# When cancer is detectable, how many sub-clones are there at that stage?

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I have read somewhere that cancer is detectable when the number of cells reaches $$10^7 - 10^9$$, which probably varies according to the specific tumor. At this early stage, what is the expected number of subclones of the tumor? Any references would be super appreciated!

p/s: To answer this, I tried running a simple code for a birth-death process, but my laptop freezes at $$10^7$$ cells. :(

## Key Statistics for Colorectal Cancer

Excluding skin cancers, colorectal cancer is the third most common cancer diagnosed in both men and women in the United States. The American Cancer Society’s estimates for the number of colorectal cancer cases in the United States for 2021 are:

The rate of people being diagnosed with colon or rectal cancer each year has dropped overall since the mid-1980s, mainly because more people are getting screened and changing their lifestyle-related risk factors. From 2013 to 2017, incidence rates dropped by about 1% each year. But this downward trend is mostly in older adults and masks rising incidence among younger adults since at least the mid-1990s. From 2012 through 2016, it increased every year by 2% in people younger than 50 and 1% in people 50 to 64.

## What can be detected in the blood?

Scientists are exploring multiple ways to detect cancer in the blood. And, so far, most of the progress has been made in developing blood tests that help to monitor whether someone’s cancer has returned or to inform treatment options.

“The hope is that similar tests could be used for both early detection and monitoring,” says Aitman.

Cancer Research UK funded researchers in Manchester are looking to develop a blood test to track cancer cells that have broken free and begun to circulate in the bloodstream. When studied in the lab, these circulating tumour cells could give useful information about a person’s cancer and help scientists monitor their response to treatment.

But looking for entire, intact cancer cells isn’t the only way to hunt for cancer in the blood. Scientists are looking for even smaller clues.

Aitman describes how in the last 10 years or so scientists have found that when cancer cells die, which happens constantly as a tumour develops and grows, they release tiny bits of DNA into the blood. This free-floating DNA (known as cell-free DNA) carries a footprint of the cancer itself, which can be analysed to detect cancer.

“Once the blood sample has been collected, the cell-free DNA found can be sequenced and we can look for changes in the genome that might indicate cancers,” says Aitman.

And it’s not just the DNA code that scientists can use to help diagnose cancer. They’re also looking for markers on the DNA that affect how and when it’s read. An example of this type of change is DNA methylation, which can be found on free-floating DNA from cancer cells.

Blood tests that pick up these clues are being developed for a variety of different cancers, each relying on different markers.

But there’s also work ongoing to develop a test that can detect multiple types of cancer. Aitman explains that scientists have uncovered DNA changes that can be found in multiple cancers, which means in theory a test could be developed to detect several different cancers at once. But in order for a test like this to be useful, it would also need to signal where the cancer is growing.

## Mitosis

The part of the cell division cycle that gets the most attention is called the M phase or mitosis. Mitosis is the process by which a single cell divides into two daughter cells. The two cells have identical genetic content of the parent cell. As we will see later, cancer cells don't always follow this rule. Mitosis is further broken down into sub-phases based on visible changes within the cells, especially within the nucleus.

The first step is prophase . In prophase, the nuclear envelope dissolves and the chromosomes condense in preparation for cell division. Just like winding up thread on a spool, the condensation of the chromosomes makes them more compact and allows them to be more easily sorted into the forming daughter cells. Also in prophase, protein fibers ( spindle fibers ) form and reach from one end of the cell to another. This bundle of fibers give the dividing cell the structure it needs to push and pull the cell components and form two new cells.

The protein strands that reach from one end of the cell to the other are called microtubules. These proteins are assembled and disassembled during the cell division process. They are the target of several different chemotherapy agents. Taxol®, a chemical derived from an extract of the yew tree, binds to the microtubules and does not allow them to disassemble. This causes the cells to fail in the mitosis process and die. Another class of chemotherapy agent, represented by vinblastine, has the opposite effect. These drugs don't allow the spindle fiber to form. The result is the same, as the cell division process is inhibited. More on Cancer Treatments.

### A Closer Look at Human Chromosomes

The image below shows the chromosomes from a human cell. The depiction of all of the chromosomes in this manner is known as a karyotype. Karyotypes are often performed on fetal tissues during pregnancy to detect chromosomal abnormalities in the unborn child. These chromosomes have been colored by the binding of fluorescent dyes. Notice that there are two copies of each chromosome. The wide range in sizes of the chromosomes is also apparent. The chromosomes are numbered in the inverse order of their size. Chromosome 1 is the largest chromosome and the smallest chromosomes are those numbered 21 and 22. The karyotype shown below is from a male and contains one X and one (much smaller) Y chromosome. The DNA in even the smallest of the chromosomes contains millions of basepairs.

In many cancer cells the number of chromosomes is disturbed so that there are either too many or too few chromosomes in the cells. Cells with too many or too few chromosomes are said to be aneuploid. More on mutation and cancer.

The image above is courtesy of Applied Imaging, Santa Clara, CA.

## Management and Treatment

### How is breast cancer treated?

If the tests find cancer, you and your doctor will develop a treatment plan to eradicate the breast cancer, to reduce the chance of cancer returning in the breast, as well as to reduce the chance of the cancer traveling to a location outside of the breast. Treatment generally follows within a few weeks after the diagnosis.

The type of treatment recommended will depend on the size and location of the tumor in the breast, the results of lab tests done on the cancer cells, and the stage, or extent, of the disease. Your doctor will usually consider your age and general health as well as your feelings about the treatment options.

