2019/2020 Tung Wah College

MED4001 Honours Year Project (BSc(MLSc) 2017 Cohort)

Literature review

The Effect of length of Formalin Fixation Duration on the

Immunohistochemical Reactivity of bcl2 in colorectal cancer

Name: Luk Ming Chu

ID: 17003965

Supervisor: Albert Li
Colorectal cancer

Colorectal cancer is a common disease in worldwide and lead to considerable amount

of deaths every year. Colorectal cancer ranks third among cancer disease in

worldwide that over 1.8 million new cases had been diagnosed in 2018 (Colorectal

cancer statistics , n.d.). More than eight hundred thousand people were killed by

colorectal cancer which is second high cause of motility among cancer disease

(Colorectal Cancer, 2018). The incidence is also increasing in different regions

especially in developed countries or cities (Rawla, Sunkara, & Barsouk, 2019)

Hungary has the highest colorectal cancer incidence rate among the world followed

by South Korea and then Slovakia (Colorectal cancer statistics , n.d.). Moreover, it

has been found that colorectal cancer is more common in Asia as Asia accounts for

more than half incidences and mortality around the world (Colorectal Cancer, 2018).

In Hong Kong, it has the highest prevalence rate among different cancers that account

for 17% of cancer case in 2017 (Colorectal Cancer, 2020). It also caused 2314

individuals’ death which account 15.9% of cancer-caused mortality in 2017 and rank

second in the cancer-caused death (Colorectal Cancer, 2020). Thus, the screening and

diagnosis of colorectal cancer attract much attention to decrease mortality and cancer

burden by early treatments. The mortality of colorectal cancer in Hong Kong decrease

as days goes by. Several advancements such as improving the screening and diagnosis

technique and therapeutic treatments may lead to this phenomenon (Key Statistics for

Colorectal Cancer, 2020).

Colorectal cancer is the abnormal and uncontrollable cell proliferation in colon or

rectum due to gene mutations. Either mutation of oncogene and tumor suppressor

gene may lead to uncontrollable cell proliferation (Mohammad Ilyas et al., 1996).

Normally, there are DNA repair system and apoptosis to prevent DNA mutation

accumulations. Both mechanisms dysfunction will lead to accumulation of mutations

thus result in cancer development (Watson, 2004). The common causes and risk

factors of colorectal cancer include family history, lifestyle and age. Nowadays, most

of the people are having an unhealthy lifestyle such as insufficient exercise, obesity

and high red meat and processed food diet (Colorectal cancer risk factors, n.d.), hence

increasing the incidence of colorectal cancer. The symptoms of colorectal cancer are

bloody stools, diarrhea, gas pains and cramps (Colorectal Cancer: A Serious, But

Preventable Disease, 2020). Fecal Occult Blood Test (FOBT), sigmoidoscopy and

colonoscopy are used to screen colorectal cancer with or without symptoms while

immunohistochemical staining is the widely used to evaluate colorectal cancer on

surgical tissues (Prevention and screening for colorectal cancer, 2017). Surgical

treatment, radiation treatment and medication can be used to treat colorectal cancer

(Colorectal Cancer: Types of Treatment, 2019).

Correlation between Colorectal cancer and Bcl-2

The development of cancer is due to inhibition of normal apoptosis, uncontrollable

proliferation or dysfunction of DNA repair mechanism (Tsamandas et al., 2007).

Apoptosis is a cancer prevention mechanism that induce cell apoptosis when DNA

mutation occur thus prevent mutation accumulation and development of cancer

(Watson, 2004). Failure of apoptosis not only favor cancer development but also

prompt the transition of colorectal cancer.

Bcl-2 is expressed in the crypts and produce 26-kd cytoplasmic protein which

contribute to the inhibition of cell death (Mohammad Ilyas et al., 1996). Bcl-2 is one

of the Bcl-2 family proteins member that control the cell survival and apoptosis. Bcl-2

protein family contains both pro-apoptotic proteins that trigger apoptosis and anti-

apoptotic proteins that prevent apoptosis. The proteins in Bcl-2 protein family can be

also categorized into different classes base on the types of Bcl-2 Homology (BH)

domains. There are total four types of BH domains which are BH1, BH2, BH3 and

BH4 (Reed, 2008). Bcl-2, Bcl-XL and Bcl-w are anti-apoptotic proteins that contains

all Bcl-2 Homology (BH) domains while only three domains which are BH1, BH2

and BH3 domains left on the pro-apoptotic protein such as Bax and BaK. The last

group such as Bid and Bad which is the upstream sentinels of apoptosis only has BH3

domains. The pro-apoptotic proteins correlate to mitochondria dysfunction,

cytochrome c release, and the downstream apoptotic activation thus activate intrinsic

apoptosis pathway (Saunders, Mir, Kapur, & Singh, 2019 ； Tollenaar et al., 1998).

