Abeloff's Clinical Oncology, 4th Edition
Part I – Science of Clinical Oncology
Section C – Diagnosing Cancer: Pathology and Laboratory Medicine
Chapter 19 – Molecular Diagnostics
Jeffrey A. Kant
SUMMARY OF KEY POINTS
Characteristic markers involved in the molecular pathogenesis of cancer have been described with increasing regularity over the last three decades. “Molecular diagnostics” in the context of cancer generally refers to the analysis of nucleic acid (DNA or RNA) in patient samples to detect markers for purposes of diagnosis or prognosis. Most of these markers are acquired during cancer development and restricted to neoplastic cells. A subgroup of germline mutations found in all body cells that predispose to cancer at different ages is significant for the management and counseling of individuals and family members.
Sensitive detection technologies facilitate the use of molecular markers to track residual cancer, thus assisting with monitoring and adjusting therapy. Molecular tools can detect agents of etiologic interest (e.g., human T-cell leukemia virus, human herpesvirus) and also assist with the selection of therapeutic cell products (e.g., high-resolution histocompatibility typing for bone marrow transplantation). After transplantation, molecular methods are used to assess engraftment. The evolving potential of molecular diagnostic assays to yield information allowing individually tailored drug selection and dosage is commonly referred to as “personalized medicine.” Because the functional manifestations of most nucleic acid changes are mediated through proteins produced (or not produced) from affected genes, the term molecular diagnostics also is used occasionally in the context of applications that involve the analysis of proteins (i.e., proteomics; see Chapter 20 ).
This chapter focuses on nucleic acid-based applications for which clinical validity and utility has been established. Molecular cytogenetic applications, including fluorescent in situ hybridization (FISH) and array comparative genome hybridization (CGH), are discussed in Chapter 19. Selective background is offered for a number of promising future molecular diagnostic applications currently under study. Clinicians in the United States should keep in mind that assays that yield information on which patient management is based must be performed in Clinical Laboratory Improvement Act (CLIA)-licensed laboratories.
Assays begin with extraction of target nucleic acid from a sample. Nucleic acid typically is obtained from the nucleus or cytoplasm of disrupted cells but may be derived from cell-free portions of the sample and even serum or plasma. Because nucleated cells are the primary source of DNA, paucicellular samples of bone marrow and blood or tumors following therapy may not reflect the state of a diagnostic sample. After taking tissue for histopathologic examination or other studies, it is crucial, if possible, to set aside a portion of the “diagnostic” specimen for nucleic acid preparation or storage, on which assays can be performed subsequently if desired. Blood, bone marrow, and body fluid samples must be anticoagulated adequately, because appreciable numbers of nucleated cells are lost in clots. Ethylenediaminetetraacetic acid (EDTA) or citrate is preferable to heparin as an anticoagulant because heparin may bind nucleic acids or other reagents and potentially interfere with molecular assays. (Washing heparinized samples can help.) Heparin is the anticoagulant of choice for cytogenetic analysis, so it often is desirable to obtain two samples or split a sample when both assays are performed (Table 19-1 ). Tissue (at least 100–200 mg) can be frozen at -80°C or in liquid nitrogen and processed subsequently for nucleic acid or protein. DNA (and RNA) can be obtained from archival paraffin-embedded specimens, but typically there is appreciable fragmentation and/or degradation, and the preferred sample for molecular diagnostic analysis is a fresh sample. Assessment of molecular markers in cell-free samples (plasma or serum) has been demonstrated and may offer advantages; tumor-associated DNA and RNA presumably reside in cell-free domains protected from degradation.    Enrichment of tumor tissue from paraffin sections via manual or laser capture microdissection is increasingly useful in evaluating solid tumors.  
