Tumor Biology and Tumor Markers

Global Burden of Cancer

Worldwide, cancer is responsible for one in six deaths, accounting for 9.6 million total deaths worldwide in 2018. The distribution and types of cancer that occur continue to change, being affected primarily by the growth and aging of populations, as well as changing prevalence of multiple risk factors associated with cancer development—many which are associated with increasing socioeconomic development. Although overall cancer incidence rate is two-to threefold higher in the developed, as compared to the developing, world, differences in overall cancer mortality are small due to higher case fatality rates for most cancers in developing countries. Europe and the Americas account for 23.4% and 21%, respectively, of the global incidence of cancer but only 20.3% and 14.4%, respectively, of the global burden of cancer mortality. Meanwhile global cancer incidence in Asia and Africa (48.4% and 5.8%, respectively) is substantially less than their contributions to global mortality, which stands at 57.3% and 7.3%, respectively.

Overall, lung cancer is the most commonly diagnosed cancer worldwide and the leader in cancer death. For males, lung, prostate, and colorectal cancer are the leading cancer by incidence globally, while lung, liver, and stomach cancer are the leading cancers by mortality. For females, breast, colorectal, and lung cancer are the leading cancers by global incidence, while breast, lung, and colorectal cancer are the leading cancers by mortality. However, the leading cancer by incidence and mortality for males and females differs significantly by country. In the United States, Canada, the United Kingdom, and Australia, lung cancer remains the most common cause of cancer mortality for males and females. When looking at developing countries such as Thailand and Mongolia, liver cancer is the leading cause of cancer mortality irrespective of sex. Finally, throughout much of Africa, Mexico, and parts of South America, prostate and breast cancer are the leading cause of cancer death in males and females, respectively. This all translates to significant global economic burden, with total costs for cancer care in the United States alone projected at a staggering $174 billion by 2020.

Aging and Cancer

The incidence of cancer increases with age; thus, cancer disproportionately affects people aged 65 years and older ( SEER.gov ). In the United States, the incidence of cancer in people 65 years old and older is nearly nine times that of people younger than 65 years of age. In people older than 70 years old, the incidence of invasive cancer is one in three for males and one in four for females. Worldwide, approximately 80% of cancers are diagnosed in people 50 years of age or older. The median age of cancer diagnosis in the United States is 66 years of age.

The proportion of the U.S. population aged 65 years old and older is growing rapidly. From 2010 to 2050, this segment of the U.S. population is expected to more than double in size, which is a recognized trend throughout the developed world. With an expanding older population, the incidence of cancer will increase, thereby raising the overall cancer burden on society. In addition, cancer care will also be of increasingly greater complexity in this population; reasons for this include more comorbidities of greater severity in the setting of declining physiologic reserve, difficulties with access to care, and lack of social support.

Cancer treatment in the elderly is less well studied, and it has been shown that the elderly population is underrepresented in clinical trials. Clearly, surgeons must more carefully weigh individuals’ operative risk in the context of the morbidity of the procedure with greater consideration for quality of life and functional status. A number of reports have demonstrated underuse of adjuvant therapy in the aging population, despite evidence that adjuvant therapies can be beneficial for this group of patients. Thus, age alone should not be the sole reason for withholding systemic therapy in these patients.

The major mechanism driving increased cancer with aging relies on the accumulation of mutations over time that confer a growth advantage to the cell. Many of these mutations occur due to chance, and thus the more cell divisions a given cell goes through (as one ages), the more chances it has for a mistake to occur. It was beautifully demonstrated that there is a striking linear relationship (Spearman rho=0.81) between the total stem cell divisions over the lifetime of a given tissue and the risk of invasive cancer in the corresponding tissue.

Obesity, Physical Activity, and Cancer

The prevalence of overweight (body mass index [BMI] of 25–29.9 kg/m ² ) and obesity (BMI ≥30 kg/m ² ) in most developed countries (and in urban areas of many less developed countries) has increased markedly during the past two decades. In the United States, more than one-third of the population is now classified as obese, and 5% of men and 10% of women have a BMI greater than 40 kg/m2. Although obesity has long been recognized as an important cause of diabetes and cardiovascular disease, the relationship between obesity and cancer has historically received less attention. Epidemiological studies indicate that obesity is a risk factor for cancers at multiple sites, including esophageal cancer, colorectal cancer, gallbladder cancer, pancreatic cancer, liver cancer, gastric cancer, postmenopausal breast cancer, uterine cancer, ovarian cancer, renal cell carcinoma, meningioma, multiple myeloma, and thyroid cancer. Globally, increased BMI is third, only behind infection and smoking, as a risk factor for cancer and contributes to up to 20% of cancer-related deaths. Encouragingly, bariatric surgery has been demonstrated to have the potential to offset some of the cancer risk in this population, with a recent study demonstrating a 33% reduction in cancer risk for obese patients undergoing bariatric surgery as compared to weight matched controls.

In concert with the obesity epidemic, decreased physical activity has been shown to be associated with an increased incidence of many different cancers. In a recent metaanalysis, the cancers where increased exercise is most protective are esophageal adenocarcinoma, gallbladder, and liver cancer, with hazard ratios (HRs) of 0.58, 0.72, and 0.73, respectively. Of 26 cancer subtypes looked at, 13 were associated with decreased rates in people participating in moderate to vigorous physical activity, most of which remained significant predictors even after adjusting for BMI. Interestingly, physical activity was associated with increased risk of melanoma (HR 1.27) and prostate cancer (HR 1.05). For some cancer types, such as colorectal adenocarcinoma, increased physical activity has been tied to decreased cancer risk in a dose-dependent manner. In addition to increased overall cancer incidence seen with increased sedentary behavior, cancer outcomes have been shown to be inferior in sedentary individuals, with cancer mortality being increased by ∼20% for individuals in the least active quartile of the population.

Mechanistically, immune, metabolic, endocrine, and inflammatory properties of excess adipose tissue appear to lead to the increased incidence of malignancy in overweight/obese individuals. For example, greater amounts of adipose tissue lead to increased circulating levels of free fatty acids. This in turn causes liver, muscle, and other tissues to increase their use of fats for energy production, thereby reducing their need for uptake and metabolism of glucose and eventually leading to hyperglycemia. This functional insulin resistance forces an increase in pancreatic insulin secretion. Epidemiological and experimental evidence suggests that chronic hyperinsulinemia increases the risk of cancers of the colon and endometrium and probably other tumors (e.g., those of the pancreas and kidney). Obesity can also lead to increased adipocyte-derived stem cell and adipose tissue deposition in the tumor microenvironment, leading to extracellular matrix (ECM) deposition, altered immune profiles as compared to nonobese subjects, as well as a rich supply of growth factors, nutrients, and cytokines beneficial to tumor growth. Adipocyte-derived stem cells promote fibrosis and can become cancer associated fibroblasts, which have been tied to more aggressive cancer biology. In obese patients, increased adiposity in the omentum has been tied to a proinflammatory state that can drive cancer at multiple sites via immune cell activation and inflammatory signaling pathways. Circulating levels of estrogens are strongly related to adiposity. For cancers of the breast (in postmenopausal women) and endometrium, the effects of overweight and obesity on cancer risk are largely mediated by increased estrogen levels. For patients with breast cancer, adiposity has been associated with both worse survival and increased likelihood of recurrence.