The pro-apoptotic proteins Bax will be directed to the mitochondria outer membrane

from cytosol and the Bak proteins in mitochondria membrane will be activated by

death signals. When both actions occur, the cytochrome c will be released from

mitochondria intermembrane space and activate caspase cascade thus initiates cell

death (Watson, 2004；Gao, Pu, Luo, & Chang, 2001). The pro-apoptotic proteins and

anti-apoptotic proteins can function independently or forming complex to induce

apoptosis or inhibit apoptosis. Reed (2008) indicated that the Bax and Bcl-2 will form
heterodimer with each other. On the other hand, the pro-apoptotic proteins also

undergo dimerization with itself and forms homodimer. Both of them have inhibition

effect to each other. For instance, Bcl-2 can inhibit Bax induced apoptosis by

dimerize to Bax or conversely. If the cell has Bax/Bax homodimers predominant, the

cell will be more susceptible to apoptosis. Vice versa. Thus, the apoptosis

susceptibility of cells depends on the ratio of Bax and Bcl-2.

Apart from that, Bid and Bad are activated by extrinsic death receptor and loss of

growth factor or glucose respectively. The activated Bid and Bad will bind to the

proapoptotic proteins such as Bax and Bak hence activate Bak and Bax and trigger

intrinsic apoptosis. On the other hand, Bcl-2 and Bcl-XL can prevent BH3 only

proteins triggered apoptosis by dimerization with Bid and Bad thus inhibit the

activation of Bak and Bax. The inhibition of Bak and Bax then prevent the

cytochrome c release thus inhibit apoptosis (Watson, 2004). Bcl-2 not only inhibit the

apoptosis that cause by Bid and Bad but also inhibit the Fas initiated apoptosis by

binding to Fas receptor. Fas is a death signal receptor which able to trigger both

extrinsic and intrinsic pathways that cause cell death and create an immune privilege

when they are inactivated. Through the extrinsic pathway, the Fas and tumour

necrosis factor (TNF) receptors will be activated thus produce a death inducing

signaling complex (DISC) by procaspase 8 recruitment The DISC then trigger the

effector caspases 3 and 7 activation causing apoptosis (Watson, 2004). Besides, Fas

also able to initiate apoptosis in mitochondria intrinsic pathway by activating caspase

8 thus cleave Bid and trigger apoptosis (Reed, 2008). The intrinsic pathway of
apoptosis triggered by Fas can only happened in some of the cells such as

hepatocytes. Bcl-2 can only inhibit the intrinsic mitochondria apoptosis by blocking

the Fas receptor or binding to Bid (Watson, 2004).

Interestingly, a minority found that Bcl-2 is not related to colorectal cancer as the Bcl-

2 expression were completely negative in half of colorectal cancer samples. Only

17.8% samples with colorectal cancer gave moderate to strong positive results

(Tollenaar et al., 1998). However, the correlation between colorectal cancer and Bcl-2

has been proved by lots of researches. Some researchers discovered that Bcl-2

overexpression exist in adenomas and colorectal cancer but predominant in adenomas

(Ilyas et al., 1996 ; Baretton et.al, 1996 ; Mosnier et.al, 1996 ; Kaklamanis et.al,

1996). Another research has also found that most dysplasia cells in adenomas has Bcl-

2 expression. The Bcl-2 expression decrease and gave negative result in half of the

adenocarcinomas samples (Bosari et al., 1995). This suggested that the Bcl-2

expression decrease when progress to carcinoma (Tollenaar et al., 1998). Thus, the

overexpression of Bcl-2 is an early sign of colorectal cancer and might be negative in

established tumors leading to the negative Bcl-2 expression in other researches (Ilyas

et al., 1996 ; Bronner et al, 1995)