Breast cancer treatments are local or systemic. Local treatments are used to remove, destroy, or control the cancer cells in a specific area, such as the breast. Surgery and radiation treatment are local treatments. Systemic treatments are used to destroy or control cancer cells all over the body. Chemotherapy and hormone therapy are systemic treatments. A patient may have just one form of treatment or a combination, depending on her individual diagnosis.

Surgery: Breast conservation surgery involves removing the cancerous portion of the breast and an area of normal tissue surrounding the cancer, while striving to preserve the normal appearance of the breast. This procedure has often been called a lumpectomy, also referred to as a partial mastectomy. Typically, some of the lymph nodes, either in the breast and/or under the arm are also removed for evaluation. Usually, six weeks of radiation therapy is then used to treat the remaining breast tissue. Most women who have a small, early-stage tumor are excellent candidates for this approach.

Mastectomy (removal of the entire breast) is another option. The mastectomy procedures performed today are not the same as the older, radical mastectomies. Radical mastectomies were extensive procedures that involved removing the breast tissue, skin, and chest-wall muscles. Today, mastectomy procedures do not ordinarily remove muscles and, for many women, mastectomies are accompanied by either immediate or delayed breast reconstruction.

### What happens after the local breast cancer treatment?

Following local breast cancer treatment, the treatment team will determine the likelihood that the cancer will recur outside the breast. This team usually includes a medical oncologist, a specialist trained in using medicines to treat breast cancer. The medical oncologist, who works with the surgeon, may advise the use of the drugs like tamoxifen or anastrozole (ARIMIDEX®) or possibly chemotherapy. These treatments are used in addition to, but not in place of, local breast cancer treatment with surgery and/or radiation therapy.

After treatment for breast cancer, it is especially important for a woman to continue to do a monthly breast examination. Regular examinations will help you detect local recurrences. Early signs of recurrence can be noted in the incision area itself, the opposite breast, the axilla (armpit), or supraclavicular region (above the collar bone).

Maintaining your follow-up schedule with your physician is also necessary so problems can be detected when treatment can be most effective. Your health care provider will also be able to answer any questions you may have about breast self-examination after the following procedures.

### Breast Examination After Treatment for Breast Cancer

After surgery

The incision line (scar) may be thick, raised, red and possibly tender for several months after surgery. Remember to examine the entire incision line.

If there is redness in areas away from the scar, contact your physician. It is not unusual to experience brief discomforts and sensations in the breast or nipple area (even if the nipple has been removed).

At first, you may not know how to interpret what you feel, but soon you will become familiar with what is now normal for you.

After breast reconstruction

Following breast reconstruction, breast examination for the reconstructed breast is done exactly the same way as for the natural breast. If an implant was used for the reconstruction, press firmly inward at the edges of the implant to feel the ribs beneath. If your own tissue was used for the reconstruction, understand that you may feel some numbness and tightness in your breast. In time, some feeling in your breasts may return.

After radiation therapy, you may notice some changes in the breast tissue. The breast may look red or sunburned and may become irritated or inflamed. Once therapy is stopped, the redness will disappear and the breast will become less inflamed or irritated. At times, the skin can become more inflamed for a few days after treatment and then gradually improve after a few weeks. The pores in the skin over the breast also may become larger than usual.

Some women have different sensations in the breast because of changes in skin sensitivity. You may feel numbness or tingling in the breast, or feel that the breast is more sensitive to clothing or tight garments. After radiation therapy, the breast may become smaller. Normally within a year after radiation therapy, most of the changes will improve.

During radiation therapy, you should continue with monthly self-examinations of the radiated breast as well as the other breast. If you notice any new developments, call your health care provider.

By immediately reporting any suspicious changes to your physician, you will not only receive early treatment if necessary, but you will also resolve your own fear and anxiety. Most breast lumps (about 80 percent) are benign. However, your self-examination may lead you to the early detection of a new or recurrent cancer. The earlier the diagnosis, the better the chances for successful therapy.

## Associated Data

Cancers evolve by a reiterative process of clonal expansion, genetic diversification and clonal selection within the adaptive landscapes of tissue ecosystems. The dynamics are complex with highly variable patterns of genetic diversity and resultant clonal architecture. Therapeutic intervention may decimate cancer clones, and erode their habitats, but inadvertently provides potent selective pressure for the expansion of resistant variants. The inherently Darwinian character of cancer lies at the heart of therapeutic failure but perhaps also holds the key to more effective control.

Cancer is a major cause of mortality throughout the world and despite the extraordinary amount of effort and money expended over the past several decades, successful eradication and control of advanced disease remains elusive 1 . In parallel, our understanding of cancer biology and genetics has changed beyond recognition 2 . The translation of cancer genomics to cancer therapy needs to accommodate the cellular complexity of the disease and address its dynamic, evolutionary character. The latter provides both barriers to success and opportunities.

In 1976 Peter Nowell 3 published a landmark perspective on cancer as an evolutionary process, driven by stepwise, somatic cell mutations with sequential, sub-clonal selection. The implicit parallel was to Darwinian natural selection with cancer equivalent to an asexually reproducing, unicellular, quasi-species. The modern era of cancer biology and genomics has validated the fundamentals of cancer as a complex, Darwinian, adaptive system 4,5 (Box 1, and additional references in Supplemental information).

### Cancer as a Complex System

Cancers exist in an extraordinary variety of taxonomically, quasi-classes, genera, species, characterised by divergent cells of origin and mutational spectra. Each cancer is individually unique.

Cancers evolve over variable time frames (

1� years) and tempos and, in any one patient, the clonal structure, genotype and phenotype shifts over time. Contemporaneously, any one cancer is, in effect, multiply different (sub-clonal) cancers occupying overlapping or distinct tissue habitats.

