence of such disease, meaning a healthy person could have their genome sequenced and compared to a cancer patient, to determine whether they too have the mutation in their genes and thus have a raised likelihood of developing that particular cancer if such gene were to be expressed and uncontrollably replicated. There are a selection of studies open with regards to helping stop the spread of pancreatic cancer in the UK in particular, such as the European Registry of Hereditary Pancreatitis and Familial Pancreatic Cancer (EUROPAC), observing the genetic causes of pancreatic cancer with aims of developing a successful screening programme in the nearby future. EUROPAC focus particularly on families where pancreatic cancer is already present and carry out investigations such as blood tests to determine as to whether the case of pancreatic cancer within the family is/ will become hereditary. There is a CA19-9 tumour marker commonly found in patients with pancreatic cancer; if this is found in a healthy patient their risks of also developing such cancer increases. Whilst understanding that you have a higher risk of developing a particular cancer allows you to become more focused on your health and therefore make more informed health-orientated decisions, discovering a gene mutation can also become psychologically draining. Adding anxieties to a person’s everyday life may stop them from living a fulfilled life, and just because their likelihood of developing such disease is higher does not mean they will acquire it. It is extremely difficult when faced with the decision of investigating your probable chances as to whether cancer is likely within your family, which is why many avoid screening programmes completely. The increased risk indoctrinates people into believing they are already sick, and that life itself is simply not worth living if cancer is around the corner.

Although screening programmes are massively impactful in stunting the spread of cancer by catching it at an early stage, another important factor of which must be stimulated is the encouragement of living a healthy lifestyle in order to prevent cancer as a whole. Schemes implemented to discourage smoking and alcohol have been deemed to successfully decrease the number of cancers within the lungs and liver – in fact by just simply introducing plainly-packaged cigarettes, the number of smokers within the UK decreased by a significant 300,000. Cancer treatment approximately costs the NHS around £30,000 per patient, and in contrast screening programmes cost £150 per patient. Economically, cancer screening programmes are so much less detrimental to the NHS. Screening and prevention programmes offered. Impact of Covid and the fall in screenings. Covid-19 has negatively affected the nation in several ways. In relation to cancer screening programmes, attendance to such appointments has massively declined.