Familial Adenomatous Polyposis

The first hereditary colon carcinoma to be described, familial adenomatous polyposis (FAP), is characterized in its classic form by the occurrence of hundreds to thousands of polyps in the colon of affected persons with the formation of invasive carcinomas at an early age. Polyps may also sometimes be found in the upper gastrointestinal tract. As long ago as 1882, siblings were described with numerous and recurring polyps,⁴² and by the mid twentieth century, FAP was established as a hereditary autosomal-dominant disorder.⁴³ A gene mutation associated with FAP was identified on chromosome 5q by linkage analysis^44,45 and positional cloning studies.^46–49 The gene, adenomatous polyposis coli (APC), is now known to encode a tumor suppressor that modulates wnt signaling through binding and stabilization of a complex of proteins that includes GSK3b, β−catenin, and axin family members. Mutations in APC truncate the protein at its carboxyl terminus and disrupt the protein signaling complex, which results in deregulation of cell cycle control, cell migration, differentiation, and apoptosis (reviewed in reference 50).⁵⁰ In addition to being responsible for FAP, APC mutation is present in approximately 60% of sporadic colon adenocarcinomas^51,52 and an approximately equal number of sporadic polyps,^51,53 suggesting that APC mutation is an early event in sporadic carcinogenesis, as well as in hereditary disease. An attenuated form of FAP with smaller numbers of tumors (<100) has been described.^54,55 This condition is referred to as attenuated FAP⁵⁴ and may be driven by the mutations in APC in the 3′ and 5′ ends rather the central portion of the gene that is affected in classical FAP.⁵⁵

Hereditary Nonpolyposis Colon Cancer

The most common form of hereditary colon cancer is hereditary nonpolyposis colon cancer (HNPCC). HNPCC is a familial syndrome that differs from FAP in that it manifests a smaller numbers of polyps compared with FAP. The earliest description of this disorder can again be traced back to the early twentieth century,⁵⁶ but the hereditary character and syndromic features of the disease were firmly established by the diligent kindred studies of Lynch and Lynch,⁵⁷ after whom the syndrome is named (Lynch syndrome). In brief, the syndrome consists of CRC that occurs in close relatives, often younger than 50 years,^58,59 without adenomatous polyposis. HNPCC is also associated with an increased frequency of tumors in the endometrium, stomach, ovary, hepatobiliary tract, upper urinary tract, small bowel, and central nervous system.⁴¹ In contrast to FAP polyps, which are frequently found on the left side of the colon, persons with HNPCC frequently have flat lesions on the right side of the colon with a distinct histologic appearance referred to as “serrated adenoma” (Fig. 17-3).

The molecular genetic underpinnings of HNPCC were deciphered beginning with the linkage to microsatellite markers on chromosome 2p.⁶⁰ The genetic basis of the disease was established when it was found that patients with the syndrome harbored mutations in the mismatch repair (MMR) genes hMLH1,^61,62 hMSH2,^63,64 hMSH6,⁶⁵ and hPMS2,^66,67 with mutations in hMLH1 and hMSH2 accounting for more than 90% of HNPCC cases. The germline mutations are heterozygous, and colon carcinogenesis results from silencing of the nonmutant allele by methylation^68,69 or by acquired allelic loss.

The phenotypic consequence of gene silencing is loss of MMR protein and the resulting inability to repair DNA replication errors, ultimately leading to accumulation of mutations contributing to carcinogenesis. At a clinical level, protein loss can be the demonstrated by immunochemistry and optimally requires four separate stains. The inability of affected cells to repair DNA replication errors results in insertions or deletions in short tandem repeats (microsatellites) that exist throughout the genome and is referred to as “microsatellite instability” (MSI).70,71 Currently, MSI is documented by testing of a standard set of five microsatellite markers recommended by a consensus panel.⁷² Results are scored on the basis of the number of unstable loci and are classified as MSI-High (MSI-H), MSI-Low (MSI-L), or MSI-Stable (MSI-S). More than 90% of HNPCC tumors are MSI-H, but approximately 15% of all sporadic tumors are also MSI-H,⁷³ and thus the test is not specific for hereditary disease.

