Classification of Tumors—Classical Histology and Modern Genetics

Carcinoma in situ (often abbreviated CIS) refers to preinvasive epithelial malignant tumors of glandular or squamous cell origin. Cancers develop incrementally, as they accumulate specific genetic lesions. Careful surveillance for cancer often detects abnormal growths in epithelial tissues that have atypical cells and an increased proliferation rate compared with normal surrounding tissues. These early stage cancers are localized to the epithelium and have not penetrated the local basement membrane or invaded the surrounding stroma (see Figure 12-1). Based on these characteristics, they are not malignant but are often called carcinoma in situ (CIS). CIS is recognized in a number of sites, including the cervix, skin, oral cavity, esophagus, and bronchus. In glandular epithelium, in situ lesions occur in the stomach, endometrium, breast, and large bowel. In the breast, ductal carcinoma in situ (DCIS) fills the mammary ducts but has not progressed to local tissue invasion. DCIS lesions are readily treatable, although the optimal therapeutic approach is controversial. CIS lesions can have one of the following three fates: they can remain stable for a long time, they can progress to invasive and metastatic cancers, or they can regress and disappear. CIS can vary from low-grade to high-grade dysplasia, with the high-grade lesions having the highest likelihood of becoming invasive cancers. The time that such preinvasive lesions remain in situ before becoming invasive is unknown.¹ Some carcinomas of the cervix appear as preinvasive lesions in situ for several years before they progress to invasive carcinoma and metastatic tumors (see Figure 12-1). Knowing how to best treat low-grade CIS lesions is challenging because the proportion that progress to cancer vs. the proportion that will never cause clinical problems is usually not known. Although most persons prefer removal of any CIS as opposed to “watchful waiting,” this topic continues to be a source of great debate.

Because our knowledge about the molecular alterations in cancer can influence the choices of therapy, it becomes increasingly important for clinicians to accurately molecularly classify each cancer. The classification, and hence the treatment decisions, of cancers was originally based on gross and light microscopic appearance and is now commonly accompanied by immunohistochemical analysis of protein expression. Increasingly, this is supplemented by a more extensive molecular analysis of the tumors. Sometimes a single gene is examined (for example, to determine if there is a characteristic chromosomal translocation diagnostic of chronic myelogenous leukemia [CML]), and sometimes a panel of genes and proteins are examined (e.g., in breast cancer) to determine if the tumor expresses estrogen receptor, progesterone receptor, and the epidermal growth factor (EGF) receptor HER2, or if there are mutations in specific genes that include response to therapy. In a research setting, and increasingly in clinical settings, expression and mutation analysis of a large number of genes can be measured using polymerase chain reaction (PCR), microarray, or advanced DNA sequencing technology. These analyses can be used to classify tumors more precisely and may predict the most effective therapy. This detailed analysis of each tumor is a form of personalized medicine that offers therapy based on a very detailed knowledge of each individual’s characteristics and their specific cancer.² This enhanced molecular characterization subdivides cancers into therapeutically and prognostically relevant smaller groups. As an example, breast cancers can now be subclassified into more than four types (luminal A, luminal B, basal-like, and others) based on their expression of specific markers, such as estrogen receptor, HER2/Neu, and other specific genes and proteins. Each subtype has a different response to therapy and a different prognosis (Figure 12-3).

Tumor Markers

During surveillance or diagnosis of cancer as well as following therapy, specific biochemical markers of tumors have proven to be helpful. These tumor markers are substances produced by both benign and malignant cells that either are present in or on tumor cells or are found in blood, spinal fluid, or urine (Table 12-3). Some tumor markers have been known for many decades. For diseases associated with a tumor marker, there is indeed a “blood test for cancer.” Tumor markers include hormones, enzymes, genes, antigens, and antibodies. Liver and germ cell tumors secrete a protein known as alpha fetoprotein (AFP) into the blood, and prostate tumors secrete prostate specific antigen (PSA) into the blood. If the tumor marker itself has biologic activity, then it can cause symptoms, a phenomenon known as a paraneoplastic syndrome. For example, the adrenal medulla normally secretes the catecholamine epinephrine (adrenaline). Benign tumors of the adrenal medulla (pheochromocytoma) can produce catecholamines (e.g., adrenaline) in vast excess, leading to rapid pulse rate, high blood pressure, diaphoresis (i.e., sweating), and tremors. Detection of elevated blood or urine levels of catecholamines helps to confirm the diagnosis, and treatment of the disease relieves the symptoms. Tumor markers can be used in three ways: (1) to screen and identify individuals at high risk for cancer; (2) to help diagnose the specific type of tumor in individuals with clinical manifestations relating to their tumor, as in adrenal tumors or enlarged liver or prostate; and (3) to follow the clinical course of a tumor. For example, a falling PSA level after radiation or surgical therapy for prostate cancer indicates successful treatment, and a later rise in the PSA level may indicate a recurrence.