In addition to the obvious role of increased physical activity in helping maintain a healthy weight, the remainder of its anticancer properties are still actively being defined. One potential mechanism for estrogen-driven breast cancers is the association between increased physical activity and decreased blood estradiol concentrations. Physical activity has also been shown to upregulate cell cycle and deoxyribonucleic acid (DNA) repair pathways in male patients undergoing surveillance for prostate cancer. In addition to potential positive effects of physical activity, increased sedentary behavior has been tied to increased insulin resistance and C-reactive protein levels.

Healthcare Disparities and Cancer

Race and socioeconomic status have been shown to play a role in both cancer incidence and mortality. African Americans have a higher risk of developing such cancers as colorectal, breast, and prostate cancer as compared to Caucasians, and, when diagnosed, the cancer phenotype tends to be more aggressive. Five-year survival for all cancers combined in the United States is 68% for whites and 61% for blacks. African Americans are more likely to be diagnosed at a later stage and have a lower stage-specific survival for most cancer subtypes. Overall, the risk of death after cancer diagnosis is 33% higher in blacks than whites. Other ethnic minorities have also been shown to have inferior cancer outcomes, with Hispanics tending to be diagnosed at a later disease stage.

In addition to race, socioeconomic status has been implicated in the incidence of cancer, as well as outcomes. A recent study looked at all counties in the United States and found a ∼50% increase in the number of cancer-related deaths in the 90 ^th percentile county for number of cancer deaths per 100,000 people as compared to the 10 ^th percentile county. Differences in socioeconomic status, geographic risk factors, and access to quality healthcare explain a significant portion of these differences. One major factor entwined with these disparities is health insurance status. In one analysis of patients aged 18 to 64 years old in the United States, those without insurance or covered by Medicaid (as compared to nonMedicaid insurance) were more likely to present with later stage disease and were less likely to undergo cancer-directed therapy (e.g., surgery or radiation therapy). In addition, cancer-related mortality was increased for these patients (HR 1.44 Medicaid and 1.47 for uninsured).

Sustained Proliferative Signaling

Cells within normal tissues are largely instructed to grow by neighboring cells (paracrine signals) or through systemic (endocrine) signals. Likewise, cell-to-cell growth signaling occurs in the majority of tumors as well. The immediate tumor cell environment (the stroma) contains residing nonmalignant cells, such as parenchymal cells, epithelial cells, fibroblasts, and endothelial cells. In addition, most tumors are characterized by infiltrating immune cells, such as lymphocytes, polymorphonuclear cells, mast cells, and macrophages. Altered differentiation of cell subsets, and/or selective recruitment and integration of nonmalignant cells results in coevolution with the tumor cells to sustain the growth of the tumor cells. Finally, basement membranes form the ECM that provides a scaffold for proliferation of fibroblast and endothelial cells. Together, tumor cells and stroma produce factors (autocrine and paracrine factors) that, in cell-bound, matrix-bound, or soluble form, directly or indirectly influence tumor development. Autocrine factors secreted by tumor cells promote growth of tumor cells but may also stimulate neighboring tumor cells. In addition, tumor cells secrete paracrine factors that act on host cells or ECMs, generating a supportive microenvironment. For example, transforming growth factor-β (TGF-β) may induce angiogenesis, production of ECM molecules, and production of other cytokines by fibroblasts and endothelial cells. Simplified, tumor growth is dependent on the response of tumor cells to paracrine and autocrine factors ( Fig. 29.5 ). These factors include angiogenesis factors, growth factors, chemokines (polypeptide signaling molecules originally characterized by their ability to induce chemotaxis), cytokines, hormones, enzymes, and cytolytic factors that may promote or reduce tumor growth ( Table 29.2 ). A classic example of hormone signaling in cancer is breast cancer, whereby in many tumors, overexpression of the nuclear estrogen receptor, leads to estrogen-dependent tumor cell proliferation. Taking away the supply of estrogen from the tumor (e.g., letrozole) or blocking the receptor (e.g., trastuzumab) leads to decreased tumor cell proliferation and cell death.

During the evolution of a tumor, its responsiveness to growth signals changes. Paracrine growth mechanisms are dominant during the early development of tumor. Tumors become resistant to paracrine growth inhibitors and gain responsiveness to paracrine growth promoters. However, autocrine growth mechanisms become more prominent as tumors further develop. The observation that metastatic tumor cells tend to spread more randomly through the body in late-stage tumors suggests that autocrine growth mechanisms may be more dominant than paracrine growth mechanisms. It is even possible for a tumor to grow completely autonomously (acrine state) and to be independent of growth factors and inhibitors.

To achieve growth self-sufficiency, growth signaling pathways are altered. This involves alteration of extracellular growth signals, of transmembrane transducers of those signals, or of intracellular signaling pathways that translate those signals into action. Growth factor receptors are overexpressed in many cancers. Receptor overexpression may enable the cancer cell to respond to low levels of growth factor that normally would not trigger proliferation. For example, the epidermal growth factor receptor family (ErbB) is a family of receptor tyrosine kinases that includes both epidermal growth factor receptor (EGFR) and HER2/neu receptor, which are overexpressed in several cancer types. EGFR mutations leading to EGFR pathway overexpression in non–small cell lung cancer may be sensitive to small molecular tyrosine kinase inhibitors, such as gefitinib ( Table 29.3 ). HER2 mutation and overexpression seen in some breast and gastric cancers (and others) leads to sensitivity to HER2 inhibition with trastuzumab ( Table 29.3 ). Another clinically relevant example is the growth factor receptor tyrosine kinase c-kit, whereby activating mutations (95% of gastrointestinal stromal tumors) leads to activation of multiple protumor signaling cascades. In the majority of c-kit mutations, therapy with the tyrosine kinase inhibitor imatinib leads to therapeutic response ( Table 29.3 ). In some cases growth factor receptors can signal independent of ligand binding. This can be achieved through structural alteration of receptors, such as truncated versions of EGFR that lack much of its cytoplasmic domain and are constitutively activated—seen in gliomas and head and neck squamous cell carcinoma.

Cancer cells can also modulate their stromal environment, including the ECM, through secretion of factors such as basic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), TGF-β, and others. ECM components, such as collagens, fibronectins, laminins, and vitronectins, may bind to two or more receptors and may also bind other ECM molecules. The matrix molecule–receptor interaction induces signals that influence cell behavior, including entrance into the active cell cycle. Cancer cells can switch the types of ECM receptors (integrins and heparan sulfate proteoglycans) they express, favoring ones that transmit progrowth signals. Similarly, fibroblasts and immune cell subsets such as macrophages undergo differentiation into cancer-associated fibroblasts and tumor-associated macrophages, respectively, that promote tumor growth and spread.

A more complex mechanism for acquisition of self-sufficiency in growth signals stems from changes in intracellular signaling pathways, some of which actively engage in crosstalk. Many of the oncogenes mimic normal growth signaling and induce mitogenic signals without stimulation from upstream regulators. For instance, KRAS mutations are seen across a wide variety of cancers, with activating mutations leading to receptor independent signaling. This constitutive activation of the KRAS signaling cascade leads to multiple protumor events (e.g., sustained proliferation, immune evasion, resistance to apoptosis, cell migration, and metastasis). KRAS targeting has been the subject of extensive, ongoing research. Another clinically relevant mutated oncogene is BRAF , for which activating mutations (e.g., V600E) lead to constitutive activation (seen in such cancers as melanoma, colorectal cancer, and thyroid cancer), upregulation of MEK and ERK signaling, and increased transcription of protumor survival and proliferation factors. BRAF signaling pathway activation can be clinically targeted with BRAF and MEK inhibition ( Table 29.3 ). Finally, negative-feedback mechanisms that help regulate normal signaling pathways can be disrupted and thereby enhance proliferative signaling.