MUTYH-Associated Polyposis

A third hereditary variant of colon carcinoma is MUTYH-associated polyposis (MAP), which is the newest polyposis syndrome (first described in 2002).⁷⁴ MAP is a disorder similar to attenuated FAP and is characterized by adenomatous polyps that may number in the hundreds but not in the thousands, usually ranging from 10 to 1000.^77–77 Polyps may also occur in extra colonic sites, usually the duodenum. Adenomas tend to occur in the proximal colon.⁷⁶ MUTYH is a glycosylase and part of the base excision repair pathway. Oxidative damage to DNA frequently results in the modification of guanine (G) to 8-oxoG. 8-oxoG can pair with adenine (A) during DNA replication, a mismatch that is recognized by MUTYH, which removes the mismatched A and restores the correct base, cytosine (C). The damaged G (8-oxoG) is then replaced with an intact G. Mutation of MUTYH at specific hot spots hinders recognition of the 8-oxoG : A mispairing, resulting in G : C to A : T transversion at sites of oxidative damage. The mutational hot spots are found in exon 7, resulting in amino acid substitution Y179C, and in exon 13, resulting in substitution G496D. Germline mutation at both hot spots (biallelic mutation) occurs in 0.3% of patients with CRC. Biallelic mutation confers 28-fold relative risk for CRC compared with control subjects. Monoallelic mutation is more common with a frequency of approximately 2% in CRC but also occurs in approximately the same frequency in control populations. Most studies suggest that monoallelic mutation confers little or no increased risk of CRC.^75,76 Among control patients without polyposis, biallelic mutation is rare.⁷⁶

Hamartomatous Polyposes

The fourth hereditary form of colon cancer is the hamartomatous polyposes, a category that includes Peutz-Jeghers syndrome (PJS) and juvenile polyposis syndrome. First described in the 1890s,78,79 PJS was named in 1954 by Bruwer and colleagues⁸³ in recognition of the work of Peutz⁸⁰ and Jeghers.^81,82 PJS is an autosomal-dominant condition characterized by multiple histologically distinct polyps (Fig. 17-3) that occur in the large and small bowel in association with melanin pigmentation of the lips, mouth, nose, anus, and extremities.⁸⁴ The condition is associated with a high risk for cancer in not only the large and small intestine but also in the esophagus, stomach, pancreas, breast, uterus, ovary, and lung.^87–87 Gene mapping has identified a locus on chromosome 19.3 p13^88,89 that encodes a serine-threonine kinase, STK11 (LKB1), a gene that is mutated in more than 90% of affected individuals.^90–94 A variety of mutations including deletions, truncations, and missense point mutations may occur over nearly the full coding region of the gene.⁸⁶ STK11 protein is multifunctional, playing a role in cell cycle arrest, wnt signaling, and the tuberous sclerosis complex pathway, with effects on mammalian target of rapamycin signaling.⁸⁴ Mutation, often associated with loss of heterozygosity,^95,96 results in loss of function, and the gene is thus considered to be a tumor suppressor gene. The overall frequency of PJS is estimated at 0.5 to 2.0 per 100,000 live births.⁹⁷

Juvenile Polyposis

Juvenile polyps are characteristically solitary peculated lesions that contain tortuous, often cystically dilated crypts, stromal granulation tissue and inflammation, and denudation of the surface epithelium.⁹⁸ These lesions frequently occur in children, and there is no evidence that solitary juvenile polyps pose an increased of risk for carcinoma.^100–100 However, multiple juvenile polyps may occur in young persons and their family members,^103–103 and the presence of >5 juvenile polyps or a single juvenile polyp in a person with a family member with juvenile polyposis now defines the condition, termed “juvenile polyposis.” This form of polyposis carries a lifetime risk of colon carcinoma of 39% and a relative risk of 34.¹⁰⁴ Fifty percent to 60% of affected individuals carry germline mutations in SMAD4 or BMPR1A.^105–107 Nearly all patients with SMAD4 mutations also have hereditary hemorrhagic telangiectasia¹⁰⁸ (overlap syndrome). Both SMAD4 and BMPR1A are involved in the transforming growth factor–β (TGF-β)/bone morphogenetic protein pathway.

Other Polyposes

Finally, a variety of polyposis syndromes with potential genetic linkage have been described and are best exemplified by hyperplastic polyposis syndrome (serrated polyposis syndrome).109,110 This condition consists of the occurrence of multiple sessile polyps variously referred to as hyperplastic polyps or serrated adenomas, frequently on the right side of the colon and numbering from 5 to >100.¹¹¹ Although familial associations of the syndrome¹¹² and linkage to high risk of colon carcinoma¹¹³ have been reported, the genetic basis is complex and associated with mutations in several driver genes, including BRAF and KRAS.¹¹⁰ No single genetic mechanism has been linked to the syndrome, and the true frequency is not established. To date, the number of reported cases is small.

