Cancer Diagnostics: Tumor Markers, Cancer Genomics, Circulating Tumor Cells and Circulating Tumor DNA
The statistical peril of developing cancer in one’s lifetime is 40.1% in men and 38.7% in women according to the American Cancer Society. The risk of dying from that cancer: 21.34% in men and 18.33% in women.
Cancer is among the leading causes of death worldwide. In 2018, there were 18.1 million new cases and 9.5 million cancer-related deaths worldwide. In 2020, an estimated 1,806,590 new cases of cancer were diagnosed in the United States and some 606,520 people will die from the disease. By 2040, new cancer cases per year are expected to grow to 29.5 million and the number of cancer-related deaths to 16.4 million.
Cancer is a complex illness which can affect any organ system in the body. A variety of factors contribute to the development and growth of cancer; from epigenetic influences like radiation, toxin exposure, obesity and stress, to a patient’s genetic background. Combinations of these influences can create and promote cancer in the body. Several mechanisms provide for cancer’s survival and growth in the human body. Some of these factors include evading cell death and senescence messaging, a limitless replication potential, the capacity for immune evasion, the ability to spread throughout the body, metabolic stress and genomic instability.
Minimally invasive cancer surveillance, diagnostics and monitoring is critically important for patient care and has been evolving over the past decade. Many practitioners, from primary care physicians to surgeons and subspecialists, utilize organized approaches to evaluate and diagnose cancer patients.
In order to understand the road to a cancer diagnosis let’s start at the beginning. Practice styles may vary, however the pathway most clinicians and surgeons take in diagnosing and staging a patient with the suspicion for cancer includes the following:
Most physicians use the VINDICATE model to develop a differential diagnosis for a given patient’s presentation. This template allows physicians to organize the case into distinct categories as they develop their assessment. The acronym VINDICATE stands for the following:
• V – Vascular
• I – Inflammatory
• N – Neoplastic/Cancerous Disease
• D – Degenerative / Deficiency Issue
• I – Idiopathic (Unknown), Intoxication (Drug induced)
• C – Congenital
• A – Autoimmune / Allergic
• T – Traumatic
• E – Endocrine
This differential diagnostic tool starts the clinician on their journey to discover what is the underlying cause of the patient’s complaint. In the case of a cancer diagnosis, the clinician initiates a variety of tests and referrals to establish the cancer type, the stage of disease and the patient’s overall clinical situation. Although every case is different, the most logical approaches refine the diagnosis through taking a complete history, performing a physical exam, obtaining a pathology specimen, and gathering appropriate lab work along with medical imaging. A thorough history includes information about physical signs and symptoms which may be reflective of a mass effect. Common signs like pain and changes in bodily function can be associated with cancer. The history will also identify familial risk factors, like close relatives with cancer diagnoses, which guide clinicians in ordering certain genetic tests.
Some solid tumors, masses or lesions are found directly upon exam (e.g.: physical exam or colonoscopy). Imaging of an asymptomatic patient may reveal a mass as well (e.g.: through an x-ray, ultrasound or mammography), or in a symptomatic patient (e.g.: abdominal pain, coughing up blood or neurological changes from the presence of a mass) where a CT/PET and/or an MRI is obtained. These scenarios, of course, can vary but in general this is how most cancer diagnoses come about. Once a solid tumor mass is identified, a biopsy and/or tumor resection (partial or complete) can be performed to further the diagnostic process by examining the macro and microscopic nature of the tissue.
Normal cancer diagnostics include a wide range of pathology tools. Direct histological observation and staining measures which can help define the type of cancer that someone is dealing with. Once pathology is confirmed, the process of staging the cancer can begin. The purpose of staging is to gather prognostic information, which will include further lab work and anatomical imaging, and from there ultimately develop a therapeutic plan.
Advanced imaging technologies such as CT scans, PET scans and MRI help stage the disease and guide therapeutic planning. Further imaging is used to monitor progression of the cancer, and identify disease stability or regression.