The number of mutations found in any cancer can vary from a handful (10�) to (more usually) hundreds of thousands. The great majority are ‘passengers’, a modest but undefined number being functionally relevant 𠆍rivers’. The mutational processes are very diverse.

Cancers acquire, via mutational (and epigenetic) changes, a variety of critical phenotype traits that compound to empower territorial expansion, via proliferative self-renewal, migration and invasion properties that are part and parcel of normal developmental, physiological and repair processes.

Advanced, disseminated or very malignant cancers appear to be almost uniquely competent to evade therapy.

Most, if not all, of this complexity can be explained by classical evolutionary principles.

Cancer clone evolution takes place within tissue ecosystem habitats which have themselves evolved over a billion years. Their complex anatomies and networked signals have evolved to optimise and integrate multi-cellular functions whilst restraining renegade clonal expansion. The balance however is delicate as the resilience of multi-cellular and long-lived animals such as ourselves depend upon the very phenotypic properties that, if not tightly regulated, drive or sustain malignancy, i.e. self-renewal coupled with stabilization of telomeres that allows extensive proliferation, angiogenesis, cell migration and invasion 6 .

Tissues provide the context for cancer cell evolution. The usually protracted time required for the clinical emergence of cancer and the resultant mutational complexity often reflect the sequential and random ‘searches’ for phenotypic solutions to micro-environmental constraints. The evolutionary progression of cancer is more often than not stalled or aborted, as revealed by the high frequencies of clinically covert pre-malignant lesions 7𠄹 . Cancer suppressive mechanisms relegate most cancers to old age where they have little effect on the reproductive fitness of their hosts.

Resource limitations and other micro-environmental constraints limit the size of tumours at multiple stages of progression. Even full-blown malignancies often exhibit Gompertzian growth 10 . The doubling time of cancer cells (

1𠄲 days) is orders of magnitude faster than the doubling time of tumours (

60� days) 10 , implying that the vast majority of cancer cells die before they can divide 11 . Thus, natural selection in tumours, like selection among organisms, often takes place through severe competition for space and resources.

Oncologists change cancer clone dynamics dramatically by introducing a new potent source of 𠆊rtificial’ selection – with drugs or radiotherapy. But similar evolutionary principles apply. Massive cell death will usually ensue providing selective pressure for the proliferation of variant cells that can, by one of several mechanisms (see below), resist therapeutic oblivion. To make matters worse, many cancer therapeutics are genotoxic surviving cells regenerating the cancer may have incurred additional mutational insults, some of which could improve their fitness and malignant potential.

These general considerations suggest that much can be gained by applying the tools and insights of evolutionary biology and ecology to the dynamics of cancer pre- and post-treatment. Here, we provide a portrait of cancer as an evolutionary process and argue that this can both explain our modest therapeutic returns and suggest alternative strategies for effective control.

## Understanding a Breast Cancer Diagnosis

If you’ve been diagnosed with breast cancer, you’ve probably heard a lot of different terms used to describe your cancer. Doctors use information from your breast biopsy to learn a lot of important things about the exact kind of cancer you have. You may also need more tests to get more details, such as the stage of the cancer or how fast it’s growing.

### Types of Breast Cancer

There are several types of breast cancer. The type of breast cancer you have depends on where in the breast it started and other factors.

### Breast Cancer Grade and Other Tests

Doctors use information from your breast biopsy to learn a lot of important things about the exact kind of breast cancer you have.

### Stages and Outlook (Prognosis)

If you have been diagnosed with breast cancer, tests will be done to find out the extent (stage) of the cancer. The stage of a cancer helps determine how serious the cancer is and how best to treat it.

You can take an active role in your breast cancer care by learning about your cancer and its treatment and by asking questions. Get a list of key questions here.

## Blood test for early detection of cancer: final study results support screening use

Final results from a study of a blood test that can detect more than 50 types of cancer have shown that it is accurate enough to be rolled out as a multi-cancer screening test among people at higher risk of the disease, including patients aged 50 years or older, without symptoms.

In a paper published in the leading cancer journal Annals of Oncology [1] today (Friday), researchers report that the test accurately detected cancer, often before any symptoms arose, while having a very low false positive rate. The test also predicted where in the body the cancer is located with a high degree of accuracy, which could help doctors choose effective diagnostic tests.

GRAIL, Inc. (California, USA), the company developing and funding the research, has now made the multi-cancer early detection test available in the USA by prescription only, and to complement other, existing screening methods such as those for breast, cervical, prostate, lung and bowel cancers [2]. Many of the cancers that the test is capable of detecting do not have screening tests available, such as liver, pancreatic and oesophageal cancers, which are among the most deadly and where early detection could make a real difference.

First author of the paper, Dr Eric Klein, chairman of the Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, USA, said: "Finding cancer early, when treatment is more likely to be successful, is one of the most significant opportunities we have to reduce the burden of cancer. These data suggest that, if used alongside existing screening tests, the multi-cancer detection test could have a profound impact on how cancer is detected and, ultimately, on public health."

The test involves taking a sample of blood from each patient and analysing it for DNA, known as cell-free DNA (cfDNA), which tumours (and other cells) shed into the blood. Genomic sequencing is used to detect chemical changes to the DNA called "methylation" that control gene expression, and a classifier developed with machine learning (artificial intelligence) uses these results to detect abnormal methylation patterns that suggest cancer is present. In addition, the machine learning classifier can predict where in the body the cancer is located. Results are available within ten business days from the time the sample reaches the lab.