Biomarkers for Colon Cancer Screening

Premalignant Lesions in the Lung

Unlike mortality reductions that have been achieved in the colon and cervix, where preinvasive and therefore nonmalignant lesions are targeted, the benefits of LDCT screening for lung cancer have been achieved through early detection of invasive tumors. A strategy to identify in situ premalignant lesions could potentially reduce lung cancer mortality to a much lower rate. Unfortunately, what are thought to be premalignant lesions in the lung are difficult to detect and may occur at unpredictable locations anywhere in the central and alveolar airways (Figs. 17-5 and 17-6), exemplifying the challenges confronting prevention efforts in some organ systems.

Molecular Changes during Lung Carcinogenesis

As in other organs, most notably the colon, molecular changes that accompany the morphologic changes of dysplasia have been investigated in considerable detail. These studies are informed by the well-established role of tobacco smoke as a carcinogen in lung cancer. The dominant carcinogenic compounds found in cigarette smoke are the polyaromatic hydrocarbons and nicotine-derived nitrosamine ketone.¹³⁷ These compounds are metabolized to highly reactive intermediates that are inactivated largely by mitochondrial glutathione transferases. However, repair is imperfect, and a small proportion of the intermediates react directly with DNA, forming bulky adducts. Unless removed by the DNA repair system, adducts can create errors of replication. Over time, molecular alterations accumulate, providing a selective advantage to mutant cells for growth and survival.

Components of this pathway have been associated with increased risk for future lung cancer. Case control studies indicate that current smokers with tumors have higher levels of adducts in blood or tissue but that nonsmokers and former smokers do not have these higher levels.¹³⁸ Bulky adducts appear to reflect ongoing smoking-related DNA damage rather than biological changes specifically associated with malignant transformation. Clinically applicable adduct levels predictive of lung cancer have not been established.

Glutathione transferase and DNA repair enzyme mutations and polymorphisms that affect DNA repair activity could represent a risk factor for lung cancer because efficient deactivation of reactive carcinogens and repair of DNA damage caused by bulky adducts are important in protecting potential precursor cells from critical mutational changes. However, single-nucleotide polymorphism (SNP) association studies in many carcinogen metabolizing and DNA repair enzymes have demonstrated only weak and inconsistent associations and have not been sufficiently informative to guide intervention in potentially affected persons.139–143

IHC Markers

IHC staining for estrogen receptors (ERs) and progesterone receptors (PRs) was one of the earliest applications to find a place in patient management. Until the early 1980s, antibodies to these proteins were not widely available. However, with the development of monoclonal antibody technology, antibodies that could identify hormone receptors were created¹⁵⁹ and found to reliably label receptors even in FFPE sections of breast carcinomas.^160,161 Unexpected results of this testing were that these receptors are typically found in the cell nucleus and that the intratumoral and intertumoral range of expression is large. Because of this finding, semiquantitative scoring that takes into account intensity of staining and percentage of cells stained has been used to establish expression levels that define positive and negative results.^162,163 These scoring systems continue to prove their prognostic value to the present time.¹⁶⁴

A second IHC test that has been successfully applied to the management of breast carcinoma is the test for HER2/neu (ERBB2) expression. HER2 is a tyrosine kinase receptor (TKR) and a member of the epidermal growth factor receptor (EGFR) family of signaling molecules. Its role in human carcinogenesis was initially discovered through DNA screening, where it was found to be amplified in 30% of the tested breast cancers.¹⁶⁵ RNA and protein were shown to be overexpressed in tumors that are amplified, as well as in a subset of nonamplified tumors.¹⁶⁶ HER2 testing became crucial when it was found that HER2 protein overexpression was a determining factor for in vitro and xenograft responsiveness to anti-HER2 monoclonal antibody treatment.^167,168 In early clinical trials, anti-HER2 IHC was used as the method for determination of HER2 status and determined eligibility for treatment with the anti-HER2 antibody drug, Herceptin.¹⁶⁹ A standardized approach to HER2 IHC testing was established several years later when expert panels agreed upon criteria for determining HER2 status (Fig. 17-7).¹⁷⁰

Recently, tests for multiple IHC markers have been combined into panels that include ER, PR, HER2, and the proliferation marker Ki-67,¹⁷¹ as well as several other markers thought to be of prognostic importance.¹⁷² These panels have been found to provide prognostic information that is comparable with that of multiplex mRNA expression assays discussed later and have the added advantage of accessibility in local laboratories and ready correlation with histologic features.

A substantial subset (~15%) of breast tumors do not express ER, PR, or HER2 and are now referred to as triple-negative tumors.¹⁷³ These lesions are notable for their aggressive histology and poor long-term outcome. This category overlaps but is not identical to the basaloid-like breast cancer category defined by microarray-based expression profiling¹⁷⁴ and includes nonhereditary tumors that frequently express perturbations of BRCA1 pathway gene expression. The triple-negative category thus has increased the importance of accuracy in both positive and negative IHC testing and reemphasizes the need for local validation and controls for accurate IHC procedures (see the Best Practices section in this chapter).