Table 19-1 -- Sample Handling Tips for Molecular Diagnostic Testing
Samples should be submitted to the laboratory in a timely fashion, particularly if RNA is to be assayed. Collection tubes are available to immediately stabilize collected samples, although weekend blood or bone marrow samples from which nucleic acid is extracted often perform satisfactorily after several days at refrigerator (not freezer) temperatures. Usually, 4 to 6 mg of DNA is obtained per 1 million nucleated cells, so nucleic acid yield is problematic only in hypocellular samples, such as those derived from cerebrospinal fluid or bone marrow after treatment. In such cases, DNA may be insufficient to perform certain assays (e.g., Southern blotting).
Polymerase Chain Reaction
Polymerase chain reaction (PCR) underlies the vast majority of molecular diagnostic assays. Isolated double-stranded DNA first is denatured to single strands, to which short single-stranded DNA oligonucleotide primers are bound, flanking the region of interest. Enzymatic extension from the primer copies the targeted region on each strand, effectively producing twice the amount of original nucleic acid target. Repeated cycles of denaturation, annealing, and extension amplify the targeted region many million-fold and facilitate the analysis of samples with low levels of target nucleic acid. One to as many as 10 to 15 assays generally can be performed for each microgram of DNA obtained.
For sensitive qualitative detection of unique DNA or RNA constructs such as chromosomal translocations, nested PCR can be per formed, in which an aliquot of the initial PCR reaction undergoes a second PCR series using a new primer set targeting sequences on both sides of the target but internal to the initial flanking primers. Nested PCR offers sensitivity with increased risks of contamination; increasingly, PCR is done on “real-time” platforms, which offer broad linear ranges and provide quantitative or qualitative results.   PCR assays can amplify DNA regions up to several kilobases. “Long distance” PCR,   which amplifies larger regions of DNA, is appealing for some applications but is not in widespread clinical use.
For PCR assays in which RNA is the starting material (RT-PCR), RNA first is incubated with the enzyme reverse transcriptase (RT) in the presence of either general or sequence-specific oligonucleotide primers. RT makes a DNA copy strand (cDNA) from the RNA template. The cDNA products then are subjected to standard PCR amplification.
DNA sequencing typically is performed from primary PCR products.   Oligonucleotide primers bound to the “ends” of forward and reverse strands of PCR fragments are copied into single strands of varying lengths, allowing the DNA sequence to be read directly from capillary or gel-based laser systems ( Fig. 19-1 ). This is accomplished by using a mixture of normal and fluorescently labeled dideoxynucleotides with termination of the copying reaction each time a dideoxynucleotide is inserted. Pyrosequencing, by contrast, determines DNA sequence over short stretches of nucleotides by measuring the amount of pyrophosphate generated after sequential addition of differing nucleotides.    Pyrosequencing in nanoliter chambers followed by software assembly of overlapping short sequences is the technical basis for high throughput sequencers capable of determining millions of nucleotides.
Figure 19-1 Direct mutation detection. Forward-strand DNA sequences are shown for missense (p.Ile127Ser) (A) and frameshift (c.153_154insT) (B) mutations in the SDHB gene. C, Allele-specific hybridization for, top to bottom, BRCA1 (185delAG, 5282insC) andBRCA2 (6174delT) mutations. Specific probes for normal (WT) and mutant sequence are used on samples amplified together for BRCA1 exons 2 (top) and 20 (middle) and BRCA2 exon 11 (bottom). Patient samples tested in duplicate are 1,4, 2,5, 3,6. Heterozygous controls are samples 7, 8, 9. Sample 10 is a normal control. Sample 11 is a minus-DNA control (except 5382, where it is another normal control).
Southern blotting is a venerable but increasingly less-used technique for assessing clonality or chromosomal translocations in hematologic or solid tumors ( Fig. 19-2 ). Restriction endonuclease-digested fragments of total sample DNA are separated in gels by size, then replica-transferred in denatured single-stranded form to a reinforced membrane where those fragments binding a labeled complementary probe can be visualized. Physical restructuring of a chromosomal region by translocation or physiologic elimination of DNA segments from immunoglobulin and T-cell receptor genes during lymphocyte development creates restriction fragments of new lengths characteristic of clonal cells that have expanded to form the cancer.