Bcl-2 and prognosis of colorectal cancer

Bcl-2 not only relates to the diagnosis of colorectal cancer but also predicts the

prognosis of colorectal cancer patients. The presence of Bcl-2 expression in tumor

cell indicate a better prognosis compared to absence of Bcl-2 expression(Melincovici

et al., 2016; Ilyas et al., 1998). It is suggested that Bcl-2 would slow down the cell

cycle as severe growth inhibition had been showed when Bcl-2 was overexpressed

(Pietenpol et. al,1994). More studies suggested that Bcl-2 expression can suppress the

cell cycle thus inhibit cell transition (Mazel et al, 1996; Huang et al, 1997). It has

been found that more than 60% metastases development and more than 90% of local

recurrence showed absence of Bcl-2 expression which indicate that Bcl-2 able to slow

down the local tumor growth and decrease recurrence (Öfner et al., 1995). Moreover,

the Bcl-2 expression has been discovered to has an inverse relationship with the tumor

size as the Bcl-2 expression in pT1 colorectal tumor is 80% and drop to 20% in pT4

colorectal tumor (Öfner et al., 1995). As a result, the presence of Bcl-2 is related to a

good prognosis of colorectal cancer.

In contrast, there were several studies pointed out that the Bcl-2 expression did not

correlate to the colorectal cancer prognosis (Tollenaar et al., 1998 ; Giatromanolaki et

al., 1999 ; Melincovici et al., 2016). Apart from that, Bhatavdaker et al. (1997)

illustrated that the increase Bcl-2 expression result in poor prognosis of colorectal

cancer. The poor prognosis is due to the resistance to apoptosis of tumor cell. Also, it

has been found that the expression of Bcl-2 affect the therapeutic effect of cancer

treatment such as radiotherapy and chemotherapy by preventing apoptosis induced by

treatment (Tsamandas et al., 2007). Bax able to bind with Bcl-2 and Bax itself thus

forming either bax/bax homodimers or bax/bcl-2 heterodimers. The Bcl-2

overexpression result in bax/bcl-2 heterodimers predominance in cells thus decrease

the susceptibility of apoptosis and enhance resistance to tumor therapy (Tsamandas et

al., 2007). Ilyas et.al (1998) had suggested that the findings of worse prognosis due to

Bcl-2 expression may due to different phases of tumor progression. The studies that

concluded in worsen prognosis did not control the tumor phase thus affect the results

as the apoptotic effect may vary in different phases of tumor development. The better

prognosis result may attributes to the loss of Bcl-2 anti-apoptotic function during

tumor development and the inhibition of cell cycle (Ilyas et al., 1998). The studies

conducted had controlled this variable and concluded that presence of Bcl-2

expression will result in good prognosis of colorectal cancer (Ilyas et al., 1998).

Immunohistochemistry staining

Immunohistochemistry staining is one of the common diagnostic methods of cancer

by using chromogen and specific antibody to spot the target antigens in histology

laboratory. It can be used to diagnose cancer when an abnormal tissue is cut from

patient under surgical process (IHC Detection, n.d.). Target specific monoclonal or

polyclonal antibody should be prepared. Target specific monoclonal primary antibody

is suggested to use as it is extracted in hybridoma of mouse or rabbit thus has higher

specificity than polyclonal antibodies (IHC Primary Antibody Selection &

Optimization, n.d.). The primary antibody will bind to target antigen specifically and

connect the target antigen to enzyme labeled secondary antibody. Signals will be

produced when substrates are added and metabolized by enzyme

(Immunohistochemistry (IHC) Principle, n.d.). Polymer-based detection method is

one of the immunohistochemical staining methods that amplify signals and increase
sensitivity by conjugating secondary antibody to a dextran labeled with lots of

enzyme (IHC Detection, n.d.).

The immunohistochemical staining results is then analysis by different methods

including qualitative method, semi-quantitative method and quantitative method.

Qualitative method is performed by visualization of slide and grading the staining

with negative, weak, moderate or strong under microscope by pathologists. This

method is simple to perform and has a fast procedure, but it is very subjective hence

cannot compare with other results (Fedchenko & Reifenrath, 2014). Thus, semi-

qualitative method is carried out to convert the qualitative results into numeric values.