There are several significant problems in using tumor marker assays to screen populations of healthy individuals for cancer. Testing large populations will always detect a few normal individuals with test results at the high end of the normal distribution (the “false positives”), which can lead to expensive and invasive additional tests, and unnecessary concern. Similarly, some individuals with disease will have test results in the normal range (“false negatives”). Furthermore, some nonmalignant conditions also can produce tumor markers. The presence of an elevated tumor marker therefore may suggest a specific diagnosis, but it is not used alone as a definitive diagnostic test. Identification of ideal sensitive and specific tumor markers that are elevated early in the course of common cancers remains a high priority because the early detection of cancer often improves the treatment outcome.

Cancer Metabolism

Cancer cells live in a distinct milieu from normal cells and have different nutritional requirements from nonproliferating cells. The successful cancer cell divides rapidly, with the consequent requirement for the building blocks of new cells. Cancers often must grow in a hypoxic and acidic environment. Cancers also are parasites, able to selectively extract nutrients from the bloodstream without any evolutionary pressure for balanced metabolism. Nonmalignant cells in the presence of adequate oxygen normally generate adenosine triphosphate (ATP) by mitochondrial oxidative phosphorylation (OXPHOS), generating 36 ATP molecules from each glucose molecule that is broken down to water and carbon dioxide. Only in the absence of sufficient oxygen do normal cells perform anaerobic glycolysis, generating only two ATP molecules per molecule of glucose, with lactic acid as a byproduct. However, even in the presence of oxygen, cancer cells perform glycolysis, not OXPHOS⁴ (Figure 12-5). Although this aerobic glycolysis was originally postulated as some form of cancer-specific mitochondrial dysfunction, it is now apparent that this is instead a highly regulated and beneficial adaptation for cancer cells. This shift from OXPHOS to glycolysis allows lactate and its metabolites to be used for the more efficient production of lipids and other molecular building blocks needed for rapid cell growth. Furthermore, many cancer genes promote this switch to aerobic glycolysis. Alterations in a number of cancer genes, including receptor tyrosine kinases, AKT, PTEN, TP53, and MYC, inhibit OXPHOS and promote the activity of glycolytic and related metabolic pathways that support the rapid growth of cancers.^4,5

Clinically the high glucose utilization of a cancer can be exploited for its detection. ¹⁸F-Fluorodeoxyglucose (FDG) is incorporated into cells in the same way as glucose, with two key differences. Because it is missing a key hydroxyl group it cannot be broken down by glycolysis and, thus, FDG accumulates in cells. Because it is tagged with ¹⁸F, it can be imaged by positron emission tomography (a PET scan). Small metastatic tumor masses that are consuming huge amounts of glucose can readily be detected with this imaging method (Figure 12-6).

Cancer Stem Cells

Many tissues, most notably the skin, intestines, and blood-forming cells, continuously renew themselves. The human gut sheds and replaces hundreds of grams of cells each day. This ongoing proliferation of these tissues with a high turnover rate depends on their regeneration from a small fraction of cells known as adult stem cells. Adult stem cells have two essential characteristics: first, they self-renew (that is, some fraction of the cell divisions creates new stem cells); and second, they are multipotent, or have the ability to differentiate into multiple different cell types. In the bone marrow, it is estimated that only 0.05% (1 in 20,000) of the blood-forming cells are stem cells, yet this small pool of stem cells can be stimulated to divide and to repopulate all the mature bone marrow–derived cells in approximately 2 weeks after bone marrow transplantation. As few as 10 stem cells are sufficient to entirely repopulate the entire bone marrow of a mouse in bone marrow transplantation experiments. Similarly, the absorptive and goblet cells lining the intestine have a life span of less than 1 week, after which they undergo cell death, or apoptosis, and slough into the intestinal lumen. They too must be replenished by ongoing proliferation of intestinal stem cells. A key feature of stem cells is that they can divide asymmetrically (i.e., during division, the cytoplasmic contents are unevenly distributed between the daughter cells); they can give rise to another stem cell and one daughter cell that ultimately terminally differentiates into diverse cell types, depending on the needs of the tissue (Figure 12-7). Multipotent bone marrow stem cells can self-renew and differentiate into all types of bone marrow–derived cells, such as red cells, lymphocytes, and neutrophils. Certain adult stem cells have a broader range of potential fates. For example, mesenchymal stem cells are able to differentiate into multiple types of cells, such as blood vessels, neurons, and muscle cells. Theoretically, such a stem cell might be able to give rise to all cell types in the body, and as such, could be useful in regeneration of diseased tissues.

