New Technological Solutions to the Staging of cancer

1.1 Cancer Types

What is Cancer:

Cancer is a terminal disease caused by the uncontrolled growth of cells. While regular cells proliferate in a controlled manner, cancer cells proliferate uncontrollably due to a loss of cell cycle control systems. Cancers are not well understood, because there are several different types, and there are different causes for each.

It is important to understand the mechanisms behind cancer, so that treatments can be developed. Cancer is one of the leading causes of death throughout the developed world. As per Globocan statistics for the year 2018, 18.1 million new cancer cases have been diagnosed. The major contributor to this number is Asia, which is responsible for 48.4 percent of the new cancer cases. Currently, lung cancer is responsible for most of the deaths and new cases in 2018. Cancer to date has taken the lives of 9.6 million victims.

Types of cancer:

Tumors can be classified as either benign or malignant. Benign tumors are not cancerous and do not spread to nearby tissue or the bloodstream. Malignant tumors are cancerous tumors that can spread to nearby tissue or enter the bloodstream.

There are several types of cancer, which can impact the different organs of the human body.2 Cancers having high incidence globally are leukemia, lung cancer, breast cancer, brain cancer, liver cancer, skin cancer, and colon cancer.

Demographic of Cancer:

Developed countries such as the United States, United Kingdom, and Canada exhibit high incidences of prostate, colorectal, lung, and breast cancers. Genetic and lifestyle factors are responsible for the cancer types observed in developed countries. Developing countries such as Afghanistan, Bangladesh, and Cambodia have increased incidences of liver, stomach, and cervical cancer types due to the prevalence of less hygienic conditions.3

Causes of cancer:

The primary causes of cancer are genetic, dietary, and/or environmental factors. Metabolic factors can be influenced by the genetic makeup of an individual. Cancer arises due to mutations in the cell’s genetic code. This genetic alteration causes the cell to pass through the process of mitosis without control. Mitosis is the process of cell division where two genetically identical cells, also known as daughter cells, are created. Since the genetic information which informs the cells to stop proliferating has been altered the cells will not stop dividing. The daughter cells produced will contain the altered DNA from the parent cell. Some mutations involved in cancer affect the oncogenes which are responsible for regulating the cell cycle. If the oncogene products mutate cells will uncontrollably proliferate. But, this is only one of the causes for cellular transformation. That is because cancer cells generally proliferate uncontrollably. That means that cancer is not a matter of speed but where the cancer spreads to in the body. This is how cancer cells gain the potential to become malignant.

1.2 Staging of cancer using Histopathology

Detection of cancer:

Tumors or lumps found in some cancer types, such as breast cancer, may be an indication that one may have cancer. Endoscopy and needle biopsies are methods of detecting cancer. Endoscopy involves using an endoscope to examine hollow organs and cavities inside the body. During the endoscopic procedure, physicians perform a needle biopsy by using a needle to obtain a tissue sample of the cancerous area. Cancer can also be detected by physical examinations, conducted by physicians, which reveal changes in the skin. Non-invasive methods such as urine samples, blood tests, imaging techniques such as CT (Computed Tomography) scans, MRI (Magnetic Resonance Imaging) scans, and many more can also be used to detect cancer. CT and MRI scans are advanced imaging techniques used for identifying the local areas in the organ affected by cancer. Early detection of cancer is very helpful for both the physician and the patient to prevent the spread of cancer to other organs. It can also provide insight to cure some cancer types.

In an endoscopy detection, the doctor uses a thin tube with a light and camera, also known as an endoscope, to view the inside the target organ or body cavity and understand the type and location of the tumor(s). The endoscope can also be utilized to spot a tissue sample for further examination.

Needle biopsies are a method to obtain tissue samples from the patient. A needle is used to collect tissue samples from the targeted location for histopathological examination (microscopic examination of tissue to study the disease). Other methods can also be used to collect tissue samples, such as Pap tests and fine needle aspirations.

The sample obtained can be studied to identify the cancer, or it can be used as a reference during the treatment of cancer. For example, when the treatment begins the tissue sample can be used as a marker of the patient’s previous health. Once they finish the treatment they can use the previously collected tissue sample to make sure that the patient’s health improved.11

Histology:

Histology is the study of the anatomy of cells by viewing the tissue samples through a compound light microscope. Histology is frequently used in cancer treatment to assist in identifying the stages of cancer. This allows the pathologist to view tissue samples in better detail. Pathologists note the density of cancer cells in the tissue sample examined and, thus, diagnose the stage of cancer carried by the patient.5

How the tissue samples are processed:

The samples obtained from the patients need to be preserved, as the processing of tissue samples increases their shelf life. The tissue samples need to be preserved for comparison purposes with future samples during the treatment process.