Adjunctive lab testing can include measuring tumor markers. Common tumor markers, sometimes called cancer markers, are found in the blood, urine, or bodily tissues. Tumor markers are substances made by cancer cells or normal cells in response to cancer in the body. These markers vary in their value and should be used in concert with imaging and clinical findings to determine a patient’s status. Other diagnostic and prognostic parameters include examining cancer cell receptors, gene mutations and amplifications. Some of the most common cancer markers, receptors and gene mutations include the following:
Markers and Cancer Type
CA15-3, CA27.29 seen in Breast cancer
CEA seen in Colorectal cancer
CA 19-9 seen in Pancreatic cancer
PSA, PAP seen in Prostate cancer
CA 125 seen in Ovarian cancer
Alpha Fetoprotein (AFP) seen in Liver, Testis and Ovarian cancer
Cancer Cell Receptors and Gene Amplifications
Progesterone receptor positive (PR+) seen in Breast cancer
Estrogen receptor positive (ER+) seen in Breast and Ovarian cancers
Androgen receptor positive (AR+) seen in prostate cancer/other hormone sensitive cancers
HER2/Neu gene amplification/overexpression seen in Breast, Ovarian, Pancreatic and gastric cancer
Programmed Death Ligand (PDL-1) seen in melanoma, GI cancers, lymphomas and other cancers
Gene Mutations and Cancer type
BRCA1/2 mutations seen in breast and ovarian cancer
KRAS gene mutation seen in some colorectal and non-small cell lung cancer
MYC gene expression seen in some leukemia and lymphoma
RAS gene mutation seen in pancreatic, lung and colorectal cancers
TP53 gene mutation seen in breast cancer, bone and soft tissue sarcomas, brain tumors and adrenocortical carcinomas
EGFR gene mutation seen in non-small cell lung cancer
BRAF gene mutations seen in melanoma, non-small cell lung cancer and colorectal cancer
BCR-ABL fusion gene seen in Chronic myeloid leukemia, acute lymphoblastic leukemia and acute myelogenous leukemia
Many tumor markers are proteins made by both normal cells and cancer cells, but they are made in higher amounts by cancer cells. Tumor markers include CA-125 (in ovarian cancer), estrogen receptor and progesterone receptor (in breast cancer), CEA (in colon cancer), PCA3 mRNA and PSA (in prostate cancer), EGFR and KRAS gene mutations (in non-small cell lung cancer).
A variety of tumor markers and characteristics which are seen in and on tumors may respond to therapies that may not be traditionally used per NCCN guideline treatments. Such markers include PD-L1, HER2/Neu protein, estrogen receptors, androgen receptors, BRCA1/BRCA2 mutations, KRAS mutations and BRAF mutations.
Advances in cancer diagnostics have led to ever-evolving insights into this insidious disease. Beyond basic pathology and tumor markers, a variety of relatively new testing tools have opened more doors for cancer monitoring and guiding therapeutics.
Oncology is a rapidly evolving field and the direction cancer therapy is moving requires focus on an individual’s tumor genetics. Personalized cancer medicine comes from studies of human genetics and the unique gene expression seen in tumors and individual cancer cells. By focusing on an individual’s tumor genetics, practitioners can provide more individualized therapies and protocols in treating their patients in an effort to get better results with less potential for side effects.
Tumor genetic studies allow the promise of precision medicine to become a reality. Molecular profiling has become standard of care for many cancer types – and required for certain therapies. We must remember no two tumors are alike and they are created by an interplay between epigenetic factors, the immune system, the tumor microenvironment and the patient’s genetics. Complicating factors are circulating tumor cells and satellite lesions, and the fact that tumor heterogeneity varies within any given mass.
Comprehensive tumor profiling assesses DNA, RNA and proteins. This provides the highest quality molecular blueprint to guide more precise and individualized treatment decisions, which are proven to extend overall survival.
Types of tumor genetic and cancer cell analysis utilized include the following:
• • Immunohistochemistry (IHC): detects the presence of specific protein markers which can assist with accurate tumor classification and diagnosis.
• • Fluorescence in situ Hybridization (FISH): detects gene deletions, amplifications, translocations and fusions. Evaluating these genetic disturbances can provide improved prognostics and help guide therapies.