Evading Growth Suppressors

Cell division is an ordered, tightly regulated process involving both stimulatory and inhibitory signals. Thus, in addition to acquiring stimulatory growth signals, tumor cells need to overcome or to neutralize growth-inhibitory signals. These signals include both soluble growth inhibitors and immobilized inhibitors embedded in the ECM and on the surfaces of neighboring cells. Similar to many of the stimulatory signals, the growth-inhibitory signals are transduced by transmembrane receptors coupled to intracellular signaling pathways that target genes regulating the cell cycle. The cell cycle can be divided into an interphase and a mitotic (M) phase ( Fig. 29.6 ). The interphase is further subdivided into two gap phases (G ₁ and G ₂ ), separated by a phase of DNA synthesis (S phase). The two gap phases involve crucial regulatory events that prepare the cell for DNA replication and mitosis. Central to cell cycle progression are the cyclin-dependent kinases that bind to the cyclin proteins. These proteins are regulated by numerous other proteins including tumor suppressors and oncogenes that induce stimulatory or inhibitory signals. Antigrowth signals can block cell division by two distinct mechanisms. Cells may be forced to exit the cell cycle into a quiescent (G ₀ ) state ( Fig. 29.6 ).

Alternatively, cells may be induced to enter a postmitotic state, usually associated with terminal differentiation. Many of the signaling pathways that enable normal cells to respond to antigrowth signals are associated with the cell cycle block, specifically with the components governing the restriction point in the G ₁ phase of the cell cycle. The restriction point marks the point between early and late G ₁ phase passage that represents an irreversible commitment to undergo one cell division. Cells monitor their external environment during this period and, on the basis of sensed signals, decide whether to proliferate, to be quiescent, or to enter into a postmitotic state. At the molecular level, many antiproliferative signals involve the retinoblastoma protein (pRb) and its two family members, p107 and p130. pRb is a key negative regulator at the restriction point. In quiescent cells, pRb is hypophosphorylated and blocks cell division by binding E2F transcription factors that control the expression of many genes essential for progression from G ₁ into S phase ( Fig. 29.6 ). In contrast, growth-stimulatory signals induce phosphorylation of pRb that does not bind E2F factors and is considered functionally inactive. Likewise, disruption of the pRb pathway liberates E2Fs and thus allows cell proliferation, rendering cells insensitive to antigrowth factors that normally operate along this pathway to block advance through the G ₁ phase of the cell cycle. For example, TGF-β prevents the phosphorylation of pRb, which inactivates pRb and thereby blocks advance through G ₁ . In some tumors, such as breast, colon, liver, and pancreatic cancers, TGF-β responsiveness is lost through downregulation of TGF-β receptors or through expression of mutant, dysfunctional receptors. In others, such as colon, lung, and liver cancers, the cytoplasmic SMAD4 protein, which transduces signals from ligand-activated TGF-β receptors to downstream targets, may be eliminated through mutation of its encoding gene. Alternatively, in cervical carcinomas induced by human papillomavirus, the viral oncoprotein E7 binds pRb and thereby induces dissociation of E2F and subsequent transcription of genes necessary for cell cycle progression. In addition, cancer cells can also turn off expression of integrins and other cell adhesion molecules (CAMs) that send antigrowth signals. In summary, the antigrowth signaling pathways converging onto Rb and the cell cycle are disrupted in a majority of human cancers. Cyclin–cyclin-dependent kinase complexes, essential for cell cycle progression, are regulated by two families of cyclin–cyclin-dependent kinase inhibitors in normal cells. However, in tumor cells, these regulatory proteins, such as the p16 member of the INK4 family, are frequently deleted, allowing tumor cells to bypass cell cycle arrest.

In addition to avoiding antigrowth signals, tumor cells may also avoid terminal differentiation, for example, through overexpression of the oncogene c- myc , which encodes a transcription factor regulating expression of cyclins and cyclin-dependent kinases or through upregulation of Id (short for inhibitor of DNA-binding/differentiation) family members. Likewise, during human colon carcinogenesis, mutations of adenomatous polyposis coli (APC), a negative regulator of β-catenin, lead to the constitutive activation of Wnt/β-catenin signaling, which serves to block the terminal differentiation of enterocytes in colonic crypts.

Resisting Cell Death

The growth of tumors is determined by the ability of tumor cells to proliferate, offset by cell death. Most if not all types of tumors are characterized by defects in cell death signaling pathways and are resistant to cell death. Cell death in tumors is caused primarily by programmed cell death, or apoptosis, which is the most common and well-defined form of cell death. Apoptosis is a physiologic cell suicide program essential for embryonic development, functioning of the immune system, and maintenance of tissue homeostasis. Apoptosis is characterized by disruption of membranes and chromosomal degradation in a matter of hours. The general apoptosis signaling pathway involves the release of cytochrome c from mitochondria that activates various caspases (a family of proteases) in sequence ( Fig. 29.7 ).

Activation of caspase cascades leads to DNA fragmentation and apoptosis. Induction of apoptosis is either death receptor dependent (extrinsic pathway) or independent (intrinsic pathway). The two best described receptor pathways are the Fas receptor and death receptor 5 that bind the extracellular Fas ligand and TRAIL, respectively. Binding of the ligands triggers activation of caspase 8 and promotes the cascade of procaspase activation, leading to release of cytochrome c from mitochondria and eventually apoptosis. The intrinsic pathway is triggered by various extracellular and intracellular stresses, such as growth factor withdrawal, hypoxia, DNA damage, and oncogene induction. Receptor-independent pathways involve translocation of proapoptotic molecules from the cytoplasm to the mitochondria, causing mitochondrial damage and release of cytochrome c . Cytochrome c is directly involved in the activation of caspase 9, which activates caspase 3, which then leads to apoptosis.

The idea that apoptosis forms a constraint to cancer was first raised in 1972 when massive apoptosis was observed in the cells populating rapidly growing, hormone-dependent tumors after hormone withdrawal. The discovery of bcl-2 oncogene as having antiapoptotic activity opened up the investigation of apoptosis in cancer at the molecular level. Bcl-2 promotes formation of B cell lymphomas through a chromosomal translocation linking the bcl-2 gene to an immunoglobulin locus, which results in constitutive activation of bcl-2 , driving lymphocyte survival. Further research has demonstrated that altering components of the apoptotic machinery allows a cell to resist death signals, providing it with a selective growth advantage. For example, functional inactivation of the tumor suppressor p53 is observed in more than 50% of human cancers in many series. p53 is a key regulator of apoptosis by sensing DNA damage that cannot be repaired and subsequent activation of the apoptotic pathway. Other abnormalities, such as hypoxia and oncogene overexpression, are also channeled in part through p53 to the apoptotic machinery and fail to elicit apoptosis when p53 function is lost. In addition, alterations in cell survival pathways can suppress or alter apoptosis. For example, the phosphatidylinositol 3-kinase/AKT pathway, which transmits antiapoptotic survival signals, is likely involved in inhibiting apoptosis in many human tumors. This signaling pathway can be activated by extracellular factors such as insulin-like growth factors I and II or interleukin-3 (IL-3), by intracellular signals from Ras, or by loss of the PTEN tumor suppressor that negatively regulates the phosphatidylinositol 3-kinase/AKT pathway. A final example is the discovery of nonsignaling decoy receptors such as the membrane-bound decoy receptors DcR1 and DcR2 in acute promyelocytic leukemia and prostate cancer. Alternatively, soluble death receptors osteoprotegerin and DcR3 neutralize death-inducing ligands such as TRAIL and FAS ligand in breast cancer, and a high fraction of lung and colon cancers, respectively. Expression of these decoy receptors dilutes the death signal mediated through death receptors.