Finally, EGFR is expressed at a high level in most non–small cell lung carcinomas, including both adenocarcinoma and squamous carcinoma, and expression correlates with gene amplification and copy number.175,176 When a scoring system was applied in a recent phase 3 trial testing the effectiveness of the anti-EGFR chimeric (mouse/human) monoclonal antibody, Cetuximab, in advanced cases of non–small cell carcinoma, only patients with the highest levels of EGFR expression (scores >200) experienced a survival benefit.¹⁷⁷ This result suggests that EGFR expression levels determined that IHC may be a reasonable selection criterion for anti-EGFR antibody treatment. Properly scored EGFR IHC expression may prove to be a valuable predictor of response to anti-EGFR monoclonal antibody treatment.

Gastrointestinal Stromal Tumors

GISTs were, until recently, a rare and ill-defined neoplasm of somewhat mysterious origin. In the early 1990s these tumors were defined as spindle cell malignancies that exhibited no evidence of cell of origin.¹⁸⁶ The status of these tumors began to change as it was recognized in the mid 1990s that GIST tumors express CD34 but not desmin¹⁸⁷ and thus displayed a distinct immunophenotype. A watershed discovery was reported by Hirota et al.¹⁸⁸ in 1998. These investigators described nearly uniform expression of KIT (CD117) in GIST tumors and suggested that they may arise from the immunophenotypically similar paraganglionic interstitial cells of Cajal. Most importantly, they identified deletions and point mutations in the juxtamembrane portion of the KIT TKR gene. These mutations were shown to activate KIT signaling, and KIT was thus a potential therapeutic target. KIT is a member of the TKR signaling family and is encoded at chromosome 4q11-q12. In GIST tumors, mutations in the KIT gene (reviewed in references 189, 190, and 191)^191–191 are located predominantly in the juxtamembrane domain (exon 11), with two thirds of tumors harboring mutations in this region of the gene. Exon 11 mutations allow adenosine triphosphate to phosphorylate receptor in the absence of ligand (stem cell factor). Phosphorylation of the receptor triggers downstream signaling of RAF, MEK, MAPK, PIK3CA, and STAT3, transforming mutated cells to malignant status. Approximately 10% of mutations are found in exon 9, which encodes the extracellular domain of the molecule. These mutations induce a change in receptor conformation mimicking that seen in the presence of ligand and resulting in receptor phosphorylation. Exon 9 mutations are commonly seen in large and small intestinal tumors but rarely in gastric neoplasms. Finally, mutations in exons 13 (tyrosine kinase domain) and 17 (activation loop) are occasionally found in GIST tumors. The effects of exons 13 and 17 mutations on the receptor are less well established, but the net result of all these mutations is receptor activation and oncogenic transformation of mutant cells. KIT mutations occur in approximately three fourths of GIST tumors.^191–191

Tumors that do not contain a KIT mutation may contain a mutation in the closely homologous tyrosine kinase receptor gene, platelet-derived growth factor receptor (PDGFR), alpha polypeptide (PDGFRA).192,193 PDGFR protein has two isoforms, α and β, and it is the α form that is mutated. Approximately 10% of GIST tumors contain mutations in PDGFRA.^191–191 The mutated loci in PDGFRA are distributed similarly to KIT, but frequencies differ. The most frequent mutation site (exon 18) encodes a portion of the tyrosine kinase domain (TK2) and occurs in 6% of all GISTs. Juxtamembrane mutations in PDGFRA are relatively infrequent, occurring in <1% of GISTs.

Introduction of TKIs that block signaling by mutant receptor has had a profound effect on the outlook for this tumor. Prior to 2000, the outlook for patients with an advanced GIST was bleak. Most patients experienced a recurrence after surgical resection, and responses to chemotherapy were poor. Only 10% of patients were disease free after extended follow-up.194,195 A dramatic reversal of this situation occurred when it was realized that GIST is exquisitely sensitive to tyrosine kinase blockade. The drug successfully used to treat GIST was originally designed to block signaling by the BCR-ABL fusion protein in chronic myelogenous leukemia. Structural similarities between the intended target of the drug and KIT led to the discovery that the TKI now known as imatinib strongly inhibits mutated KIT.¹⁹⁶ Subsequent clinical trials demonstrated high response rates and prolonged progression-free survival (PFS),^199–199 leading to FDA approval of the drug as discussed elsewhere in this volume. KIT testing is now used routinely to identify persons likely to respond to the drug.