Figure 19-2 Immunoglobulin heavy-chain gene assessment by Southern blot. Typical three-restriction enzyme analysis (using enzymes Bgl II, Bam HI, and Hind III) of five patient specimens (patient 1: lanes a, h, m; patient 2: lanes b, j, o; patient 3: lanes c, k, p; patient 4: lanes d, i, n; patient 5: lanes e, l, q). Note the different order of patients for Bgl II versus the other enzymes. A single germline band is seen in all enzyme digests for the normal control (patient 1). Lanes f and g are mixtures of 10% and 4% β-cell lymphoma DNA in normal DNA (note faint rearranged alleles above and below the germline band on the Bgl II digest); these serve as positive and sensitivity controls. Two rearranged alleles for both immunoglobulin heavy-chain genes are seen in patient samples 2 and 3. Patient 2 demonstrates an additional faint band, with enzymes Bgl II and Hind III supporting clonal evolution. Note also the reduced intensity of the germline allele for patient 2, indicating that this sample contains a high fraction of neoplastic cells. Patients 4 and 5 are normal for all enzyme digests.
Southern blot assays have a number of disadvantages. They require moderate amounts of nucleic acid, typically several micrograms per lane evaluated. Southern blots also are labor-intensive, technically demanding, and typically take 5 to 7 days to deliver an answer.
Other DNA Assays
Numerous di, tri-, and tetranucleotide sequences, known as simple tandem repeats (STRs) or microsatellites throughout the genome, demonstrate polymorphisms of varying lengths on maternally and paternally inherited alleles. Such markers are useful in segregation (or linkage) analysis to follow at-risk chromosomes passed within families, in the assessment of bone marrow engraftment, and in the comparison of the relative abundance of STRs in tumor and normal tissues to determine allelic loss (loss of heterozygosity [LOH]). The human genome also features large numbers of single-nucleotide polymorphisms (SNPs) that can be used to assess allelic loss as well as gain or loss of chromosomal regions.
Microarray “chips” composed of high-density assemblies of oligonucleotides or cDNA probes on glass or silica surfaces can be used to assess the array of transcripts or SNPs in normal or tumor DNA.   A particularly powerful application of chips is expression profiling of tumors to provide a relative assessment of RNA transcript levels from many genes in a tumor population. Powerful software analysis programs assemble such data into patterns that indicate the origin or behavioral profile of a tumor.  
Clinical applications exist for (1) hematolymphoid neoplasms for which a large variety of molecular tests have evolved over two decades and (2) selected solid tumors. While molecular diagnostic assays that drive management of patients with solid tumors are still limited, with improved understanding of pathogenesis and related development of new therapies, they will be a major area of future growth.
Most molecular oncology assays are methods developed in individual laboratories. External proficiency surveys are available for some hematolymphoid and solid tumor assays, but standardization is limited. This lack of standardization results from a combination of factors, including low test volumes and limited incentive for commercial development, the tendency of academic laboratories to be early developers of new testing, and the fact that it is cheaper to perform laboratory-developed assays even when commercial assays are available.
Molecular assays for hematolymphoid neoplasms are useful if the information provided by histology or immunophenotypic assessment is insufficient for diagnosis. If the plan is to use a molecular marker to monitor disease following treatment, it is useful to confirm that that marker is present in diagnostic material, or store sample nucleic acid for future analysis. Molecular assay results typically are best evaluated in the broader context of clinical, histologic, immunophenotypic, and other information to arrive at the best picture of a patient's disease. Typically, this is done by a hematopathologist. Properly chosen assays can help in both the diagnosis and classification of neoplasms. Clonality assays, which are first-line tests for suspected hematolymphoid neoplasms, are directed at populations that demonstrate immunoglobulin heavy chain (IgH) or T-cell receptor (TcR) gene rearrangement or a specific chromosomal translocation.