Many cancers, like normal tissues, are heterogeneous, with differences in cell shape, size, behavior, and protein expression. There are two models to explain this heterogeneity within tumors. In the clonal evolution model, all the cancer cells divide regularly, and heterogeneity arises from proliferation, mutation, epigenetic changes, differences in local environment, and natural differentiation of cells. In this model, most of these cells are still robust cancer cells, capable of forming complex tumors in experimental animals. In contrast, in the cancer stem cell model, heterogeneity arises because there is a rare cancer stem cell whose offspring do not have stem cell properties, but undergo a limited number of divisions while generating heterogeneity through epigenetic and environmental alterations. However, similar to the adult tissue stem cells, only the cancer stem cell, if transplanted, is postulated to be capable of forming complex and heterogeneous tumors. The typical cancer stem cell experiment separates cancer cells into different pools depending on some measurable characteristic such as cell surface proteins, and then assays how many cells must be injected into an experimental mouse to form a cancer. In some cases, these tumor-initiating cells are very rare, with only 1 in 10,000 human colon cancer cells able to re-form a complex and heterogeneous colon cancer in mice (Figure 12-8).⁶ However, these tumor-initiating cells are not always rare; in human melanomas, one in four cells can initiate a complex tumor in the appropriate mouse model.⁷ This is an experimentally difficult area of research, because human cancers are examined in mice where there are multiple barriers to transplantation, including immune responses, as well as species differences in the various growth factors, cytokines, and microenvironment that can all influence the results. Therefore most progress is likely to be made by studying cancer stem cells arising in mouse tumors rather than human tumors.

Enthusiasm for the cancer stem cell model arises, in part, from its therapeutic potential. Most of the drugs now used in treating cancer can kill a large fraction of cancer cells but may not touch the cancer stem cell. If less than 1 in 100,000 cells in a cancer is responsible for perpetuating a cancer, then perhaps we should be looking for drugs that target those rare cells instead of using more toxic drugs that initially shrink tumors but do not kill the cancer stem cells.

What Are the Driver Mutations?

To understand how genetic mutations cause cancer, first it is important to distinguish between oncogenes and tumor-suppressor genes. Table 12-4 compares the two types of cancer genes. Oncogenes are mutant genes that in their normal nonmutant state direct synthesis of proteins that positively regulate (accelerate) proliferation. Conversely, tumor-suppressor genes encode proteins that in their normal state negatively regulate (halt, or “put the brakes on”) proliferation. Hence, they also have been referred to as anti-oncogenes.

In its normal, nonmutant state, an oncogene is referred to as a proto-oncogene. An example of a proto-oncogene would be a growth factor (e.g., epidermal growth factor) or a growth factor receptor (e.g., epidermal growth factor receptor). Other positive regulators of proliferation are in the signal transduction pathway that transmits the signal from the growth factor receptor to the cell nucleus. Normally, Ras is a proto-oncogene (Figure 12-11).

Oncogene Addiction

There are a number of common driver mutations in cancer. Cancers that arise because of these mutations often depend on these mutant genes and proteins for their continued growth and survival. If the mutations and abnormal proteins can be returned to their normal states, the cancers often stop growing and even regress. The cancers are addicted to their mutant cancer genes, a concept known as oncogene addiction. This also provides a key example of how targeted cancer therapy can work—for example, if the oncogene is a protein kinase (e.g., BCR-ABL, EGFR, HER2, or BRAF) that can be inhibited by a drug, then the cancer can be deprived of the function of the oncogene. Treating the addiction treats the cancer.¹¹

Mutation of Normal Genes into Oncogenes

Point Mutations

Several types of genetic events can activate oncogenes (Box 12-1 and Figure 12-12). Perhaps the most common events are small-scale changes in DNA such as point mutations, the alteration of one or a few nucleotide base pairs (see Chapter 4). This type of mutation can have profound effects on the activity of proteins. A point mutation in the ras gene converts it from a regulated proto-oncogene to an unregulated oncogene, an accelerator of cellular proliferation. Activating point mutations in ras are found in many cancers, especially pancreatic and colorectal cancer.¹² Specialized tests, such as direct DNA sequencing, can detect such point mutations in clinical samples.