Nonapoptotic types of cell death that can promote tumor growth include necrosis, autophagy, ferroptosis, and mitotic catastrophe. Necrosis is normally induced by pathophysiologic conditions such as infection, inflammation, and ischemia. Necrosis is characterized by unregulated cell destruction associated with the release of proinflammatory signals. Inflammatory cells, in particular those of myeloid origin that are recruited to the tumor environment, actively promote tumor growth through induction of angiogenesis, immune suppression, tumor invasiveness, and metastasis formation. Autophagy is triggered by growth factor withdrawal, hypoxia, DNA damage, and differentiation and developmental triggers. Degraded intracellular organelles give rise to metabolites that allow survival of tumor cells in stressed, nutrient-limited environments. Autophagy is particularly important for growth of RAS-mutated cancers such as pancreatic, lung, and colon cancers. Ferroptosis is a recently discovered form of cell death that results from iron-dependent lipid peroxide accumulation. When iron-involving oxidative phosphorylation in mitochondria produces reactive oxygen species (ROS), along with ATP, that exceeds the cell’s antioxidation capacity, the oxidative stress response damages lipids and proteins, causing cell death. Finally, aberrant mitosis caused by failure of the G ₂ checkpoint to block mitosis when DNA is damaged can lead to cell death, known as mitotic catastrophe. Defects in nonapoptotic cell death pathways have been linked to cancer. For example, deletion of the autophagy-regulating gene beclin-1 is seen in high percentages of ovarian, breast, and prostate cancers. In addition to cell death, cells can undergo permanent growth arrest, called senescence, when repair of damaged DNA fails. Senescent cells lose their clonogenicity, but defects in the senescent program contribute to tumor development.

Enabling Replicative Immortality

Acquired disruption of cell-to-cell signaling by itself does not ensure expansive tumor growth on its own. This is due to the intrinsic programmed decline in replication potential that limits multiplication of normal somatic cells. This program must be disrupted for a clone of cells to develop into a macroscopic tumor. Normal cells have a finite replicative potential. Once a cell population has progressed through a certain number of doublings, they stop growing but remain viable, a process termed senescence .

With the exception of stem cells, activated lymphocytes, and germline cells, normal cells have a limited replicative potential. Stem cells give rise to progenitor cells that can progress through a certain number of doublings with an increasing degree of differentiation. Fully differentiated cells do not have replicative potential. The number of doublings is controlled by telomeres, the ends of chromosomes that are composed of several thousand repeats of a short 6–base pair sequence element. Telomeres prevent end-to-end chromosomal fusion. However, each DNA replication is associated with a loss of 50 to 100 base pairs of telomeric DNA from the ends of every chromosome. The progressive shortening of telomeres through successive cycles of replication eventually causes them to lose their ability to protect the ends of chromosomal DNA. When the critical length is bridged, the unprotected chromosomal ends participate in end-to-end chromosomal fusions, yielding a karyotype disarray that almost inevitably results in the death of the affected cell. Telomeric attrition is negated by the enzyme telomerase that elongates telomeric DNA. Telomerase activity is high during embryonic development and in certain cell populations, such as stem cells in adults. However, many tumors are characterized by elevated telomerase activity. Alternatively, telomeres are maintained through recombination-based interchromosomal exchanges of sequence information. Thus, by maintaining a telomere length above a critical threshold, the tumor cells have unlimited proliferative potential and are considered immortal.

Evidence has recently been obtained for the existence of cancer stem cells (CSCs), or cancer-initiating cells, that give rise to tissue-specific progenitor cells and phenotypically diverse cancer cells with limited replicative potential. The definition of CSCs is still a subject of some debate, but a CSC subclass has now been described in most types of cancer. Characteristic of CSCs is the exponentially enhanced ability to seed new tumors, relative to the nonCSC population, in immunodeficient mice. These cells are extraordinarily rare within the tumor and usually make up less than 10%, and often less than 5%, of neoplastic cells. CSCs may generate tumors through self-renewal as well as through differentiation into multiple cell types. Various cell surface markers have been used to define CSCs, such as CD44, CD133, and CXCR4. A recent study identified Lgr5 as a key marker of intestinal colorectal CSCs in mice and showed that CSCs are critical for metastasis formation. In this study, targeting of the CSC population led to the Lgr5 ^- nonCSC population transitioning into Lgr5 ⁺ stem cells, and thus the CSC population can be repopulated even after eradication. Interestingly, CSCs possess similar transcriptional profiles with normal tissue stem cells, further supporting their designation as stem-like. Furthermore, evidence suggests that CSCs are more resistant to traditional therapeutic modalities, such as chemotherapy and radiation.

Inducing Angiogenesis

Based on the observation that many individuals who died of noncancer-related causes had in situ tumors at the time of autopsy, physicians and scientists concluded that these microscopic tumors are in a dormant state. The reason for tumor dormancy is that the body blocks the tumor from recruiting its own blood supply to provide tumor cells with the required oxygen and nutrients. The growth of new blood vessels, angiogenesis, is a highly regulated process to ensure supply to all cells within an organ. Surprisingly, the microscopic tumors lack the ability to induce angiogenesis, and only an estimated 1 in 600 acquire angiogenic activity. Research pioneered by pediatric surgeon Judah Folkman has demonstrated that naturally occurring endogenous angiogenesis inhibitors prevent tumors from expanding. The angiogenesis inhibitors keep the tumors in check by counterbalancing the angiogenic signals. These signals are mediated by soluble factors and their receptors on endothelial cells as well as by integrins and adhesion molecules mediating cell-matrix and cell-cell interactions. Angiogenic activity is induced by growth factors such as vascular endothelial growth factor (VEGF), basic and acidic FGF, and PDGF. Each binds to transmembrane tyrosine kinase receptors displayed primarily by endothelial cells that are connected to intracellular signaling pathways. Angiogenesis inhibitors are associated with specific tissues or circulate in the blood. The first inhibitor, IFN-α, was reported in 1980, and additional endogenous inhibitors have been identified since then. These include thrombospondin, tumstatin, canstatin, endostatin, and angiostatin. Evidence for the importance of inducing and sustaining angiogenesis in tumors is overwhelming. For example, the switch of dormant human tumors into fast-growing tumors in immune-compromised mice is associated with an angiogenesis gene signature. Most telling are the results of clinical studies with the antiVEGF antibody bevacizumab (Avastin), the first angiogenesis inhibitor approved by the Food and Drug Administration for treatment of colon cancer. Bevacizumab significantly prolongs the survival of some patients with advanced cancer. It should be noted, however, that a minority of tumors are nonangiogenic, while others may contain a mixture of both angiogenic and nonangiogenic areas. Both primary and metastatic tumors may be nonangiogenic, and are most commonly observed in lung, liver, and brain lesions.