PDGFRA mutations are not equivalent to those in KIT. More than half of PDGFRA mutations encode a D842V amino acid substitution that is resistant to imatinib, whereas other mutations in PDGFRA are sensitive.²⁰⁰ Alternative drugs that show activity against the D842V resistance mutation such as midostaurin (PKC412)²⁰¹ are under investigation.

Colon Carcinoma

Other Mutations

Additional mutations of predictive importance are NRAS and PIK3CA, which occur at frequencies of <5%211,213,227 and 10% to 15%,^213,225,227 respectively, in colon carcinoma. PIK3CA mutations overlap those found in other genes, but NRAS mutations are nonoverlapping with mutations in other genes, including KRAS and BRAF. Mutations in either PIK3CA or NRAS are associated with poor response to EGFR monoclonal antibody therapy.^213,225 Finally, initial results of comprehensive molecular analysis by the TCGA have been reported and identify large numbers of mutations that are likely to become targets of specific molecular inhibitors.²²⁷ Comprehensive molecular testing is expected to contribute greatly to colon carcinoma management in the near future.

Lung Cancer—A Heterogeneous Tumor

For many years, molecular characterization of lung cancer was impeded by the extreme heterogeneity of this disease as reflected in the most recent revision of the anatomic classification of lung carcinoma.²²⁸ Since the 1920s, lung tumors have been divided into small cell (“oat cell”) lung carcinoma (SCLC) and non–small cell lung carcinoma (NSCLC) that differ not only morphologically but express different classes of cell surface molecules, possess different mutational profiles, and exhibit different biological behavior. Small cell carcinomas express a number of markers that are also found on neural cells and in current classifications are categorized as one of several different types of “neuroendocrine” carcinomas. The vast majority of NSCLC does not express neuroendocrine markers but instead expresses large quantities of Erb-B cell surface TKR. SCLC is highly genetically unstable and appears to be driven predominantly by seven transmembrane receptors. In NSCLC, the level of genetic instability is more variable, and the TKRs are major tumor cell drivers. Finally, SCLCs are uniformly aggressive and are particularly sensitive to mitotic inhibitors.

More recently, a high degree of heterogeneity has been observed within the category of NSCLC. The two main histologic categories are adenocarcinoma, a tumor characterized by the formation of acinar or papillary structures, and squamous carcinoma, which is characterized by the formation of keratin pearls and intercellular bridges. These tumor types are also distinguished by variation in expression of specific keratin subgroups and other lineage specific markers, most notably TTF1, which is expressed by adenocarcinoma but not squamous carcinoma. Finally, within histologic categories, there is molecular variation that is reflected by differences in driver protein expression and activation that can be therapeutically exploited. Most of these differences involve activation of TKRs and their signal transducers. The first of these receptors to be defined as a therapeutic target was EGFR.

EGFR

The original isolation of EGF from mouse salivary gland in 1962,²²⁹ the demonstration of its binding to cell membrane receptor,²³⁰ and sequencing of the receptor²³¹ established the role of intercellular signaling in epithelial cell growth and maintenance. By the 1990s, overexpression of EGFR had been demonstrated in squamous carcinoma. Monoclonal antibodies to EGFR that were effective in localizing EGFR in situ became available in the late 1990s and documented the broad expression of EGFR in adenocarcinoma.¹⁷⁵ EGFR expression in NSCLC was found to be closely linked to gene copy number.¹⁷⁶

Further studies of EGFR were driven by of the discovery of a subset of patients who were mostly nonsmokers with adenocarcinoma who responded dramatically to EGFR TKIs (Fig. 17-9). Simultaneously, two groups reported that mutations in the EGFR tyrosine kinase domain were present in a subset of adenocarcinomas and that their presence correlated with sensitivity to TKI.^232,233 The mutations most commonly consisted of either single base substitution in exon 21 (c.2573G>C, p.L858R) or in-frame deletion within exon 19 ranging from 9 to 18 base pairs in the region coding for the adenosine triphosphate binding pocket of the tyrosine kinase domain. These mutations resulted in strong constitutive activation of the encoded EGFR protein.

Activating mutations of EGFR result in tumor susceptibility to TKI therapy.²³⁴ However, responses to therapy are temporary, and hopes of a long-lasting therapy are generally unrealized because of resistance to targeted therapy that inevitably arises in treated patients. Two common mechanisms of resistance to EGFR TKI are MET amplification^237–237 and a point mutation in exon 20 of EGFR (c.2369 C>T, p.T790M),²³⁸ the effect of which is depicted in Figure 17-8. As a result of the point mutation in EGFR exon 20, a bulky methionine side chain is thought to sterically hinder binding of TKIs. Tumors cells harboring this mutation may exist in low numbers in untreated tumors and emerge through selective pressure exerted by targeted therapy. In addition, insertions in exon 20 of EGFR are associated with primary resistance to TKI therapy,²³⁹ which also is thought to be due to steric hindrance of TKIs.