The first step in tissue processing consists of placing the tissue sample in formalin, which is a fixing agent. Formalin (40% formaldehyde and 60% water) causes the sample to harden. The tissue sample is placed in the fixing agent for 48 hrs.12

Next, the tissue sample is dehydrated by replacing all the water in the sample with ethanol, which replaces paraffin wax to allow the tissue sample to be more miscible with water. Paraffin wax is used to harden the tissue sample and keep the sample together. Since paraffin wax is immiscible with water, ethanol is used. The tissue sample is placed in different concentrations of ethanol for varying times to dehydrate the tissue sample using ethanol.

In the third step, the organic solvent xylene, a clearing agent, is used to replace the ethanol to make the paraffin wax more miscible, able to be mixed together, with the tissue sample. The tissue sample is placed in xylene at differing times.

Finally, the tissue sample is carefully placed in a mold of paraffin wax. The xylene is replaced with paraffin wax to create a paraffin embedded tissue sample. The xylene is used to make the paraffin wax miscible with the tissue sample. The xylene has to be replaced to create a paraffin embedded tissue sample.

The embedded tissue sample is then sectioned by the microtome, which is a machine that segments the tissue samples into pieces of certain thicknesses according to their tissue type. The sliced tissue sample is deparaffinized, which involves the use of xylene and ethanol to wash out the paraffin wax from the tissue sample. The deparaffinized tissue sample is rinsed with cold water to rehydrate it.6

The tissue sample, at this point, is ready to be stained using the H&E (hematoxylin and eosin) stain, which allows the pathologist to stage the cancer case with clarity. The hematoxylin shades the nuclei blue. The eosin shades the cytoplasmic parts of a cell pink. Every other part is shaded with a combination of the colors differing in the shade. This stain is used to help the pathologist differentiate between the nuclei and cytoplasmic components.7

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Figure Number 1: This figure shows the process of preserving a tissue sample extracted through aforementioned methods.

How the tissue samples are studied:

Tissue samples undergo gross examinations. Pathologists investigate the sample by looking at, measuring, and/or touching the tissue. They note the size, color, consistency, among various other characteristics of the tissue sample. Abnormalities identified during gross examination are further studied using the microscope. Gross examinations help pathologists decide the margins of dissection for the sample obtained from the intended method of collection.4

How pathologist diagnose cancer using histology:

The structure and function of the cell architecture is closely related in normal body tissue cells, and the characteristic structures of cells from a range of body tissues is well characterised. Cancer cells have an irregular shape and size. The nucleus also appears larger in cancer cells than in regular cells. In addition to that, the nuclei in normal cells are round. But in cancer cells, they have an irregular outline.8 The color of the nucleus is also much darker in cancer cells than in regular cells.13 The nucleus of a cancer cell also has a very irregular shape. This irregular shape is caused by bumps called blebs.14 The microscopic image of tissue samples displaying the normal to invasive malignant carcinoma is given in Figure 2. We can see clearly in Figure 2, that as the pictures move from A to D the cells become increasingly unorganized and have enlarged nuclei. The nuclei in Figure 2 are colored dark purple. These irregularities can be identified by histologists as markers that the patient has cancer.

Figure Number 2: These pictures are all histology slides stained with H&E staining. Picture A shows normal cells while picture C shows malignant cells. As you can see the cells become progressively disorganized and the pictures go from A to D and the nuclei coloured purple also enlarges. (Reproduced with permission from Teresa Araujo) 15

Staging of cancer using pathological method:

The severity of cancer and its significant proliferation is represented by four unique stages. The four stages of cancer staging and a description of each stage are listed below.

In Situ (‘in its original place’): Cancer patients diagnosed at an early stage carry cancer cells which have not spread beyond where they have been formed. A simple surgery can be performed to remove the tumor from the patient. Most cases of “in situ” cancer are found to have benign tumors. Benign tumors do not spread to nearby tissue or enter the bloodstream.

Local: Cancer patients at the local stage, contain cancer cells that have spread across the organ on which the tumor is located by the pathologists. This can be treated in the same manner as cancers in the stage of in situ.

Regional: In the stage of regional, the cancer has spread to the nearby tissue. Hence this stage of cancer is much more difficult to cure. Multiple methods of curing cancer may need to be applied to cure the patient. For example, surgery and chemotherapy may need to be implemented. Most of the time many known methods are simply used in combination to save the patient.

Distant: The cancer has spread to other organs or other organ systems.