The ability to induce and to sustain angiogenesis seems to be acquired in a discrete step (or steps) during tumor development through a switch to the angiogenic phenotype. Tumors appear to activate the angiogenic switch by changing the balance between the total angiogenic stimulation and the total angiogenic inhibition. This occurs in most cases when the angiogenesis stimulators overwhelm the angiogenesis inhibitors. In some tumors, these changes may be linked. It is likely that such disruption in the angiogenic balance is under control of the genetic makeup of the individual tumor cell and its microenvironment. Angiogenesis inducers and inhibitors may be genetically controlled by tumor suppressor genes such as p53 , whereas oncogenes (e.g., RAS ) may downregulate transcription of endogenous inhibitors or activate inducers. For example, Bcl-2 activation leads to significantly increased expression of VEGF and angiogenesis. Another dimension of regulation is through proteases, which can control the bioavailability of angiogenic activators and inhibitors. Thus, a variety of proteases can release basic FGF stored in the ECM, whereas plasmin, a proangiogenic component of the clotting system, can cleave itself into an angiogenesis inhibitor form called angiostatin. Another angiogenesis inhibitor, endostatin, is an internal fragment of the basement membrane collagen XVIII. Finally, hypoxia and other metabolic stressors, mechanical stress from proliferating cells, or inflammatory immune responses can trigger angiogenesis. The coordinated expression of proangiogenic and antiangiogenic signaling molecules and their modulation by proteolysis appear to reflect the complex homeostatic regulation of normal tissue angiogenesis and of vascular integrity. Different types of tumors use distinct molecular strategies to activate the angiogenic switch.

Endothelial cells are key in the formation of new blood vessels through production or expression of angiogenesis-promoting factors. These factors include proinflammatory cytokines such as IL-6, VEGF, and hematopoietic growth factors such as colony-stimulating factors that recruit and activate bone marrow–derived progenitor cells. Among the progenitor cells are myeloid precursors that further promote the proinflammatory responses at the tumor and actively contribute to angiogenesis by producing matrix metalloprotease-9, a critical regulator of tumor angiogenesis through the induced release of VEGF. Bone marrow–derived endothelial precursors foster tumor blood vessel assembly.

Activating Invasion and Metastasis

Progressing tumors give rise to distant metastases that are the cause of 90% of human cancer deaths. For tumors to successfully metastasize, primary tumor cells have to break off from the primary tumor; enter the vasculature (intravasation), extravasate at distant sites, and colonize destination organ sites. Invasion and metastatic growth of tumor cells do not appear to be random processes. Paget observed in 1889 that breast carcinoma often metastasized to the liver, lungs, bone, adrenals, or brain. He hypothesized that tumor cells (the “seed”) would grow only in selective environments (the “soil”), where conditions supported tumor growth, hence the so-called seed-and-soil hypothesis. Since then, additional studies have confirmed this hypothesis. For example, malignant melanoma metastasizes to the brain, but ocular malignant melanoma frequently metastasizes to the liver. Prostate cancer metastasizes to the bone and colon carcinoma to the liver.

Whereas metastatic spread is in part determined by circulation patterns, the retention of disseminated tumor cells in distant organs and successful development suggest the existence of specific molecular interactions. Molecular analysis has provided several theories to explain preferential outgrowth of tumor cells. One theory, the growth factor theory, proposes that tumor cells in the blood or lymphatics invade organs at similar frequency, but only those that find favorable growth factors multiply. Transferrins, for example, are iron-transferring ferroproteins required for cell growth that have additional mitogenic properties beyond their iron-transporting function. Increased concentrations of transferrin are found in lung, bone, and the brain and are associated with elevated levels of transferrin receptors on metastasizing tumor cells. Another theory, the adhesion theory, proposes that endothelial cells lining the blood vessels in certain organs express adhesion molecules that bind tumor cells and permit extravasation. A third theory is that chemokines secreted by the target organ can enter the circulation and selectively attract tumor cells that express receptors for the chemokines. Examples include the chemokine receptor-ligand axis between elevated levels of CXCR4 on breast cancer cells and CXCL12 secreting bone marrow, liver, lymph nodes, and lung, which explains why these organs are preferred sites for breast cancer metastasis. A similar phenomenon was observed for melanoma cells that were found to express elevated levels of the receptors CXCR4, CCR7, and CCR10 compared with normal melanocytes. Lymph nodes, lung, liver, bone marrow, and skin express the highest levels of the ligands for these receptors and are the preferred sites for metastatic spread of melanomas. Because chemokines are now known to affect angiogenesis and expression of cytokines, adhesion molecules, and proteases in addition to inducing migration, it appears that chemokines and their receptors play an essential role in the successful outgrowth of tumors at preferential sites. While the exact mechanism for the so-called organotropism is still unclear, recent discoveries have shown that primary tumors induce the formation of microenvironments in distant organs that permit the homing and outgrowth of tumor cells before their arrival. These premetastatic niches are initiated by tumor-secreted factors and tumor-derived extracellular vesicles that in concert trigger a cascade of events involving increased vascular permeability in microvessels in organs, and recruitment of various bone marrow-derived cell subsets that aid in local tissue remodeling and recruitment of cancer cells.

Detailed analysis of primary tumors indicates that gene functions mediating metastatic activities are present early in the disease. These functions result from genetic or epigenetic alterations. The genes can be grouped into classes, such as metastasis-initiating genes that control invasion, angiogenesis, circulation, and bone marrow mobilization. Similarly, metastasis progression genes control extravasation, survival, and reinitiation, whereas metastasis virulence genes regulate organ-specific colonization. These intrinsic properties of the tumor together with its cellular origin determine the organ specificity and temporal course of metastasis formation.

The epithelial-to-mesenchymal transition (EMT) program plays an essential role in progression from primary to metastatic cancer. The EMT program is a cell-biologic program, orchestrated by certain transcription factors, in which epithelial cells convert into more mesenchymal cell states. The extent of EMT depends on both extracellular signals and intracellular gene circuitry. The EMT program is not only essential at multiple stages during embryonic morphogenesis, but also in wound healing, tissue fibrosis, and cancer progression. Recent work suggests that cells in EMT acquire similar stem-like properties to CSCs, and thus EMT could give rise to CSCs. Cancer cells use EMT to become invasive and to metastasize. During EMT, cancer cells downregulate the expression of cellular adhesion molecules, such as epithelial (E)–cadherin, and become spindle shaped, thus allowing them to invade surrounding tissues, to intravasate into the bloodstream, and to metastasize. Once cells in EMT arrive at distant sites of metastasis, they extravasate and undergo a process of mesenchymal-epithelial transition, whereby these cells revert back to their original epithelial phenotype for clonal expansion and establishment of metastasis.

Both intravasation and extravasation are characterized by changes in ECMs and their interactions with tumor cells. The cell-cell and cell-matrix interactions are mediated through CAMs, primarily by members of the immunoglobulin and calcium-dependent cadherin families, the hyaluronan receptor CD44, selectins, and integrins, which link cells to ECM substrates. Studies have shown that the molecules mediating adhesion are also capable of signal transduction. As such, changes in expression of adhesion molecules will alter signaling pathways, and conversely, signaling molecules can directly affect the function of adhesion molecules in tumor cells.