The semi-quantitative result can be performed based on one variable or several

variables (Fedchenko & Reifenrath, 2014 ; Klopfleisch, 2013). The combined semi-

quantitative method is more common than simple semi-quantitative method as it can

analyze the result more comprehensively. The criteria of combined semi-quantitative

method include the positive cells percentage and the staining intensity of the target

(Fedchenko & Reifenrath, 2014). Both criteria will be scored and calculated thus

obtained a value for statistical analysis or drawing a conclusion. The scoring system is

designed by researches or technicians as there is no standard analysis method for most

of the antigens or antibodies except estrogens and progesterone that had develop

standard analysis method such as Allred score and H-score scoring system

(Fedchenko & Reifenrath, 2014; Fitzgibbons et al., 2014). Though semi-quantitative

method is more objective than qualitative method, automated analysis method of IHC

is the most efficient, objective and precise way to analysis immunohistochemical

staining result (De Matos et al., 2006). It has been found that the time required for

manual analysis is much longer than automated analysis. Also, the numeric values

generated by automated analysis software such as Image J and CellProfiler and

ImmunoRatio is more objective and precise than the scoring system based on human

visualization of slide by observer (De Matos et al., 2006 ; Rizzardi et al., 2012).

Besides, the blurred boundaries between upper and lower moderate staining intensity

leads to the disagreeing results between observers (De Matos et al., 2006 ). Moreover,

the scoring systems of each researcher and pathologist are different thus make the

comparison between results difficult. The digital value generated by software can deal

with all the stated issues. Additionally, automated analysis can process lots of samples

in a shorter time. Lastly, automated method can detect minor differences between

samples which is indistinguishable by human naked eye (De Matos et al., 2006 ;

Rizzardi et al., 2012). However, automated analysis method is less common than

semi-quantitative method due to higher cost of maintenance and underdeveloped

system. Some of the softwares do not have the cell isolation and cell morphology

interpretation ability thus cannot substitute manual analysis of immunohistochemical

staining result. Although both of the results have their advantages and disadvantages,

it was found that both semi-quantitative method and automated analysis of IHC image

are strongly agree with each other. Thus, both of the methods give reliable and precise

result (Rizzardi et al., 2012). Several criteria can be considered when choosing the

analysis method. If numerous samples need to be handled within short period and

required objective results for comparison or statistical analysis with no cost limit,

automated analysis method is preferred. If there is a cost limit and the automated
analysis method is not available due to its limitation, manual method should be

adopted.

Immunohistochemical staining is easy and inexpensive to perform thus is widely used

in clinical laboratory. Also, the staining slide can be preserved for a long time as the

staining will remain permanently (Furrer, Sanschagrin, Jacob, & Diorio, 2015).

Furthermore, the detection is performed in the histologic lesions tissues or abnormal

tissue area, thus false positive result that caused by non-specific antibody binding can

be identified (Webster, Miller, DuSold, & Ramos-Vara, 2010). As a result, it is now

widely used in research and clinical diagnosis. For instance, it can be used to diagnose

the infectious agent by applying the antibodies that specific to the DNA or RNA of

infectious agent. Besides, it is applied to confirm brain trauma by detecting amyloid

precursor proteins. Moreover, by detecting muscle proteins that located in

extracellular matrix, nucleus and other area of muscles, some muscle diseases can be

diagnosis. Lastly, it can be used to diagnose cancer, predict the prognosis and therapy

response and effect by analyzing the expression of the tumor markers such as

oncogene and tumor suppressor genes (Duraiyan et.al, 2012). Before

immunohistochemical staining, tissue should be fixed to minimize the loss of cellular

components for later analysis. The most common fixatives used in laboratory is

formalin.

Formalin fixation
In clinical laboratory, tissues that require staining should all undergo tissue

processing, including tissue fixation, dehydration, clearing and embedding etc. Tissue

fixation is a very important process to preserve proteins for the later analysis. It is also

necessary to prevent autolysis, drying, shrinkage, distortion and putrefaction by

killing infectious agent. Hardening tissues and mordanting effect are also the aims of

tissue fixation (Pikkarainen, Martikainen, & Alafuzoff, 2010). Formalin is an

universal fixatives that is widely used in routine histology laboratory.

Formalin is made by dissolving 37%-40% of formaldehyde to water. It is a non-

coagulating and additive fixative that cross linking the proteins and DNA. It preserves

proteins by forming a methylene bridge between proteins and formaldehyde (Sean,

2018). It is a golden standard of fixatives due to fast penetration, well preservation of

architecture and components of tissue, hardening tissue and low cost. However, this

universal fixative is a limiting factor in immunohistochemistry staining (Webster,

2010).