ALK and Other Large-Scale Rearrangements

Point mutations and small deletions are not the only molecular mechanisms driving lung cancer. Overexpression of oncogenes may also be driven by gene activation through chromosomal rearrangements. Rearrangements that fuse gene promoters to the tyrosine kinase domain of growth factors, growth factor receptors, or transcription factors may result in inappropriate signaling from the fusion products. Examples of activation by gene fusion in lung cancer involve ALK, ROS1, and RET. In 2007, Soda et al.²⁴⁴ discovered a transcript derived from a patient with NSCLC that could transform T3T fibroblasts. Further investigation demonstrated the transcript to be fusion of a gene called echinoderm microtubule-associated protein-like 4 (EML4) with the anaplastic lymphoma kinase (ALK) gene (Fig. 17-10), which is also rearranged in large cell lymphomas. This rearrangement has been detected in 4% to 7% of pulmonary adenocarcinomas, with an increased proportion seen when cohorts are selected based on clinicopathological features including lack of smoking history.^245–248 The driver of tumorigenesis in this rearrangement is thought to be ALK, which is not typically expressed in adult lung tissue. Juxtaposition of a 5′ partner with a promoter results in high-level expression of the kinase domain of ALK and leads to inappropriate signaling by this molecule.^244,249

Less than 5 years after its initial discovery, more than 10,000 lung cancers have been tested for the presence of this rearrangement. More than 13 molecular variants of EML4–ALK fusion have been detected,246,250 and three other rare activating gene partners have been identified, including TRK-fused gene (TFG),²⁵¹ KIF5B,²⁵² and KLC1.²⁵³ A novel targeted therapy agent, crizotinib, has been found to be effective against tumors driven by this molecular marker²⁵⁴ and received approval for treatment of these specific tumors by the U.S. Food and Drug Administration. Numerous other ALK inhibitors are in initial clinical trials.

Melanoma

Several targetable mutations have been found in melanoma in recent years involving the BRAF, NRAS, and KIT genes (see Table 17-3). Of these, the most common is BRAF.²⁵⁶

Tissue Collection and Processing

Tissues obtained for solid-tumor molecular testing may come from surgical resections or biopsies (fine-needle aspirates and cores). Most often, the tissue needed for molecular testing is derived from diagnostic material, unless special arrangements are made to specifically collect tissues for molecular studies. Issues regarding specific specimen types are discussed here (also see Chapter 25).

Enriching for Tumor Cells

Because nonmalignant cells invariably accompany (“contaminate”) all clinical samples, specimens must be enriched for tumors to a level that is consistent with the analytical sensitivity of the platform that will be used for testing. As a rule of thumb, a minimum of 100 viable tumor cells in a 10-µm section is required for a single PCR-based assay. The quality of the biopsy specimen may be more important than quantity for critical molecular studies.285,286

Although tumor enrichment methods may vary from laboratory to laboratory, it is essential that tumor enrichment methodology be appropriately matched to the testing platform. At least five different procedures may be used for tumor cell enrichment depending on the amount of contaminating normal tissue that is present in the specimen: (1) enface sectioning of a trimmed block; (2) coring of paraffin blocks in regions enriched for tumor cells; (3) macrodissection; (4) manual microdissection; and (5) laser capture microdissection. Macrodissection can refer to the practice of scraping of tumor cells from a glass slide, which typically is done without direct microscopic visualization. Manual microdissection, by contrast, involves the use of some form of microscopic assistance to identify tumor cells. Manual microdissection is typically performed using a dissecting microscope and usually involves counterstaining of slides to identify regions of interest, which can be removed from the slide with a scalpel, needle, or glass micropipette. Laser capture microdissection involves melting of supporting matrix using a laser beam created with an epifluorescence lens and removal of the target cells for extraction and quantitative testing of DNA and other analytes. Although it is more precise than manual microdissection, laser capture microdissection is slower, more costly, and generates lower yields of extraction products.

Sanger Sequencing

Sanger sequencing has been the gold standard for many years and, like many other assays, is based on the dideoxy-chemistry illustrated in Figure 17-11. The sequencing reaction is read as a color code that distinguishes oligonucleotides of variable length with four specific color labels. A ladder of nucleotides is created that can be identified by their electrophoretic mobility and color of terminal fluorescent nucleotide. The identification of abnormal (mutant) peaks in chromatograms of this ladder can be facilitated by computer programs that not only create the chromatogram but compare them with reference sequences (RefSeq) and identify abnormalities that represent mutations and polymorphisms.