E–cadherin is the prototype cadherin responsible for cell polarity and organization of epithelium. In normal cells, extracellular domains of E-cadherin on opposing cells couple and form cell-cell junctions. The cytoplasmic cell adhesion complex is linked to the actin cytoskeleton through catenins (α, β, and γ). E-cadherin function is lost in most epithelial tumors during progression to tumor malignancy and may in fact be a prerequisite for tumor cell invasion and metastasis formation. Mechanisms that include mutational inactivation of the E-cadherin or β-catenin genes, transcriptional repression, or proteases of the extracellular cadherin domain induce loss of E-cadherin function. This prevents catenins from binding and leads to their accumulation in the cytoplasm. Inactivation of nonsequestered β- and γ-catenin is dependent on the presence of the tumor suppressor gene APC and an inactive Wnt signaling pathway. However, when APC function is lost, as is the case in many colon cancers or in the case of Wnt activation, β-catenin is not degraded but instead translocates to the nucleus, where transcription of genes involved in cell proliferation and tumor progression is activated, such as c- myc , cyclin D1, CD44, and others.

Changes in expression of CAMs in the immunoglobulin superfamily also appear to play critical roles in the processes of invasion and metastasis. Neuronal-CAM, for example, undergoes a switch in expression from a highly adhesive isoform to poorly adhesive (or even repulsive) forms in Wilms tumor, neuroblastoma, and small cell lung cancer. In invasive pancreatic cancer and colorectal cancers, the overall expression of neuronal-CAM is reduced.

Selectins are a family of transmembrane molecules consisting of endothelial, leukocyte, and platelet selectins that normally mediate blood cell–endothelial cell interactions. However, alterations in the expression level of selectins or their ligands, such as the endothelial- and leukocyte-selectin ligand CD44, have been associated with increased invasiveness and poor survival in several malignant neoplasms, such as breast cancer and colorectal cancer.

Changes in integrin expression are also evident in invasive and metastatic cells. For invading and metastasizing cells to be successful, they need to adapt to changing tissue microenvironments. This is accomplished through shifts in the spectrum of integrin α and β subunits displayed on the cell surface by the migrating cells. The large extracellular domain of integrins can bind to ECM molecules (such as collagens, laminin, and fibronectin), to ligands associated with vascular and coagulation physiology (such as thrombospondin and factor X), or with other CAMs. In addition, integrins may exhibit different specificities when expressed on different cell types. Thus, carcinoma cells facilitate invasion by shifting their expression of integrins from those that favor ECM present in normal epithelium to other integrins that preferentially bind the degraded stromal components produced by extracellular proteases.

The second general parameter of the invasive and metastatic capability involves extracellular proteases that regulate ECM turnover. It has become clear that tumor progression may involve an increased expression of proteases, decreased expression of protease inhibitors, and inactive zymogen forms of proteases that are converted into active enzymes. Expression of the protease tenascin, which neutralizes adhesion to fibronectin, is increased ten fold in invasive breast carcinoma compared with normal breast tissue. Matrix metalloproteases are overexpressed in melanoma, invasive breast carcinoma, and invasive squamous cell carcinoma. Matrix-degrading proteases are characteristically associated with the cell surface by synthesis with a transmembrane domain, binding to specific protease receptors, or association with integrins. One imagines that docking of active proteases on the cell surface can facilitate invasion by cancer cells into nearby stroma, across blood vessel walls, and through normal epithelium cell layers. That notion notwithstanding, it is difficult to unambiguously ascribe the functions of particular proteases solely to this capability, given their evident roles in other hallmark capabilities, including angiogenesis and growth signaling, which in turn contribute directly or indirectly to the invasive and metastatic capability. A further complexity derives from the multiple cell types involved in protease expression and display, including stromal and inflammatory cells such as neutrophils and macrophages.

The activation of extracellular proteases and the altered binding specificities of cadherins, CAMs, selectins, and integrins are clearly central to the acquisition of invasiveness and metastatic potential. The clonal and genetic diversity of tumors permits adhesion and detachment from the same matrix. Some tumor cells within a primary tumor may have the correct genotype and phenotype to permit both detachment from the surrounding tissue and entry into blood vessels or lymphatic vessels. Likewise, extravasation may be mediated by a few tumor cells that express the required receptors for certain ECM molecules. In general, those mutations that confer escape from homeostatic control mechanisms in the host or that give the tumor cell a growth advantage over others are favorably selected. Thus, tumor clones that best complement the environment with expression of particular ECM receptors may thrive because this provides an advantage over other clones. However, the regulatory pathways and molecular mechanisms that govern these changes are incompletely understood and appear to differ from one tissue environment to another.

Avoiding Immune Destruction

In the early 1900s, it was proposed by Paul Ehrlich that the frequency of cancerous transformations would be very high if it were not for the defense system of the host. This concept was later substantiated in the 1950s and 1960s. Burnet hypothesized that the development of T lymphocyte–mediated immunity during evolution was specific for elimination of transformed cells. He further proposed that there is a continuous surveillance of the body for transformed cells, hence the term immunosurveillance . It was not until the early 2000s that immune-mediated elimination of tumor cells was conclusively demonstrated in animal models. Extensive studies on immune infiltrate in primary human cancers have established that memory T cells, particularly of the T helper (CD4 ⁺ ) type 1 subtype, and cytotoxic (CD8 ⁺ ) T cells are prognostic factors for disease-free and overall survival at all stages of clinical disease. Alternatively, tumors infiltrated with abundant myeloid cells, particularly macrophages, correlate with worse prognosis in many types of cancers, such as pancreas and breast. Data from mouse and human studies combined suggest that immune surveillance of cancer does exist, mediated through immune cells and soluble factors. Whereas the immune system may eliminate most transformed cells, some cells manage to escape and may develop into tumors.

The continuous pressure of the immune system in an immunocompetent host determines to a great degree if and how tumors evolve, a process called immunoediting ( Fig. 29.8 ). In this process, the immune system plays a dual role in the interactions between tumor and the host. On one hand, the immune system effectively eliminates highly immunogenic tumor cells. At the same time, however, the immune system fails to eliminate tumor cells with reduced immunogenicity, thereby selecting for tumor variants that have acquired immune evasion mechanisms. Over time, this selection leads to outgrowth of tumor cells that fail to induce an effective immune response. As such, the interactions between an intact immune system and tumor cells evolve through three phases, referred to as the elimination phase, the equilibrium phase, and the escape phase. The recognition and elimination of transformed cells is a concerted effort between innate and adaptive immunity, representing the two arms of the immune system. Local disruption of tissue that occurs as a result of expansion of transformed cells is associated with release of chemokines and proinflammatory cytokines such as interferons, IL-1, IL-6, and tumor necrosis factor-α that trigger innate immunity.

The innate immune system represents the first line of defense against transformed cells (and microorganisms). The most important outcome of these initial events is the production of IFN-γ by activated innate immune cells. IFN-γ has direct antitumor effects and further boosts tumor cell lysis by innate immune cells. The resulting availability of tumor antigen triggers an adaptive immune response. Key in this process is the uptake of tumor antigen by antigen-presenting cells, primarily dendritic cells. The dendritic cells migrate to tumor-draining lymph nodes and stimulate T and B lymphocytes. The development of adaptive immunity represents the second line of defense against tumors and, together with innate immunity, could completely eliminate the tumor. However, this does not always occur and may lead to what is referred to as the equilibrium phase . This phase is characterized by a balance between tumor growth and tumor elimination, as the name suggests. Antitumor immunity leads to destruction of immunogenic tumor cells, whereas tumor cells with reduced immunity go unnoticed.