The immunoreactivity of antibodies decreases due to the mask of epitopes by the

cross-linkage between proteins and formaldehyde. Moreover, the cross linkage

between protein and formaldehyde are non-selective thus bind to unrelated proteins

and decrease immunoreactivity (Ramos-Vara, 2005). Besides, the three-dimensional

structure of proteins will be altered by cross linkage hence the target proteins cannot

be recognized by antibodies (Sean, 2018). It has found that the neutral buffered

formalin is the worst fixative for immunohistology among different fixatives such as
ethanol, methanol and Bouin’s solution etc (Arnold, 1996). It has also proved that

prolong formalin fixation leads to decrease signals in immunohistochemical staining

due to excessive cross linkage. For instance, the immunoreactivity of estrogen

receptor has found to be greatly decreased after 57 days formalin fixation (Arber,

2002). The intensity of prostate specific antigen, carcinoembryonic antigen and

thyroglobulin immunohistochemical staining showed dramatical decrease after 14

days formalin fixation. The signals of many antigens such as LN1, LN2 and LN3

which are lymphocyte antigens decrease significantly after 3 days formalin fixation.

The false negative result even occurred in the staining of intermediate filament

proteins vimentin, neurofilaments and desmin after 1-day formalin fixation (Leong &

Gilham, 1989).

On the other hand, the prolong fixation effect was evaluated using antigens of

animals’ tissue or surgical biopsy tissues and concluding that most antigens did not

affected by prolong formalin fixation. The evaluated antigen include amylin in

pancreas, multiple myeloma oncogene-1 (MUM-1) and lymphoid tissue antigens

such as cluster of differentiation 3 (CD3), CD20, CD79a, and B Lymphocyte Antigen

36 (Webster et al., 2010). Furthermore, no massive drop of staining intensity of some

breast cancer antigen including Ki-67, p27, and vimentin in 154 days formalin

fixation (Arber, 2002). Furthermore, immunoreactivity of Canine parvovirus was

investigated in heart, spleen, and small intestine and the immunoreactivity varying

among different tissues. The immunoreactivity of Canine parvovirus is most

susceptible to prolong formalin fixation in heart, followed by small intestine. The

signals kept strong until week 10 in spleen tissue. Thus, the effect of prolong fixation

is likely affected by different variables such as types of tissue, antigens and antibodies

(Webster et al., 2010). The immunoreactivity of Bcl-2 had decrease in breast cancer

cells after prolong formalin fixation (Hoetelmans et.al, 2001). However, no

experiment has investigated the susceptibility of Bcl-2 immunoreactivity in colorectal

cancer. As a result, the effect of prolong formalin fixation of Bcl-2 in colorectal

cancer is unknown and will be investigated in this experiment.

In conclusion, the incidence of colorectal cancer is continuously increasing among the

world including Hong Kong (Colorectal Cancer, 2020). It causes lots of death every

year and brings huge burden to medical system. To decrease the mortality and burden

on hospital, early diagnosis is one of the methods as early treatment of cancer has a

higher chance of recovery and low chance of recurrence. Therefore, early sign of

colorectal cancer brings much attention to diagnose colorectal cancer (Key Statistics

for Colorectal Cancer, 2020). Bcl-2 is an apoptosis inhibitor which can be a marker of

colorectal cancer and correlates with prognosis (Mohammad Ilyas et al., 1996).

Overexpression of Bcl-2 leads to accumulation of mutation, thus develop cancer

(Watson, 2004). Furthermore, Bcl-2 expression is related to a better prognosis

compared with the absence of Bcl-2 patients by decreasing metastasis and recurrence

(Pietenpol et. al,1994). Thus, the detection of Bcl-2 is very important in early

diagnosis and prognosis of colorectal cancer. In hospital, immunohistochemistry

staining is one of the commonly used technique to detect antigens on cancer tissues.

However, the routine used formalin fixative may decrease the signals of staining
especially when the fixation time is prolonged (Ramos-Vara, 2005). This may lead to

false negative result that some of the disease might be misdiagnosed. It has found that

the effect of prolong formalin fixation varies among different antigens, antibodies and

tissue types (Webster et al., 2010). It has found that the immunoreactivity of Bcl-2

has decreased due to prolong formalin fixation in breast cancer (Hoetelmans et.al,

2001). But none of the research has evaluate the effect of prolong formalin fixation of

Bcl-2 in colorectal cancer. Thus, the effect of prolong formalin fixation of Bcl-2 in

colorectal cancer is investigate in this experiment.

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