Gene Rearrangement Assays
Gene rearrangement assays look for a significant clonal β- or T-lymphoid population. Each tumor cell contains the same rearranged IgH or TcR gene, and Southern or PCR-based assays can detect down to 1% to 5% tumor cells. IgH gene rearrangement typically is assessed first for B cells, because this gene undergoes initial rearrangement during B lymphoid development. Light chain genes, particularly the kappa gene, also may be assessed. T-cell receptor beta or gamma chain genes commonly are examined for T-lymphoid cells. Southern blot testing is more common for beta genes, although PCR assays exist for both. Clinical assays for rearrangement of lambda light chain, T-cell receptor alpha, or T-cell receptor delta chain genes are uncommon.
Although Southern blot analysis is performed by a steadily diminishing number of laboratories, it is still the gold standard for B and T cell clonal assessment. In this technique, a labeled probe that recognizes the IgH or TcR gene is hybridized to restriction-endonuclease digested sample DNA. New hybridizing bands of different size indicate clones with rearranged IgH or TcR alleles (see Fig. 19-2 ). Rearrangement seen with two (or more) restriction enzymes for the sample is scored as positive. Clonal evolution within a neoplasm sometimes may be observed as new hybridizing bands in the presence of previously demonstrated ones (see Fig. 19-2 ).
Increasingly, PCR assays alone ( Fig. 19-3 ) are performed to look for clonal rearrangements of immunoglobulin and TcR genes. Rela tively conserved “framework” areas of variable (V) region gene segments serve as targets for upstream PCR primers; similarly conserved regions in joining (J) or adjacent sequences are targeted by downstream primers. Advances in PCR assay detection sensitivity and standardization have accompanied the European BIOMED-2 study, which developed and tested multiplexed groups of PCR primers that together detect the large majority of immunoglobulin and TcR gene rearrangements. At this time, BIOMED-2 reagents are available in the United States only labeled as “research use only, not for use in diagnostic procedures.”
Figure 19-3 Polymerase chain reaction (PCR) and reverse transcriptase (RT-PCR) analysis in hematolymphoid neoplasia. A, Immunoglobulin heavy chain gene PCR analysis. Testing is done at two or more different DNA concentrations for each of five patients. Lanes a and n are molecular size standards; lane m is a minus-DNA control to rule out potential PCR contamination. Patients 4 and 5 are known reactive (polyclonal) and lymphoma (monoclonal) samples, respectively. Patient 1 has a monoclonal process involving most cells in the specimen; patient 2 has a polyclonal process, and patient 3 has a subtle monoclonal process in a polyclonal background. B1, T-cell receptor gamma chain gene PCR analysis, straight electrophoresis. Patients 3 and 4 are known lymphoma and reactive samples. Patient 1 is polyclonal; patient 2 has a monoclonal process in a polyclonal background. B2, T-cell receptor gamma chain gene PCR analysis, homoduplex analysis. Samples are denatured following PCR amplification, allowed to renature and run on a polyacrylamide gel. Homoduplex bands at the “leading edge” of migration represent clearcut clonal specimens (lanes b,c); samples with polyclonal T-cell populations do not show distinct bands (lanes a,d,f,g,h,i). Lanes j (10% clonal + 90% polyclonal DNA) and k (100% polyclonal) are controls. A low-predominance clonal population in sample E is of uncertain clinical significance. C, Allele-specific amplification for JAK2 V617F mutation. A common 3′ primer and two 5′ primers are used in a multiplex reaction; one primer amplifies all species in this area of the JAK2 gene(upper band), whereas the other amplifies only species with the V617F nucleotide variant. Lane a is a molecular size ladder; lane b is empty; lanes c and f are positive patient samples; lanes d and e are negative patient samples; lane g is positive and sensitivity control with 2% JAK2 V617F in normal DNA; lane h is a normal patient; and lane i is a minus DNA control. Note: Size separations are shown on gels; capillary electrophoresis is an alternate analysis method.