The third and final sub-study of the Circulating Cell-free Genome Atlas (CCGA) study reported today investigated the performance of the test in 2,823 people already diagnosed with cancer and 1,254 people without cancer. It detected cancer signals from more than 50 different types of cancer and found that across all four cancer stages (I, II, III, IV), the test correctly identified when cancer was present (the sensitivity or true positive rate) in 51.5% of cases. The test's specificity (the true negative rate) was 99.5%, meaning that the test wrongly detected cancer (the false positive rate) in only 0.5% of cases.

Sensitivity of the test was 67.6% overall across stages I-III in 12 pre-specified cancers that account for two-thirds of cancer deaths in the USA each year (anal, bladder, bowel, oesophageal, stomach, head and neck, liver and bile duct, lung, ovarian and pancreatic cancers, lymphoma and cancers of white blood cells such as multiple myeloma), and it was 40.7% overall in more than 50 cancers.

For all cancers, detection improved with each cancer stage with a sensitivity rate of 16.8% at the early stage I, 40.4% at stage II, 77% at stage III and 90.1% at stage IV - the most advanced stage when symptoms are often showing.

The sensitivity varied by type of cancer. In solid tumours that do not have any screening options, such as oesophageal, liver and pancreatic cancers, overall sensitivity of the test was twice that for solid tumours that do have screening options, such as breast, bowel, cervical and prostate cancers: 65.6% compared to 33.7%. Overall sensitivity in cancers of the blood, such as lymphoma and myeloma, was 55.1%.

In addition, the multi-cancer early detection test correctly identified the tissue in which the cancer was located in the body in 88.7% of cases.

Dr Klein said: "We believe that cancers that shed more cfDNA into the bloodstream are detected more easily. These cancers are also more likely to be lethal, and prior research shows that this multi-cancer early detection test more strongly detects these cancer types. Cancers such as prostate shed less DNA than other tumours, which is why existing screening tests are still important for these cancers."

In order to understand how the test would perform when used to screen populations, the researchers estimated its positive predictive value (PPV) - the proportion of cases correctly identified as cancer among those with a positive result - as well as the negative predictive value (NPV) - those correctly identified as not having cancer. The PPV was 44.4% among people most likely to develop cancer, those aged 50-79, and the NPV was 99.4%.

Dr Klein concluded: "These data add to a growing body of literature that supports the use of next-generation sequencing for the detection of cell-free DNA in blood samples as a tool for earlier detection of common cancers that account for a significant number of deaths and other health problems worldwide. In addition, a screening test that requires only a simple blood draw could provide an option for communities that have poor access to medical facilities. I'm excited about the potential impact this approach will have on public health."

Researchers are continuing to collect additional data from the test in large, prospective studies in the USA (STRIVE, PATHFINDER and REFLECTION studies) and the UK (SUMMIT study), and to examine its feasibility for screening populations [3]. GRAIL has also established a partnership with the UK's National Health Service to investigate the multi-cancer early detection test's clinical and economic performance in approximately 165,000 eligible patients, starting later this year.

A strength of the CCGA study is that, overall, it includes a total of 15,254 participants from 142 clinics in North America, helping to ensure the results can be generalisable to a diverse population. The participants in this final sub-study had not been included in the earlier, development stages of the test to ensure accurate estimations of performance. Limitations of this sub-study include: if blood samples were collected from cancer patients after they had had a biopsy, this could increase the proportion of cfDNA in the blood compared to before the biopsy CCGA is a case control study and may not fully reflect how the test would perform in population screening conditions (this is being evaluated in the PATHFINDER study) and some inaccuracy occurred in the detection of the tissue of cancer origin for cancers that are driven by the human papilloma virus (HPV), such as cancers of the cervix, anus, and head and neck.

Editor-in-chief of Annals of Oncology, Professor Fabrice André, Director of Research at the Institut Gustave Roussy, Villejuif, France, said: "Early detection of cancer is the next frontier in cancer research as it could save millions of lives worldwide. Developing technologies that address this issue is the first step. Next steps will include the development of new therapeutic interventions. In parallel, major efforts related to population awareness must continue or all these efforts will not lead to transformation of outcomes."

[1] "Clinical validation of a targeted methylation-based multi-cancer early detection test using an independent validation set", by Eric Klein et al. Annals of Oncology. doi: https:/ / doi. org/ 10. 1016/ j. annonc. 2021. 05. 806

[2] The multi-cancer detection test is recommended for people over the age of 50 as this is when more cancers are likely to start to occur. It is available in the USA apart from in the state of New York.

[3] ClinicalTrials.gov numbers: NCT03085888, NCT03934866, NCT04241796. The REFLECTION study does not yet have a number.

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.

## Early Detection, Diagnosis and Staging

Screening for oral cancer should include a thorough history and physical examination. The clinician should visually inspect and palpate the head, neck, oral, and pharyngeal regions. This procedure involves digital palpation of neck node regions, bimanual palpation of the floor of mouth and tongue, and inspection with palpation and observation of the oral and pharyngeal mucosa with an adequate light source mouth mirrors are essential to the examination. Forceful protraction of the tongue with gauze is necessary to visualize fully the posterior lateral tongue and tongue base.

The clinician should review the social, familial, and medical history and should document risk behaviors (tobacco and alcohol usage), a history of head and neck radiotherapy, familial history of head and neck cancer, and a personal history of cancer. Patients over 40 years of age should be considered at a higher risk for oral cancer. 3

Diagnosis can be delayed by several months or more if the clinician treats the patient’s complaints empirically with drugs instead of providing a thorough physical examination and workup. Patients with complaints lasting longer than 2-4 weeks should be referred promptly to an appropriate specialist to obtain a definitive diagnosis. If the specialist detects a persistent oral lesion, a biopsy should be performed without delay.