Sanger sequencing is readily accomplished in DNA extracted from FFPE tissue that has been amplified by PCR provided that the amplified sequence is short (<300 base pair). This method has the advantage that it provides unbiased sequence results that will detect virtually any mutation in the targeted region. However, the analytic sensitivity is limited, with tumor cell concentration of approximately 50% required for accurate results. Because of the PCR amplification, the amount of DNA starting material required is usually small, depending on the number of DNA templates selected for amplification.

PCR-Based Mutation-Specific Assays

Mutation-specific techniques depend largely on the observation that mismatch of a 3′ terminal nucleotide in PCR primer results in failure of PCR amplification so that primers can be created that are highly selective for a single nucleotide. This is the basis for the ARMS that can be used to detect mutations in KRAS codons 12 and 13. In ARMS, mismatch at the 3′ prime end of the primer results in minimal amplification of the normal base but successful amplification of the sequence-containing mutant base. Elongation of the amplified segment displaces a fluorophore from its quencher, resulting in a quantitative increase in signal that can be read on a variety of real-time PCR instruments. The number of PCR cycles needed to detect signal above a background referred to as Cycle threshold (Ct) is used to quantify fluorescence. Differences in Ct between exogenous control DNA and test DNA (ΔCt) reflect the quantity of mutant DNA in each sample. In one variation of this process, ARMS/Scorpions, primer, and probe are combined in a single molecule.

Only a single mutation per amplification reaction is detected with PCR-based assays, resulting in the need for a considerable amount of DNA and increasing the cost of the overall analysis. In currently available kits for KRAS analysis, seven primer/probe sets are needed for testing each target allele at codon 12 and 13. Thus although only a small amount of DNA is needed for each reaction, the amount of DNA required for the entire analysis can escalate quickly, and an upper limit of clinical feasibility is quickly reached, especially with smaller specimens. The advantages of simplicity and speed of turnaround make this platform desirable for performing large numbers of tests for a small number of mutations.

High-Resolution Melting Analysis

High-resolution melting analysis (Fig. 17-12) measures differences in melting-point temperatures between matched and mismatched DNA doublets. Mismatching causes a reduction in melting-point temperatures that can be quantified using software that is readily available in quantitative PCR instrumentation. The advantage of melting-point analysis is that it is sensitive and requires relatively small amounts of DNA (<5 ng). It also has excellent coverage, detecting any mutational abnormality that may be present in the amplified segment. However, any perturbation of DNA structure, including untargeted SNPs and adducts, may produce aberrant melting-point curves, and nonspecific background for this method is relatively high. The specificity of this reaction is therefore low without confirmation by a second technology, typically direct sequencing, which can negate the rapid turnaround and relative low cost of the assay. Many investigators regard this test as a screening assay that requires confirmation of abnormal results by a second assay.

Pyrosequencing

Pyrosequencing does not rely on truncation of the transcription reaction but rather on the release of the pyrophosphate during nucleotide chain elongation, which initiates a chemiluminescent response that can be detected with appropriate instrumentation. For this technology, DNA is immobilized and reagents needed for the addition of successive nucleotides are added to the template. Additions of nucleotide are registered by the release of a pyrophosphate, which induces a fluorescent signal in a reaction catalyzed by luciferase.

Pyrosequencing is relatively inexpensive and sensitive and largely unbiased in that it detects all possible mutations in a target sequence, provided the release of nucleotides is cycled to identify all possible alterations. Sensitivity is higher than Sanger sequencing and approximates the sensitivity of allele-specific and multiplex methods (~5%). However, the technology is somewhat complicated by short read lengths and difficulties in interpretation when multiple nucleotides of the same species are added in sequence (homopolymers). Turnaround times are relatively short, making this technology popular.

Multiplex Assays

MASSarray (Sequenom, San Diego, Calif.) and SNaPshot (ABI, New York, NY) are based on similar chemistry but use different methods to measure the outcome of sequencing reactions. For MASSarray, a mass biased probe is used, which is detected by mass spectrometry.²⁸⁸ For SNaPshot, a fluorescent probe is used, and standard sequencing capillary gel electrophoresis is used to detect mutant or wild type peaks. PCR reactions are performed with multiplex primers, and panels of primer sets are constructed to detect mutations at multiple sites. The chemistry is based on single base extension from a probe complimentary to the region of interest. Primers are positioned immediately adjacent to the mutated base, and addition of a dideoxynucleotide at the mutation site results in the formation of a labeled oligonucleotide. Probe sets can be designed at different lengths for specific alleles so that they can be multiplexed into panels of up to eight or nine analytes per panel. Amplified probe sets can be measured by the appropriate instrumentation, either mass spectroscopy (MASSarray) or capillary gel electrophoresis (SNaPshot, Fig. 17-13). The amplification incorporated into this technology results in a high level of sensitivity so that only 10% concentration of tumor cells is required for mutation detection.