Over time, genetic instability and heterogeneity of the tumor cells may give rise to tumor variants better able to withstand the immunologic pressure. Contributing to the failure of the immune system are tumor-induced immune suppressor mechanisms. Once this point has been reached, referred to as the escape phase, the immune system can no longer contain the tumor, and the tumor grows progressively. During the last decade, multiple mechanisms have been identified through which tumors escape from elimination by the immune system. These mechanisms include host-related factors, tumor-related factors, and a combination of both. Among host-related factors are treatment-related immunosuppression, acquired or inherited immunodeficiency, and aging. The list of tumor-related escape mechanisms includes loss of major histocompatibility complex alleles, reduced antigen processing or presentation, decreased expression of costimulatory molecules required for T cell recognition, secretion of immunosuppressive factors (TGF-β, IL-10), stimulation of suppressor cells, and mechanisms that actively induce tolerance or apoptosis in activated immune cells. In addition, when looking across a large number of tumors over 33 cancer types, six unique immune profiles can be identified: TGF-β dominant, immunologically quiet, lymphocyte depleted, inflammatory, IFN-γ dominant, and wound healing. These different classes reflect different cancer outcomes, and differing tumor evolutions in the presence of the host. A thorough discussion of tumor immunology and immunotherapy is found in Chapter 30 of this edition.

Deregulating Cellular Energetics and Metabolomics

Generally, reprogrammed and/or rewired cellular metabolism provides for the energy needs of growing tumors. Otto Warburg first described the anomalous predilection of cancer cells to limit their energy metabolism to glycolysis, even in the presence of oxygen (aerobic glycolysis), an observation termed the Warburg effect . Because glycolysis has a roughly eighteen fold lower energy yield compared with aerobic metabolism through mitochondrial oxidative phosphorylation, cancer cells must dramatically upregulate the rate of glycolysis (up to 200-fold higher than normal cells) to keep up with the rapid metabolism of rapidly dividing neoplastic cells. One mechanism used by tumor cells to increase the rate of glycolysis is to upregulate the expression of glucose transporters, such as GLUT1. Clinically, the Warburg effect is used to diagnose or to stage cancers, such as visualizing glucose uptake by positron emission tomography with ¹⁸ F-fluorodeoxyglucose.

Different classes of metabolic reprogramming activities have been identified: (1) transforming activities, (2) enabling activities and (3) neutral activities. Transforming activities are directly tied to cell transformation, with the most studied examples being mutations of isocitrate dehydrogenases 1 and 2 ( IDH1, IDH2 ), succinate dehydrogenase, and fumarate hydratase. IDH1 and IDH 2 mutations lead to a now mutated IDH enzyme not able to properly do its normal job in the citric acid cycle, and instead it converts alpha ketoglutarate to the oncometabolite 2-hydroxyglutarate. Succinate dehydrogenase and fumarate hydratase catalyze reactions in the citric acid cycle, and mutations lead to oncometabolite succinate or fumarate buildup. Succinate, fumarate, and 2-hydroxyglutarate all interfere with dioxygenase function, which leads to multiple downstream effects, including impaired DNA and histone demethylation (see Cancer Epigenetics). This mechanism cooperates with other drivers to help promote cancer growth.

Enabling activities refers to activities that are altered in cancer cells but are not involved in transformations as previously described. For instance, mutant KRAS leads to increased nutrient acquisition and macromolecule synthesis, all of which are crucial for continued tumor growth and viability. Indeed, suppressing of these pathways inhibits KRAS-driven tumorigenesis. Finally, neutral activities involve metabolic programs in the cancer cell that are not necessarily needed for continued cancer growth and survival.

All this has led to excitement in trying to determine if aspects of the cancer metabolic cycle can be targeted to suppress tumor growth and in the emergence of the cancer field of metabolomics. Examples include drugs targeting IDH1 and IDH2, now in clinical trials. In addition, drugs targeting amino acids crucial for cancer progression, such as arginine in hepatocellular carcinoma (HCC), are under investigation. It is likely that we have only scratched the surface on this exciting, and evolving area of research.

Genomic Instability and Mutation

Genomic alteration is emerging as a key enabling characteristic to many of the traits outlined before. Under normal physiologic conditions, the genome is maintained under extraordinary fidelity by important caretaker genes. Alterations of this important cellular machinery can result in loss of the ability to detect DNA damage, loss of the ability to directly repair damaged DNA, and inability to inactivate or to intercept mutagenic molecules before DNA damage can occur. Mutant copies of some caretaker genes can predictably result in an increased incidence of certain types of cancer. TP53 is an important tumor suppressor gene, and it plays a key role in orchestrating the detection and resolution of mutations; hence, it is often referred to as the guardian of the genome. However, TP53 is the most commonly mutated tumor suppressor gene in cancer. Once proper maintenance of the genome is lost, cells are free to accumulate multiple genetic alterations, any of which can convey the key characteristics for tumor progression discussed previously. Two recently described classes of tumor genomic rearrangements include chromothrypsis and chromopexy. In chromothrypsis, a massive chromosomal rearrangement event occurs in a localized genomic region, while in chromopexy there is coordinated rearrangement across multiple chromosomes. This can lead to tumor beneficial gene mutations and subsequent tumor progression.

Tumor-Promoting Inflammation

Tumor-promoting inflammation is also emerging as a key enabling characteristic of many types of cancer. As previously described, immune cells may play an important role in antitumor defense. However, paradoxically, these same immune cells can also enhance tumor progression. It has long been recognized that many tumors are densely infiltrated by various immune cells. Similar to a chronic wound, leukocytes in the tumor produce various growth factors, which can promote tumor growth, angiogenesis, and therapeutic resistance. Often, inflammation is an early event in tumorigenesis and can incite the conversion of a premalignant lesion into a full-blown cancer. For example, leukocytes can elaborate various ROS that can cause damage to the DNA of nearby cells, thus fostering the progression to malignancy.

As discussed more later in this chapter (Chronic Inflammation), inflammatory changes can lead to upregulation of transcription factors NF-κB, STAT3, and/or hypoxia-inducible factor-1 $\alpha$ (HIF1 $\alpha$ ), which work to mediate cytokine and chemokine expression (e.g., IL6), as well as inflammatory enzymes such as COX-2, leads to inflammatory changes in the tumor microenvironment. Leukocytes, macrophages, mast cells, T cells, and dendritic cells are recruited and further mediate the immune response. Tumors can alter the functionality of infiltrating immune cells, causing functional leukocytes to become anergic or even immunosuppressive. For example, macrophages isolated from tumors, such as pancreatic cancer, are potently immunosuppressive and prevent antitumor immune responses by T cells; however, monocytes from the blood of these same cancer patients are capable of promoting immune responses, suggesting that the tumor environment can alter the functionality of infiltrating leukocytes to escape immune-mediated elimination.