TNM Method of Staging of Cancer

The most prominent method of staging cancer is called the TNM (Tumor Nodes Metastasis method) method, which is widely used by pathologists. This method of staging is very useful, as it assigns numbers instead of words. The numbered stages are easier to explain to patients and can be understood more easily. Hence, TNM is widely used. A description of each stage of the TNM staging method is given below:

Stage 1: The cancer is found in a small space. The cancer has not spread to the nearby tissue. Cancer at this stage can be cured without surgery.

Stage 2: At this stage, cancer has not spread to the surrounding tissue. But the tumor has just grown bigger. Sometimes stage 2 may mean that the cancerous cells may be near some lymph nodes. Lymph nodes contain lymphocytes (white blood cells), which can fight infection and disease.

Stage 3: The cancer has spread to the organ’s tissue. In addition, the cancer cells have also spread to some nearby lymph nodes. This stage of cancer cannot be fought with a simple surgery. Stage 3 is usually treated by both surgery and chemotherapy.

Stage 4: In stage 4, cancer has metastasized, cancer has spread away from the tumor and entered the bloodstream, and has entered the bloodstream. It indicates that the cancerous cells have access to the entire body, as the blood carries the cells throughout the body. At this stage, the cancer is referred to as secondary cancer or metastatic cancer.

The TNM type of staging is useful, as it informs the doctor of the patient’s case. The physician will progress to treat the patient as per their diagnosed stage of cancer. Cancer in early stages can be cured, as the cancerous cells have not spread to other organs or tissues. In contrast, cancer cases in the regional and distant stages become increasingly difficult to treat, as the cancer has spread to other organs.9

1.3 Problems Associated with Histopathology

Subjective Decisions:

Staging is very subjective as each pathologist may have a different view of histology and the cell architecture. However, it is important that this process is made objective, so that diagnosis is not influenced by human error, and becomes more consistent across all patients.

1.4 Possible Solutions

Quantify:

Quantifying many variants in cancer staging can be very useful to remove human subjectivity, and ensure each stage of cancer is consistently reported and classified. This will also lead to correct treatment being administered. If a specific treatment is given to each different case according to their stage, the cancer may be well managed without any complications.

In quantifying methods biomarkers in the body are used as a measurement. Biomarkers are molecules such as proteins, nucleic acids, lipids, carbohydrates. The qualitative (structure or sequence of molecules) and quantitative difference in biomarkers can be used to stage cancer accurately.

Spectroscopy:

Spectroscopy is the study of the emission and absorption of light by matter. In spectroscopy, scientists pass light through an object placed in a spectrophotometer. The material will absorb different frequencies of light depending on the nature and concentration of its various molecules. Unique spectra are created using the absorption characteristics of molecules.

For example, Fourier Transform-Infrared Spectroscopy (FTIR) is a form of spectroscopy that is being used. FTIR uses infrared rays which contain many different frequencies of light to quickly analyze the information between normal and cancerous cells. Normal and cancer cells show qualitative and quantitative changes in various regions of spectra. Quantitative observations in the FTIR spectra are with respect to absorbance by each type of molecule present in the cells. Computational analysis of these qualitative changes can be used initially to differentiate normal and cancer cells in a given tissue sample. In addition, these changes can be used to stage cancer for various patients.

Digital grading:

Digital grading for cancer is a method that is currently being developed. This method of grading cancer involves creating a spectrum of both normal cells and cancerous cells in a given tissue sample. This spectrum is based on the nature and concentration of biomolecules in the cells. Spectra generated by the normal and cancerous cells are analyzed. Computational techniques based on mathematical models are used to reveal the differences between normal and cancer cells. This will allow pathologists to stage the cancer more accurately. Analysis of qualitative and quantitative changes using computational methods on normal and cancer cells lead to digital staging.10

Advantages of Digital Grading of Cancer:

Digital grading will permit doctors and pathologists to get a clear idea of the severity of cancer in a patient. Digital grading uses a scale of 1 to 100. Digital grading will also allow physicians to properly arrange specific treatment plans since digital grading stages cancer very accurately. The digital grading enables the physicians to plan a treatment tailored to each and every patient. Since the physicians know exactly what standing the patient is in, between the 1 to 100 scale, they can plan the treatment specifically. For example, if the patient’s stage is anywhere between 80 and 100 physicians need to give a combination treatment to the patient. Digital grading will also enable physicians to monitor the progress shown by various methods of treatment.

1.5 Summary

Mortality rates have increased due to cancer in a span of years. Currently, pathologists are staging cancer using histopathological methods that are rather subjective. Subjective methods of staging cancer may result in many errors in diagnosis. Novel technologies such as optical staging, when applied, will make the process of cancer staging objective. Objective methods will lead to precise treatments and lower mortality rates. This type of cancer research is needed since it will improve cancer staging and treatment.