The sensitivity of PCR assays typically is no more than 85% to 95% of those achieved with Southern blot.   Hematolymphoid neoplasms negative by PCR arise, presumably because PCR primers do not bind equally well to all V or J region gene segments that may participate in clonal rearrangement. The analytic sensitivity of PCR assays usually is modestly better and, in some specialized applications, markedly better than Southern assays. Samples can be analyzed rapidly if desired, and the low levels of DNA required for testing expand the range of evaluable samples, including, importantly, paraffin-embedded specimens. Immunoglobulin and T-cell receptor genes typically are tested for suspected B and T lymphoid proliferations, respectively. Immunoglobulin heavy chain gene-negative cases may show rearrangement of light chain genes or kappa-deleting elements.  
Low-level “clones” detected by PCR and Southern blot assays must be interpreted cautiously. These may represent low levels of neoplastic disease, but they also may be expansions of normal B or T cells responding to discrete antigens or other stimuli. The polyclonal ladder pattern seen in IgH PCR assays (see Fig. 19-3 ) is an example of this phenomenon. It is important to interpret low-predominance clonal populations in the broader context of a case. Conditions that enhance immune reactivity such as autoimmune disease may provide particular challenges. Important interpretive elements of clonality assays, and especially PCR assays, are given in Table 19-2 .
Table 19-2 -- Interpretive Cautions for Clinicians Ordering Hematolymphoid Clonality Assays
IgH, immunoglobulin heavy chain; PCR, polymerase chain reaction; RT, reverse transcriptase; TcR, T-cell receptor.
Molecular detection of a translocation confirms diagnosis and provides a sensitive marker for monitoring tumor burden. Molecular cytogenetic assays, such as FISH, usually are appropriate substitutes for DNA- or RNA-based assays to identify translocations in diagnostic samples. However, the ability of PCR methods to detect very low levels of tumor or minimal residual disease (MRD), makes molecular assays the standard of practice for monitoring disease ( Table 19-3 ; Fig. 19-4 ). Either blood or bone marrow usually can be followed; bone marrow may have slight advantages. Nested PCR/RT-PCR methods offer high detection sensitivity, but as endpoint assays cannot accurately determine levels of tumor. Most MRD assays employ real-time quantitative PCR or RT-PCR to follow the level of translocation relative to a reference transcript or gene found uni formly in tumor and normal cells as a quality indicator. Results often are provided as a log10 reduction or increase relative to a laboratory baseline for patients with full-blown disease.   Any value that increases or decreases by 1 log (less in some laboratories) over a prior sample is significant. Calibrators are being developed to standardize such assays among laboratories.
Table 19-3 -- Comparison of Methods for Minimal Residual Disease Assessment
FISH, fluorescence in site hybridization; PCR, polymerase chain reaction; RT, reverse transcriptase.
Figure 19-4 A, BCR/ABL1 transcript assessment by quantitative real-time RT-PCR. Numbers on the X axis indicate PCR amplification cycles. This panel shows amplification (the PCR cycle number at the rising curve crosses a threshold) for samples with high, 22–27 intermediate, 30–31 and lower 35–38 levels of BCR/ABL1 major breakpoint transcript. B, Internal housekeeping transcript (GUS, beta glucuronidase) level in samples. Note that most are similar over a three-cycle range. One sample (arrow) is partially degraded. The ratio ofBCR/ABL1 to GUS transcript levels versus known standards is used as a relative quantitative measure of how much BCR/ABL1 is present, and the logarithm reported of the fold reduction versus a baseline for patients with untreated CML.