The many signs and symptoms of oral cancer are usually divided into early and late presentation. They can be so diverse that the differential diagnosis may not lead to oral malignancy. Table 1 summarizes the signs and symptoms.

#### Early

Progressive swelling or enlargement Unusual surface changes

Sudden tooth mobility without apparent

Paresthesia, dysesthesia of the tongue

Chronic earache (chronic serous otitis

#### Table 2: Comparison of Toluidine Blue Uptake with Microscopic Diagnosis

Because patients may be at risk for developing multiple primary tumors simultaneously or in sequence, the entire visible mucosa of the upper aerodigestive tract must be examined. In addition, lymph nodes in the head and neck area-particularly along the jugular chain-must be palpated. Approximately 90% of patients with squamous cell carcinoma in a lymph node in the neck area will have an identifiable primary tumor elsewhere, and about 10% will have cancer in the neck lymph node (4) as an isolated finding (“unknown primary”). Thus, most cancers in the neck node represent a metastasis from a primary tumor located in the head and neck region this primary site must be identified. (5,6)

Toluidine blue (vital staining) also is a useful adjunct to clinical examination and biopsy. The mechanism is based on selective binding of the dye to dysplastic or malignant cells in the oral epithelium. It may be that toluidine blue selectively stains for acidic tissue components and thus binds more readily to DNA, which is increased in neoplastic cells.

Toluidine blue has been recommended for use as a mouthwash or for direct application on suspicious (9 ) lesions its value comes from its simplicity, low cost, noninvasiveness, and accuracy (Table 2). In addition, it can help to determine the most appropriate biopsy sites and to surgically delineate margins. Meta-analysis of toluidine blue staining in oral cancer screening found that its sensitivity ranged from 93.5% to 97.8%, and specificity from 73.3% to 92.9%. (7)

The disadvantages of toluidine blue include the risk of obtaining a false negative reaction in a case where the patient is not followed up adequately. In contrast, the infrequent false-positive only subjects the patient to a biopsy. No in vivo observations or reports have suggested a mutagenic effect from this stain. (8)

#### Diagnosis

Currently, the most effective way to control oral cancer is to combine early diagnosis and timely and appropriate treatment. Because more than 90% of all oral cancers are squamous cell carcinomas, the vast majority of oral cancers will be diagnosed from lesions on the mucosal surfaces.

The clinician’s challenge is to differentiate cancerous lesions from a multitude of other red, white, or ulcerated lesions that also occur in the oral cavity. Most oral lesions are benign, but many have an appearance that may be confused with a malignant lesion, and some previously considered benign are now classified premalignant because they have been statistically correlated with subsequent cancerous (10) changes. Conversely, some malignant lesions seen in an early stage may be mistaken for a benign (11) change. Any oral lesion that does not regress spontaneously or respond to the usual therapeutic measures should be considered potentially malignant until histologically shown to be benign. A period of 2-3 weeks is considered an appropriate period of time to evaluate the response of a lesion to therapy before obtaining a definitive diagnosis.

A definitive diagnosis requires a biopsy of the tissue. Biopsies may be obtained using surgical scalpels or biopsy punches and typically can be performed under local anesthesia. Incisional biopsy is the removal of a representative sample of the lesion excisional biopsy is the complete removal of the lesion, with a border of normal tissue. The clinician can obtain multiple biopsy specimens of suspicious lesions to define the extent of the primary disease and to evaluate the patient for the presence of possible synchronous second malignancies. Useful adjuncts include vital staining, exfoliative cytology, fine needle aspiration biopsy, routine dental radiographs and other plain films, and imaging with magnetic resonance imaging (MRI) or computed tomography (CT). Table 3 presents a suggested protocol for patient evaluation.

Most carcinomas of the oral cavity do not need a “panendoscopy” for definitive diagnosis. Such a procedure, which consists of direct laryngoscopy, esophagoscopy, and bronchoscopy, is usually performed as a diagnostic and staging procedure in patients with carcinoma of the oropharynx.

#### Imaging the Oral Cavity

A diagnostic imaging evaluation consisting of either computer tomography (CT) scanning or magnetic resonance imaging (MRI) is also used to assess the extent of local and regional tumor spread, the (12,13) depth of invasion, and the extent of lymphadenopathy. CT is superior in detecting early bone invasion and lymph node metastasis, but MRI is preferred for assessing the extent of soft tissue involvement and for providing a three-dimensional display of the tumor. MRI is also the preferred technique for imaging carcinoma of the nasopharynx or lesions involving paranasal sinuses or the skull base.

#### Table 3: Patient Work Up

2 – Head and neck examination: direct visualization mirror examination manual palpation toluidine blue staining

3 – Laboratory tests: CBC liver function

4 – Radiology: CT or MRI of head and neck chest x-ray dental films bone scan when indicated

5 – Pathology incisional biopsy excisional biopsy fine needle aspiration biopsy molecular markers flow cytometry

6 – “Panendoscopy:” define T-stage draw schematic tumor map evaluate for second malignancies

7 – Pre-therapy consultation with: radiation oncology medical oncology head and neck surgery reconstructive surgery dental oncology speech pathology psychosocial service

Diagnostic imaging often detects subsurface masses and intraosseous lesions. Although imaging of pathologic lesions does not produce a definite diagnosis, it frequently helps to define the extent of the

tumor. For example, patients who have an unexplained neck node and a negative head, neck, and oral examination may undergo CT scanning followed by a biopsy of the nasopharynx or base of tongue that reveals a suspicious area or tissue change.