• • Next-Generation Sequencing (NGS): rapidly examines and detects DNA mutations, copy number variations and gene fusions across the genome. Clinical NGS involves analysis of raw genomic data and rapid clinical interpretations for consideration by the treating clinician. There are three general ways in which NGS can aid a clinician. The first is with diagnosis; tumor subtypes that only a few years ago were defined by morphologic criteria are now defined by genetic mutations. The second is finding an appropriate “targeted therapy”, which can personalize the treatment. There are an increasing number of therapies which have indications for use based on DNA sequencing results. The third point at which clinicians stands to benefit is when a patient stops responding to a targeted therapy with known resistance mutations.
• • Sanger Sequencing: examines strands of DNA to identify mutations by analyzing long contiguous sequencing reads
• • Pyro Sequencing (PyroSeq): detects and quantifies mutations, methylation, etc. through sequencing by synthesis
• • Fragment Analysis (FA/Frag. Analysis): detects changes in DNA or RNA to indicate the presence or absence of genetic markers
Beyond assessing tumor genetics there is a branch of diagnostic and prognostic testing which slowly grew over the past 20 years. This testing involves looking for and analyzing cancer cells and cancer cellular material which has broken away from the primary tumor. These cells are referred to as circulating tumor cells and the cellular material in question is referred to as circulating tumor and/or cancer cell DNA.
Circulating tumor cells (CTCs) have long been assumed to be the substrate of cancer metastasis. In recent years, we have begun to leverage the potential of CTCs found in minimally invasive peripheral blood specimens to improve diagnostics, monitoring and managing care for cancer patients. Over the past several years it has been shown that individual patients who have a sustained and/or elevated circulating tumor cell level are at increased risk for disease progression and recurrence. A high CTC count has been also associated with poor prognosis in several cancers including breast, lung, and prostate cancer.
In fact, the overall survival and progression-free survival can be reflected by the number of circulating tumor cells present in a patient
Liquid biopsies capture whole tumor cells in the blood. The whole cell can provide a lot of information by revealing the genome and transcriptome of the CTC and what downstream proteins could be targeted by anticancer agents.
These cells not only reveal the presence of a tumor; they also indicate that a cancer is progressing or spreading. Most clinicians and researchers agree circulating tumor cells are the seeds allowing a cancer to spread. This has been challenging to prove because circulating tumor cells are rare; they are heterogeneous and may look different from the collection of tumor cells they were shed from. However, their distinct cellular morphology, genetic characteristics and larger size make them easy to identify against other circulating cellular material. They have also been found in early-stage disease, leading some to believe the primary tumor may not have presented itself alone, but with other surrounding satellite tumors. These satellites may pose future risk of recurrence and/or new tumor growth.
The CTC count is usually low in non-metastatic cases, and the CTC detection cutoff has regularly been set at ≥ 1 CTC/7.5 mL of blood in most studies which validated this technology. Cells from solid tumors circulating in blood (CTCs) determine the risk of blood-borne metastases. It is therefore crucial to monitor the response of these cells in order to tailor therapies systematically.
Circulating tumor DNA (ctDNA) is found in the bloodstream and refers to DNA that comes from cancerous cells and tumors. Most DNA is found inside a cell’s nucleus. As a tumor grows, cells die and are replaced by new ones. It is during these phases of rapid growth and death in which cancer cells shed DNA.
Researchers and clinician foresee a time when ctDNA and circulating tumor cells will be used in combination with other more traditional diagnostics to offer the best picture of what’s happening with a cancer at any given time inside a patient.
Tumor cell DNA acquires multiple genetic changes during tumor development leading to the tumor’s heterogeneous state. Therefore, ctDNA may not be an exact match to the shedding tumor. The ctDNA is also not an exact match to the individual’s DNA; finding DNA with genetic differences aids in tumor detection. Diagnosing the type of tumor using ctDNA may reduce the need for getting a sample of the tumor tissue (tumor biopsy) and certainly a better option if the biopsy procedure comes with great risk or simply isn’t possible.