Timing and Pattern of Tumor Growth and Distant Spread

As discussed previously, depending on cancer type and location, metastatic risk and specific site of likely distant organ spread differs. Different cancers undergo progression from normal cell to clinically evident mass in different time horizons. For instance, in an elegant analysis of pancreatic cancer specimens, it was conservatively estimated to take nearly 12 years from initiation of tumorigenesis to formation of the parental pancreatic adenocarcinoma clone. It was estimated that another 7 years would pass prior to the index lesion clinically presenting in the patient and 2 to 3 more years until distant metastatic spread (a total of >20 years from initial founding mutation until distant metastatic spread). Estimates for colorectal carcinoma place the time between initial index mutation and growth into a clinically detectable lesion at ∼18 years. This number likely varies significantly between tumor types and within tumor types, but, overall, the time it takes for a clinically detectable tumor to form is on the order of many years, if not decades, in some cases.

When it comes to pattern of distant metastasis, four different metastatic patterns have been described ( Fig. 29.9 ). One, simple linear evolution model, whereby clones arise sequentially from the primary tumor with metastases occurring late from the most recent subclone. Second, early dissemination with parallel evolution model, whereby tumor cells disseminate early and evolve in parallel with the primary lesion. Third, late dissemination from a single primary tumor subclone, whereby a late arising subclone is able to seed multiple metastases. And finally, late dissemination from multiple subclones, whereby multiple subclones from the primary are able to undergo metastatic spread and do so relatively later on during the tumor evolution time course. Overall, the late dissemination model is favored by the literature. A recent study of colorectal cancer evolution found that distant lesions were most similar to each other, while still retaining similarity to the primary tumor, thereby favoring the late dissemination model. An analysis of matched primary and metastatic breast cancer samples demonstrated that on average the metastases disseminated at 87% of the molecular age of the primary lesion, again favoring late dissemination. Despite this, some studies do support early (or both early and late) dissemination, and it is likely that across all cancers these two theories are not mutually exclusive.

Another subject of much debate has been regarding dissemination from a single or multiple subclones. In a recent study of colorectal cancer, in two thirds of cases, the distant organ metastasis was more similar to the primary tumor clone than the metastatic lymph node clone, thus supporting dissemination from multiple subclones theory in the majority of cases. This type of spread directly contradicts the classic tumor node metastasis (TNM) paradigm that is taught beginning in medical school, and may be partly to explain why there has been a lack of therapeutic success for recent trials of extended lymph node dissection in breast cancer and melanoma. Finally, there is potential that lymph node metastases could in theory seed distant organ metastases outside of the classic lymphatic to thoracic duct to subclavian vein pathway. In fact, recent work in murine models has demonstrated the ability for lymph node metastases to leave lymph nodes by way of local blood vessels, and then lead to distant metastatic spread.

Cancer Genetics

Malignant transformation is the process by which a clonal population of cells acquires alterations that confer a growth advantage over normal cells. Many of these alterations occur at the genetic level, involving the gain of function by oncogenes or the loss of function by tumor suppressor genes (either of which may be referred to as a cancer driver gene). A classic multistep model for colorectal tumorigenesis has been described ( Fig. 29.10 ). Designation as an oncogene or tumor suppressor gene relates to the directionality of effect, without implications about molecular detail. Indeed, the original name for what came to be known as tumor suppressor genes was in fact antioncogenes. A recent analysis looking at over 9000 tumors across 33 cancer subtypes identified 299 distinct mutated cancer driver genes. Many driver mutations of oncogenes (e.g., KRAS and HER2 ) as well as loss of function mutation of tumor suppressor genes (e.g., TP53) were discussed previously in this chapter (Tumor Biology, Sustaining Proliferative Signaling and Resisting Cell Death subsections).

Genetic mutations that are inherited from one’s parents and are present in all cells of the body are called germline (or constitutional) mutations; in contrast, somatic mutations are acquired during an individual’s lifetime and cannot be passed on to one’s children. Somatic mutations, which account for most mutations in cancer, may be caused by exposure to carcinogens in the form of radiation, chemicals, or chronic inflammation (see below in this chapter for more detailed discussion of carcinogens).

A tumor that arises in an individual may be classified as either hereditary or sporadic. In hereditary cases, a germline mutation is responsible for the predisposition to neoplasia. The index case or proband is the individual who is first diagnosed as having the syndrome, even if earlier generations are later recognized as also having had the syndrome. If the patient with a tumor does not have an inherited predisposition and the tumor’s genetic mutations are all somatic, the tumor is classified as sporadic. In some hereditary cancer syndromes, the germline mutation causes a tendency for the cell to accumulate somatic mutations.

Although hereditary cancer syndromes are rare, their study has provided powerful insights into more common forms of cancer ( Table 29.4 ). Key germline mutations in hereditary cancers are often the same as somatic mutations present in sporadic cancers. TP53 gene mutations, if inherited, cause Li-Fraumeni syndrome. Familial adenomatous polyposis (FAP) is caused by a germline mutation in the APC gene. More than 80% of sporadic colorectal cancers also have a somatic mutation of this same gene. Similarly, mutation in the RET proto-oncogene is responsible for the predisposition to development of the familial form of medullary thyroid cancer (MTC). Somatic mutations of RET are found in about 50% of sporadic MTCs.

Predisposition in familial cancer syndromes is generally inherited in an autosomal dominant fashion ( Table 29.4 ). Notable exceptions include ataxia-telangiectasia and xeroderma pigmentosum, which are transmitted in an autosomal recessive manner. Not all inherited genetic mutations have complete penetrance. There is almost complete penetrance of colorectal cancer in FAP and of MTC in multiple endocrine neoplasia type 2 (MEN2). In contrast, penetrance is less than 50% for pheochromocytoma in neurofibromatosis. Penetrance can also vary considerably for different characteristics of the same syndrome. The exact factors determining penetrance for a given mutation remain largely unknown, but factors felt to be commonly implicated include interplay between genes and the environment, sex, and age.

There are a number of features of hereditary cancers that distinguish them phenotypically from their sporadic counterparts. The former tend to cause the development of multifocal, bilateral cancer at an early age, whereas in the latter, cancer occurs later and is usually unilateral. Hereditary cancers will display clustering of the same cancer type in relatives and may be associated with other conditions, such as mental retardation and pathognomonic skin lesions.

Selected Familial Cancer Syndromes

Retinoblastoma

Retinoblastoma is a pediatric retinal tumor that holds an important place in the history of cancer genetics because the causative gene, RB1 , was the first tumor suppressor gene to be cloned. Most cases are detected by the age of 5 years (95%, with 66% detected by 2 years), but bilateral disease is manifested earlier, usually within the first year of life. It is associated with extraocular malignant neoplasms including sarcomas, melanomas, and tumors of the central nervous system. Distinct sporadic and hereditary forms of retinoblastoma have long been recognized, with predisposition conferred by a germline mutation in approximately 40% of cases. Knudson reasoned that the germline mutation is necessary, but not by itself sufficient, for tumorigenesis because some children with an affected parent do not develop a tumor but later produce an affected child, indicating that they are carriers of the germline mutation. Most affected children with an affected parent develop tumors bilaterally. He further hypothesized that hereditary retinoblastoma requires two mutations, one of which is germline and the other somatic. In children with unilateral disease and no family history, both mutations are somatic. The hereditary and nonhereditary forms of the tumor require the same number of events—the “two-hit” hypothesis ( Fig. 29.11 ). The RB1 protein product is a key regulator of the cell cycle, and its loss results in failure of retinoblasts to differentiate properly.