Both CT and MRI have limitations as well as advantages, a fact that frequently makes them complementary rather than competitive studies. The advantages of CT include its rapid acquisition time (2-3 seconds per section), patient tolerance, relatively low cost, and superior osseous detail compared with MRI. However, the soft-tissue contrast resolution of CT is relatively poor, which makes it difficult to distinguish between tumor and normal muscle. CT also may require the administration of intravenous contrast material to differentiate vessels from lymph nodes, thereby increasing the risk of an allergic reaction. In addition, CT is frequently degraded by scattered artifacts because of metallic dental appliances. (14)

MRI’s several advantages over CT have helped it evolve into a reliable alternative for imaging normal and pathologic head and neck anatomy. The superior soft-tissue resolution of MRI allows high-contrast differentiation between neoplasms and adjacent muscle. In addition, MRI can be obtained in multiple planes (sagittal, axial, coronal, and oblique), which is often helpful in assessing tumor volumes during and after therapy. Finally, the need for intravascular contrast administration is avoided because patent vessels have absent signal, or “signal void,” within their lumen, which easily distinguishes them from surrounding soft tissue structures.

However, MRI is not without its drawbacks. Because all the images within a given MRI sequence are obtained simultaneously rather than sequentially, patient movement during an MRI is less well tolerated than with CT. In addition, although the soft-tissue contrast is superb with MRI, fine-bone detail is inferior to that obtained with CT.

#### Cytology

Under certain conditions, exfoliative cytology (cell scrapings) serves as an adjunct to clinical diagnosis, as it enables more extensive screening and provides microscopic material if there is a delay in or contraindication to biopsy. However, cytologic smears are used infrequently, and patients are not treated on the basis of cytologic findings alone. Smears are most helpful in differentiating inflammatory conditions, especially candidiasis, from dysplastic or neoplastic surface lesions. In addition, cytology may be helpful in detecting field change in oral cancer, especially if this method is used in conjunction with vital staining. Cytology may also be helpful when ulcerations following radiation are suspicious and biopsy is delayed.

Fine needle aspiration biopsy of subsurface masses is also an accepted diagnostic test, one that has increased in popularity over the past few years. This technique is extremely useful in evaluating clinically suspicious changes involving salivary glands and lymph nodes. It expedites diagnosis and

staging and avoids incisional or excisional biopsies that may interfere or complicate definitive treatment. When used by a skilled clinician, fine needle aspiration can often be the best way to establish a definitive diagnosis of unexplained masses of the neck or salivary glands. It is also valuable in following up cancer patients with suspicious enlargements. 15

#### Staging of the Disease

The stage of the disease depends on several factors, including the size of the primary lesion, local extension, lymph node involvement, and evidence of distant metastasis. Tumor size, the organ or tissue affected, and the extent of spread are considered to be the best indicators of the patient’s prognosis. Table 4 summarizes the most widely accepted staging protocol, the tumor-node­ metastasis (TNM) classification of oral cancer. This system has 3 basic clinical features: the size (in centimeters) of the primary tumor the presence, number, size, and spread (unilateral or bilateral) to the local lymph nodes and the presence or absence of distant metastasis.

#### Table 4: Tumor-Node-Metastasis (TNM) Staging System for Oral Carcinoma

Primary Tumor (T)

TX Primary tumor cannot be assessed

T0 No evidence of primary tumor

T1 Tumor 2 cm or less in greatest dimension

T2 Tumor more than 2 cm but not more than 4 cm in greatest

T3 Tumor more than 4 cm in greatest dimension

cortical bone, tongue, skin of neck)

through cortical bone, into deep

tongue, maxillary sinus, skin)

#### Regional Lymph Nodes (N)

NX Regional lymph nodes cannot be assessed

N0 No regional lymph node metastasis

N1 Metastasis in a single ipsilateral lymph node, 3 cm or less in greater dimension

N2 Metastasis in a single ipsilateral lymph node, more than 3 cm but not more than 6 cm in greatest dimension in multiple ipsilateral lymph nodes, none more than 6 cm in greatest dimension in bilateral or contralateral lymph nodes, none more than 6 cm in greatest dimension

N2a Metastasis in single ipsilateral lymph node more than 3 cm but not more than 6 cm in greatest dimension

N2b Metastasis in multiple ipsilateral lymph nodes, none more than 6 cm in greatest dimension

N2c Metastasis in bilateral or contralateral lymph nodes, none more than 6 cm in greatest dimension

N3 Metastasis in a lymph node more than 6 cm in greater dimension

#### Distant Metastasis (M)

MX Presence of distant metastasis cannot be assessed

The individual clinical parameters in the TNM classification system are grouped to determine the appropriate disease stage (Table 5) stages are ranked numerically from 0 (which has the best prognosis) to IV (the worst prognosis). In general, oral staging classifications do not use histopathologic findings except to determine the definitive diagnosis.

#### Table 5: TNM Clinical Stage Grouping 16

Schematic drawings of the tumor (tumor maps) are frequently prepared to document the site and size of the tumor at the initial time of diagnosis. This initial documentation is later complemented by histopathologic findings and imaging preformed during the treatment phase.

Although the risk of distant metastasis is generally low in patients with oral cancer, there is a (17) correlation between the incidence of distant metastasis and tumor (T) and neck (N) stage. When they do occur, the most frequently involved organs are the lungs, bone, and liver. Patients with advanced T or N stages may be at risk for developing metastases outside the head and neck region a limited workup (chest x-ray, CBC and liver function tests, bone scan) to exclude such a metastasis may be indicated.

After completion of the initial workup, the final T, N, M (metastasis), and overall stage assignment should be formally determined and documented prior to treatment. Because rehabilitation planning starts with staging and treatment, a multidisciplinary approach is essential (see Chapters VII and VIII).