BOX 12-1   TYPES OF GENETIC LESIONS IN CANCER

1. Point mutations

2. Subtle alterations (insertions, deletions)

3. Chromosome changes (aneuploidy and loss of heterozygosity)

4. Amplifications

5. Gene silencing (DNA methylation, histone modification, microRNAs)

6. Exogenous sequences (tumor viruses)

Gene Amplification

Another type of genetic abnormality that can activate oncogenes is gene amplification (see Figures 12-12, B, and 12-13). Amplifications are the result of duplication of a small piece of a chromosome over and over again, so that instead of the normal two copies of a gene, tens or even hundreds of copies are present (see Chapter 4). Gene amplification results in increased expression of an oncogene, or in some cases, drug resistance genes. The N-myc oncogene is amplified in 25% of childhood neuroblastoma cases and confers a poor prognosis.¹⁷ The epidermal growth factor receptor erbB2 is amplified in 20% of breast cancers.¹⁸ Individuals whose cancers have erbB2 amplification respond well to drugs specifically targeted to this oncogene.¹⁹

Tumor-Suppressor Genes

Tumor-suppressor genes are genes whose major function is to negatively regulate cell growth and prevent mutations. Tumor suppressors may normally slow the cell cycle, inhibit proliferation resulting from growth signals, or stop cell division when cells are damaged. Examples of several tumor suppressors are given in Table 12-5. One of the first discovered tumor-suppressor genes, the retinoblastoma (Rb) gene, normally strongly inhibits the cell division cycle (see Chapter 1). When it is inactivated, the cell division cycle can proceed unchecked. Rb is mutated in childhood retinoblastoma, and in many lung, breast, and bone cancers as well.

Whereas oncogenes are activated in cancers, tumor suppressors must be inactivated to allow cancer to occur (see Table 12-5 and Figure 12-14). A single genetic event can activate an oncogene because it can act in a dominant manner in the cell. However, we have two copies, or alleles, of each gene, one from each parent. It therefore takes two hits to inactivate the two alleles of a tumor-suppressor gene. The first allele of a tumor suppressor is often inactivated by point mutations. For example, the Rb gene may be inactivated on one chromosome by a point mutation (e.g., the copy inherited from the father). Because the other copy of the retinoblastoma gene (in this example, the one from the mother) is intact, a functional RB protein can still be made and therefore the cell division cycle can be regulated appropriately. If the remaining gene is mutated or silenced, then all RB function is lost and another step toward cancer occurs (see Chapter 4).

Turning Off Genes Without Mutation

Epigenetic Silencing

Abnormal gene silencing is emerging as a major cause of cancer. Gene expression can be regulated in a heritable manner (i.e., passed from a parent to a child or from a single cell to its progeny) by an “epigenetic” mechanism called silencing. Inheritance of silencing occurs during cell division and does not require mutations or changes in DNA sequence (also see Chapter 6). More simply, the same DNA sequence can produce dramatically different phenotypes depending on chemical modifications that alter the expression of genes. Epigenetic silencing is caused by reversible chemical modification (methylation [addition of a methyl group] or acetylation [addition of an acetyl group]) of histones and related chromatin components, as well as methylation of cytosine residues in DNA (known as DNA methylation) (see Figure 12-14, Figures 6-2, 6-3, and Chapter 16). Whole regions of chromosomes are normally shut off by silencing, so that the pattern of gene expression is different than that seen in other cells with the same genes. In this way, the progeny of liver cells remain liver cells, and skin cells remain skin cells. Notably, global changes in epigenetic silencing can turn these cells back into stem cells.²¹

Changes in gene silencing contribute to the development of cancer.²² Many cancers have increased methylation of DNA in the promoter region of tumor-suppressor genes (in CpG islands or C-cytosine and G-guanine, p-phosphodiester bond, rich sequences that are often located near promoter regions; see Chapters 4 and 13). They also have associated changes in the modification of histones in the chromatin, including methylation of lysines 9 and 27 in histone H3. In addition, overexpression of chromosome silencing proteins known as the polycomb complex, and loss of expression of histone deacetylases (enzymes that remove acetyl groups from histone proteins) SIRT1 is seen in human cancers. These changes in chromatin-modifying genes alter the promoter regions of genes leading to their silencing. The boundaries of the normally silenced regions can also spread in cancer cells, thereby inactivating previously active genes. In either case, silencing can shut off critical tumor-suppressor genes in the absence of mutations in the gene. Early in the development of cancer, these changes in gene expression can lead to a selective advantage for affected cells, perhaps leading to their immortalization and clonal expansion. Silencing of tumor suppressors may be a faster way to create cancer cells than mutational or genetic loss of tumor suppressors.²³ Conversely, loss of silencing can contribute to inappropriate expression of oncogenes. Chemotherapeutic drugs that can regulate gene silencing, including histone deacetylase (HDAC) inhibitors and 5-azacytidine (which reverses the effects of DNA methyltransferases [DNMTs]), have proven effective in reactivating silenced tumor-suppressor genes in the treatment of selected cancers²⁴ (see Figure 12-14).

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