Analyzing the genome of tumor cells, ctDNA and DNA fragments can help doctors determine how effective a treatment may be. Some examples include:
• Monitoring treatment and seeing a decrease in the quantity of ctDNA may suggest the tumor is shrinking and treatment is successful.
• Monitoring periods with no symptoms (remission of cancer) in which lack of ctDNA in the bloodstream may indicate the cancer has not returned.
Cancer patients usually have a high level of Cell-Free (cfDNA) in their serum or plasma. Cell-free DNA refers to all non-encapsulated DNA in the blood stream that is a result of cellular necrosis or apoptosis. This material can be from both normal tissue turnover along with that of cancer cells. Since tumor cells divide faster than normal cells, cfDNAs are seen in higher amounts in patients with cancer. Clinicians can tell the difference between the tumor DNA and normal cellular DNA when examining blood samples. One way to distinguish the two is that cfDNA are typically longer strands of DNA while ctDNA are generally shorter length fragments. Specifically, the ratio of total cfDNA to ctDNA is smaller in the cancer patient. A high level of known cancer mutations is found amongst the ctDNA of cancer patients. Gene mutations seen in TP53, EGFR, KRAS, PIK3CA, and BRAF genes were some of the most common. Temporal analysis also suggests that these mutations grew in number as patients received treatment for their cancer; demonstrating chemotherapy and targeted therapy most likely pressured the tumor cells to ‘adapt or die’. The importance of patient monitoring is critical and can now rely on blood based labs that assess disease progression and ‘genomic pivots’ the cancer is making to evade current treatments.
Future therapies may target circulating tumor cells as well as circulating cancer cell DNA and possibly use them to make personalized cancer vaccines. They will also be used to develop targeted immunotherapies which have been specifically designed for the cancer patient.
Overall, the journey to a cancer diagnosis, staging and disease monitoring requires many steps and involves a variety of specialists and technologies working synergistically over time. Our fundamental understanding of cancer has grown dramatically over the past 20 years, we understand it to be a multifaceted and complex illness. Each cancer diagnoses carries with it the opportunity for us to learn more, not only about the disease but also the patient and ultimately the unique interaction between the two. We have seen a vast number of diagnostic tests and precision genomic testing become available in the field of oncology and personalized medicine over the past 20 years. Today there are numerous companies worldwide who are utilizing and refining cutting edge diagnostics by harnessing the information held within the genome of the tumor, the individual circulating cancer cell and the genetic material from both. By utilizing information about a given patient’s cancer, at the microscopic and molecular level, we will be able to fine tune our diagnostic and prognostic capabilities for patients and offer more individualized therapeutics. By unlocking this information, we may find patients qualifying for clinical trials, novel targeted therapies, personalized immunotherapy, chemotherapy and repurposed medications which could prove lifesaving. Through advances in Artificial Intelligence programs, like Deep Learning technology, one will be able to forecast the likelihood of future mutations, make a more accurate overall prognosis for a given patient with cancer and ultimately know real response rates to a variety of therapeutic agents and interventions.
Dr. Devlin practices Integrative Oncology and Functional Medicine and is one the founders of the International Organization of Integrative Cancer Physicians (IOICP) which is a non-profit cancer research and educational foundation, he currently sits on the IOICP educational advisory board. Dr. Devlin is a board-certified and fellowship trained physician. Dr. Devlin holds a Master’s Degree in Biochemistry and has pursued Doctoral studies in Pharmacology with an emphasis on the evaluation of novel anti-neoplastic agents. Dr. Devlin has been honored to lecture nationally and internationally on a variety of medical topics and is regularly sought to provide his expert opinion through direct consultation, speaking engagements and medical-legal review.
For more information about appropriate testing and specialty laboratories please contact Dr. Sean Devlin at one of his offices listed below:
Dr. Devlin can be reached directly at Gerber Medical Clinic in Reno, Nevada at 775-826-1900, Highland Springs Wellness Center in Grass Valley, California at 530-274-2274 and Advanced Dermatology and Cosmetic Surgery in Lake Tahoe at 775-588-5000.