#### Disease Progression

Oral squamous cell carcinoma spreads primarily by local extension and somewhat less often by the lymphatics. The extent of tumor invasion depends upon the anatomic site, the tumor’s biologic aggressiveness, and host response factors.

The lymphatic system is the most important and frequent route of metastasis. Usually the ipsilateral cervical lymph nodes are the primary site for metastatic deposits, but occasionally contralateral or bilateral metastatic deposits are detected. The risk for lymphatic spread is greater for posterior lesions of the oral cavity, possibly because of delayed diagnosis or increased lymphatic drainage at those sites, or both. Cervical lymph nodes with metastatic deposits are firm-to-hard, nontender enlargements. Once the tumor cells perforate the nodal capsule and invade the surrounding tissue, these lymph nodes become fixed and non mobile.

Metastatic spread of tumor deposits from oral carcinoma usually occurs in an orderly pattern, beginning with the uppermost lymph nodes and spreading down the cervical chain. Because of this pattern of spread, the jugulo-digastric nodes are most prone to early metastasis. Carcinomas involving the lower lip and floor of the mouth are an exception, as they tend to spread to the submental nodes. Although lymph node metastasis is not an early event, as many as 21% of individuals with oral cancer present at diagnosis with nodal metastasis.
(This proportion exceeded (18) 50% in a study of patients evaluated at admission to cancer centers. )

Hematogenous spread of tumor cells is infrequent in the oral cavity but may occur because of direct vascular invasion or seeding from surgical manipulation. Perhaps 10-34% of patients present with (3)

distant metastasis this risk increases with advanced disease. Among the most common sites for distant metastasis are the lungs, liver, and bones. These patients cannot be cured and are treated with palliative intent, usually involving chemotherapy, radiotherapy, or both. (3)

Approximately 30% of patients will present initially with highly confined localized disease stages (T 1 or T ). These patients are treated with curative intent, usually involving surgery, radiation therapy, 2 or both. Only about 20-40% of patients will develop a local or regional tumor recurrence. However, over subsequent years, these “cured” patients appear to be at higher risk for developing a second malignancy than for developing a recurrence of their initial tumor. Tumor recurrences most often occur during the first 2 years after therapy later recurrences are rare. Second malignancies, on the other hand, will be observed at a steady rate-perhaps 3-5% per year. Thus, with sufficient follow-up time, second malignancies or other medical diseases become greater problems than recurrence of the primary disease. The use of drug therapy to decrease the rate of second malignancies is being actively investigated.

Patients with locoregionally advanced disease (T 3 , T 4 , N 1 , N 2 3 , and N ) are also treated with curative intent. Given the advanced stage of their disease, surgery and radiation are utilized unless patients are considered inoperable or have unresectable disease. Despite this aggressive bimodality therapy, the majority of these cancers will recur within the first 2 years of follow-up, most commonly either locally or regionally. Some of these patients may have metastases outside the head and neck area, events that might be predicted by their initial T and N stages. Investigational therapy in this group of patients, therefore, must focus primarily on delivering more effective locoregional care. However, should locoregional control be improved, chemopreventive strategies will need to be pursued in this group of patients as well since, in principle, oral cancer patients are at risk for developing second primary malignancies in the oral cavity, pharynx, and respiratory and digestive tracts.

#### Multiple Carcinomas

Individuals with one carcinoma of the head and neck region have an increased risk of developing a (19) second malignancy the frequency of that event varies from 16% to 36%. When a second malignancy occurs at the same time as the initial lesion, it is called a synchronous carcinoma. Metachronous neoplasms, on the other hand, are additional primary surface epithelial malignancies that develop in a later time period than the original tumor. About 40% of second malignancies of the upper aerodigestive tract arise simultaneously and represent a synchronous tumor. The remaining multiple cancers in this population represent metachronous disease and usually develop within 3 years (19) of the initial tumor. Second primary tumors are the chief cause of death in patients with an early ­stage diagnosis. (20)

The tendency to develop multiple carcinomas in the upper aerodigestive region is known as “field (21) cancerization.” Prolonged and diffuse exposure to local carcinogens, particularly tobacco combined with alcohol, appears to increase the malignant transformation potential of exposed epithelial cells (22)

in the upper aerodigestive tract and lungs. The overall risk for developing a second head and neck malignancy is 10 to 30 times higher in populations that use tobacco and alcohol than in the general population. (23)

#### B. Emerging Trends Early Detection

At the present time, the most effective approach to reducing morbidity and mortality from oral cancer is early detection. However, progress in this area requires changes in public and professional knowledge, attitudes, behaviors, and practices (see Chapter IX for a full discussion).

#### Immunohistochemical Techniques

The use of immunohistochemical techniques to establish a definitive diagnosis has expanded during the past decade and continues to be refined. These diagnostic tests help to establish a definitive diagnosis when, by routine histopathology techniques, a lesion appears morphologically benign or its classification is in doubt. Research on the biochemical, genetic, and cellular levels should yield information that will identify high-risk groups for many types of cancer including oral cancer.