NextGen Sequencing

Advances in sequencing technology are making possible the complete sequencing of the cancer genome158,289,290 through such programs as TCGA, described elsewhere in this volume. It is now nominally possible to sequence the entire genome of a single tumor for individual patients at an accessible charge. Demand for genome-based selection of targeted anticancer agents is expected to rapidly increase. There is thus a developing need for flexible deep sequencing platforms to provide high-density sequencing data with broad coverage in a clinical setting. Two such platforms are currently available, Ion Torrent and MiSeq. Ion Torrent exploits the release of a hydrogen ion by DNA polymerase during sequencing (see Fig. 17-11) and measures changes in pH using an integrated semiconductor array for nonoptical sequencing.²⁹¹ The alternative MiSeq platform is a “sequence by synthesis” approach that utilizes serial additions of fluorescent-labeled nucleotides to immobilized products across a two-dimensional array that allows for parallel sequencing of thousands of nucleotide strands simultaneously. Published performance characteristics of MiSeq compare favorably with Ion Torrent.²⁹² Both platforms must be thoroughly validated for each target mutation before introduction into a clinical setting. These systems are being rapidly introduced in kit format and are expanding sequencing capabilities of clinical laboratories from single gene or small multiplex sequencing to large-scale parallel sequencing of hundreds of genes simultaneously. It is expected that in the near future, complete sequence coverage of all relevant genomic treatment targets will be available to treating physicians at a local clinical laboratories.

Assays for Gene Rearrangements

Large-scale genomic rearrangements are not easily detected by conventional gene sequencing, and specialized methods have been used for decades to demonstrate innumerable complex abnormalities for which nomenclature is now standardized²⁹³ and uniformly applied in searchable databases (http://cgap.nci.nih.gov/Chromosomes/Mitelman). Large-scale genomic changes could potentially be identified by molecular methods such as array CGH, SNP arrays, or NextGen sequencing. However, at the present time, array CGH and SNP arrays test only copy number,²⁹⁴ and NextGen sequencing is not yet sufficiently refined to routinely detect rearrangements in a clinical setting. Only FISH is validated for application to clinical samples to identify complex rearrangements. FISH has the advantages of maintaining histologic architecture, accuracy on small sample sizes, and a wide availability in clinical laboratories. The large number of FISH tests currently in active use cannot be fully addressed here because of space limitations. However, lung cancer provides an example of the relevance of discovery and clinical testing for large-scale gene rearrangements to targeted therapy, as discussed in the next section.

RNA Assays

Despite the vast breadth of RNA technology that has been developed and applied for research and biomarker discovery purposes during the past few decades, only a small proportion of this technology has been validated for clinical oncology applications. Possible explanations for this lag are numerous and include the extreme lability of mRNA in tissue and blood and the consequent special requirements for its optimal preservation; promiscuous expression of RNA by tumor; perturbations of posttranscriptional modification; the extreme complexity of expression pathways and the complicated informatics needed to assess them; and, finally, the questionable value added to clinical management provided by currently available RNA-based tests.

This situation could change with the development of specific targeted agents that require an RNA companion diagnostic and by improvement in sensitivity, specificity, and preanalytical requirements of RNA-based testing platforms. As previously mentioned, predictive RNA testing has been most widely tested in the Mammaprint and OncoDx platforms. Mammaprint was originally based on expression microarray testing of a 70-gene panel that required high-quality RNA. Formalin fixation and paraffin embedding produce chemical changes³⁰¹ in RNA, and RNA may degrade and shear in storage.³⁰² An antidote is to shorten the template to 50 to 100 bases and to use random primers rather than reverse transcribing from the polyA tail of mRNA. Mammaprint has adapted to the need for FFPE testing by incorporating these steps in a new multiplex PCR based-technology.³⁰³ OncoDx has used an RT-PCR–based approach to measure a multigene panel.³⁰⁴ Both of these platforms require careful normalization with consistently expressed housekeeping genes to ensure quality of results. Finally, a promising new platform that uses florescent nano bar codes³⁰⁵ linked to sequence-specific probes is being tested with promising early results. A major advantage of this system is that there is no requirement for PCR.