1.6 References

  1. “Latest global cancer data: Cancer burden rises to 18.1 million new cases and 9.6 million cancer deaths in 2018.” World Health Organization. September 12, 2018. https://www.who.int/cancer/PRGlobocanFinal.pdf
  2. “Breast Cancer Facts & Figures 2017-2018.” American Cancer Society. https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/breast-cancer-facts-and-figures/breast-cancer-facts-and-figures-2017-2018.pdf
  3. Ramesh Jagannathan. “Characterization of Drug-like Chemical Space for Cytotoxic Marine Metabolites Using Multivariate Methods.” ACS Omega. March 18, 2019 https://pubs.acs.org/doi/10.1021/acsomega.8b01764
  4. “What happens to biopsy and cytology specimens?.” American Cancer Society. July 30, 2015 https://www.cancer.org/treatment/understanding-your-diagnosis/tests/testing-biopsy-and-cytology-specimens-for-cancer/what-happens-to-specimens.html
  5. William C. Shiel Jr. “Medical Definition of Histology.” MediceneNet. December 27, 2018.

    https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=11&cad=rja&uact=8&ved=2ahUKEwjdtt_Ik67lAhWX9Z4KHU-0A7AQFjAKegQIABAB&url=https%3A%2F%2Fwww.medicinenet.com%2Fscript%2Fmain%2Fart.asp%3Farticlekey%3D7318&usg=AOvVaw2M-60jMI-ZfWVN-NHaSaDv

  6. Geoffrey Rolls. “An Introduction to Specimen Processing.” Leica Biosystems.https://www.leicabiosystems.com/pathologyleaders/an-introduction-to-specimen-processing/
  7. Cancer.Net. “After a Biopsy: Making the Diagnosis.” Cancer.Net. March/2018 https://www.cancer.net/navigating-cancer-care/diagnosing-cancer/reports-and-results/after-biopsy-making-diagnosis
  8. Laura Mgrdichian. “Scientists Measure Differences Between Normal and Cancer Cell Surfaces.” Phys.org. May 5, 2009 https://phys.org/news/2009-05-scientists-differences-cancer-cell-surfaces.html
  9. “Cancer Staging.” National Cancer Institute. March 9, 2015 https://www.cancer.gov/about-cancer/diagnosis-staging/staging
  10. Shaul Mordechai, Jed Goldstein, Shmuel Argov, Hugo Guterman, Jagannathan Ramesh. “Novel optical method for diagnosis and staging of premalignant and malignant human colonic tissues.” Justia Patents. May 14, 2004 https://patents.justia.com/patent/20050017179
  11. “Cancer and tissue studies.” Canadian Cancer Society. https://www.cancer.ca/en/cancer-information/diagnosis-and-treatment/tests-and-procedures/cell-and-tissue-studies/?region=on
  12. Phil Bryant. “Tissue sampling, processing and staining.” weebly.com. December 10, 2019. https://tissuesampling.weebly.com/formalin-fixation.html
  13. Rajesh Kumar, Rajeev Srivastava, and Subodh Srivastava. “Detection and Classification of Cancer from Microscopic Biopsy Images Using Clinically Significant and Biologically Interpretable Features.” hindawi.com,. July 12, 2015. https://www.hindawi.com/journals/jme/2015/457906/
  14. Laura Elizabeth Mason. “Cancer Cells vs Normal Cells.” technologynetworks.com, August 8, 2018. https://www.technologynetworks.com/cancer-research/articles/cancer-cells-vs-normal-cells-307366
  15. Araujo T, Aresta G, Castro E, Rouco J, Aguir P, Eloy C, et al. (2017) Classification of breast cancer histology images using Convolutional Neural Networks. PLoS ONE 12(6): e0177544. https://doi.org/10.1371/journal.pone.0177544

Cover Page Reference

16. Jennifer Gordetsky and Jonathan Epstein. “Grading of prostatic adenocarcinoma: current state and prognostic implications”. Diagnostic Pathology 11 (1). DOI:10.1186/s13000-016-0478-2

1.7 Acknowledgements

I would like to dedicate this article to my grandfather, Mr. P.V. Muralidharan. I would also like to acknowledge my parents and my teachers for helping me throughout this journey.

About the author

Harish Ramesh is a student enrolled in Middlefield Collegiate Institute, located in Markham, Ontario. He is currently studying grade 9. He studied in Canada for grades 1 to 8. In science Harish is very interested in space and medical sciences. In 2019, he published an ebook titled “Thrilling Short Stories” in Amazon.