#### Imaging Techniques

Imaging techniques continue to improve at a rapid rate. Newer imaging techniques hold promise for (24) clinical staging of T 2, T 3 and T 4 1 lesions, but T lesions are typically too small to be visualized. Improvements that increase definition will promote earlier detection of nasopharyngeal, submucosal, and bone lesions. One such technique appropriate for lymph nodes is positron emission tomography, which may help to define tumor activity in clinically negative areas. (25)

#### Biochemical and Genetic Factors

No matter which diagnostic technique is used, there is the possibility of a false-negative diagnosis. However, studies are under way to identify key markers that should improve accuracy. The development of monoclonal antibodies that have high sensitivity and specificity for epithelial dysplastic and malignant cells would enhance accuracy of diagnosis in some cases where the usual or typical cellular characteristics of precancer or cancer are not apparent. Such antibodies might also minimize errors about “tumor free” margins of surgical resections, thereby reducing a potential source for recurrence. In addition, assuming that an antibody was specific for a particular cellular tumor antigen, binding of cytotoxic chemotherapeutic agents for killing tumors and sparing normal cells would be a logical and possibly feasible follow-up to surgery and radiation therapy to improve cancer control.

Additional knowledge about various cell markers that reflect growth and suppressor protein presence or activity may also prove to be of great value in predicting cell behavior. Genetic/chromosome evaluations may serve a similar purpose in the identification and treatment of tumors.

Current research is exploring the genetics of biochemical processes that may affect the development of oral cancer. Included are gene mutations such as tumor suppressor gene amplification and overexpression of proto-oncogenes c-myc, EGFR and cyclin D1, as well as loss of heterozygosity of specific chromosome loci. Cellular alteration of response to growth factor and Beta’s (TGF-beta) growth suppressor effect on tumor cells may become important as well.

#### Clinical Photodetection

Photodynamic therapy, also known as PDT, and photodetection of cancer may be useful in the oral cavity. Two important variables that must be considered are the uptake of the dye and the dye contrast by normal and neoplastic tissue after injection. (26)

#### Early Detection

The role that health care professionals who are not physicians or dentists play in oral cancer screening is poorly defined. Potential participants include dental hygienists, physician’s assistants, and nurses. There has been some assessment of the role of hygienists, but very little for physician’s assistants or nurses. The medical and dental professions need additional information on the most effective ways to provide early detection screening for all patients, including medically underserved populations. In addition, health care professionals need to know how to instruct patients on oral self-examination techniques. Most practitioners are aware that such instruction is reasonable and practical for breast cancer but are unaware of its role in the early detection of oral cancer.

Similarly, most of the general public is poorly informed about the risk of oral cancer and ways to prevent this disease. In a recent NIH study, only 25% of surveyed adults could identify one sign of (27) oral cancer. Much public attention is paid to the dangers of cigarette smoking, where the major emphasis is on lung cancer and cardiovascular disease, less on increased cancer risk in the upper airways and oral cavity. In recent years more information has been directed toward oral cancer risks in smokeless tobacco abusers than in cigarette smokers.

Most people have little interest in estimating their oral cancer risk based on age, sex, race, or even habits such as drinking or smoking. The portion of the public that regularly receives medical and dental care tends to assume it is routinely and adequately screened for all types of disease, including all forms of cancer. These people are generally unaware that to screen properly for oral cancer requires a head, neck, and oral examination. Thus, the failure of a primary care doctor to perform those procedures would likely go unnoticed by the average patient. Similarly, many patients are no doubt unclear as to who should be responsible for screening them for oral cancer.

Although members of the public have been informed to some degree regarding the general warning signs of cancer, they may not know the early signs of oral cancer. Not surprisingly, far too many oral cancer patients do not seek care until their tumors are advanced, which suggests that a much better job must be done of informing patients when and how to seek help.

#### Diagnosis

Fine needle aspiration biopsy is an accepted procedure for diagnosing many subsurface lesions such as salivary gland tumors and nodal disease. However, it is often used inappropriately on many other occasions the clinician retrieves nondiagnostic tissue. Increased practitioner training on properly applying the procedure and using CT scanning to guide tissue retrieval is needed.

Another problem is that many clinicians lack a clear understanding of the criteria for ordering the various types of imaging available today, many of them quite costly. Inappropriate and indiscriminate use of imaging results in millions of dollars wasted annually. In general, except for unusual and occult lesions, sophisticated imaging is not required for early detection, but it may be essential later to enhance clinical staging and treatment. Clinicians also frequently order CTs and MRIs but do not indicate the extent of anatomy essential for staging thus, the study needs to be repeated.

Because of the well-recognized phenomenon of “field cancerization” in the head and neck region, it is important to refer patients who are diagnosed with a primary squamous cell carcinoma or epithelial dysplasia of the oral cavity for evaluation of a synchronous tumor. In addition, an annual evaluation for detection of metachronous disease should be reinforced for these patients. Such patients should be monitored routinely for high-risk behaviors, including continued tobacco and alcohol consumption, because these behaviors adversely influence survival after the occurrence of a second cancer. Finally, the use of consultations and tumor board services is essential, even in what may be deemed “early cancer.” (28)

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## Overdiagnosis and overtreatment

Screening mammograms can often find invasive breast cancer and ductal carcinoma in situ (DCIS, cancer cells in the lining of breast ducts) that need to be treated. But it’s possible that some of the invasive cancers and DCIS found on mammograms would never grow or spread. (Finding and treating cancers that would never cause problems is called overdiagnosis.) These cancers are not life-threatening, and never would have been found or treated if the woman had not gotten a mammogram. The problem is that doctors can’t tell these cancers from those that will grow and spread.

Overdiagnosis leads to some women getting treatment that’s not really needed (overtreatment), because the cancer never would have caused any problems. Doctors can’t always tell which cancers will be life-threatening and which won’t ever cause problems. Because of this, all cases are treated. This exposes some women to the side effects of cancer treatment, even though it’s really not needed.

Still, overdiagnosis is not thought to happen very often. There’s a wide range of estimates of the percentage of breast cancers that might be overdiagnosed by mammography, but the most credible estimates range from 1% to 10%.