High-sensitivity PCR testing is not suitable to screen for disease. Low level false-positive results for BCL2/IgH and BCR/ABL translocations have been described in normal individuals and raise cautions for interpreting minimal residual disease.   Such results presumably indicate small populations of cells with a translocation that have not progressed through additional steps necessary to manifest neoplasia.
Assays in Bone Marrow Transplantation
The human leukocyte antigen (HLA) histocompatibility locus on human chromosome 6 is an exceptionally diverse region with well over 1000 alleles identified.   HLA matching of donors and recipients for bone marrow transplantation at class I and class II loci is a complex and important molecular diagnostic application in clinical oncology. Millions of potential donors have been pretyped through programs such as the National Marrow Donor Program in the United States. Molecularly discrete alleles that share the same serologic (or antigenic) activity have been identified, and it is clear that high-resolution molecular typing to match donors and recipients offers benefits over low-resolution serologic typing for graft survival and graft-versus-host disease. The best results with high-resolution typing are seen in those patients with the fewest class I or class II mismatches. High-resolution DNA-based HLA typing is performed first with sequence-specific PCR primers or oligonucleotide probes, often followed by DNA sequencing when allele families are identified.
STR markers are used to assess engraftment and potential mixed chimerism following bone marrow transplantation.   These identity markers permit unambiguous determination of donor and recipient alleles and low levels of mixed chimerism down to a few percentage points. This information is useful in making decisions for pancytopenic patients regarding levels of immunosuppressive therapy as well as treatment for infection or recurrent disease.
Applications that target specific acquired mutations of selected genes are increasing in number. The JAK2 V617F variant, found in half or more of patients with chronic myeloproliferative disorders (CMPD), and rarely in other neoplastic myeloid disorders, has dramatically altered diagnostic approaches to patients suspected of CMPD. It can be detected readily in blood; bone marrow specimens may offer increased sensitivity. Heterozygous versus homozygous status has not yet been convincingly shown to be clinically significant, but patients with polycythemia vera appear to have homozygous mutations more frequently. Should V617F become a therapeutic target, quantitative assays for MRD assessment will likely follow.
Studies have revealed a range of interesting markers in patients with a normal karyotype who have AML. More than one of these markers may occur in the same clone, and prognostic studies should be assessed with that knowledge. Duplications—and, to a lesser extent, point mutations within the FLT3 gene are associated with a poorer prognosis  ; point mutations in selected regions of theNPM1 gene are associated with either a normal or improved prognosis.    Expression levels of several other genes (CEBPA, MLL, BAALC, ERG) also have been reported to affect prognosis.      IgH gene variable region mutational status, perhaps combined with clinical stage, is among the most useful prognostic markers in chronic lymphocytic leukemia.   Patients with unmutated (<2% variation from consensus germline sequences) IgH genes in the chronic lymphocytic leukemia (CLL) clone do more poorly than those with mutated (>2% variation) genes.
Sequence analysis of the ABL kinase domain often is pursued to guide alternate therapies in patients who develop resistance to Gleevec.   RNA or protein-based assays that assess expression of drug resistance proteins such as multidrug resistance-associated P glycoprotein (PGP) or breast cancer resistance protein (BCRP) remain under investigation.  
Other Applications in Hematolymphoid Neoplasia
PCR is performed to look for viral agents associated with bone marrow failure or neoplasia—for example, parvovirus B19, human herpes virus types 6 and 8, human T-cell leukemia virus (HTLV), and Epstein-Barr virus (EBV). Quantitative levels of viruses are increasingly requested to distinguish between infection and rejection in immunocompromised patients following bone marrow transplantation. Some reports suggest that quantitative PCR assessment of EBV viral DNA levels may be a useful indicator to follow for possible emergence of post-transplant lymphoproliferative disease following solid organ transplantation. Southern blot assessment of clonal or polyclonal integration of EBV viral DNA also may be useful in assessing potential cases of cases post-transplant lymphoproliferative disease for neoplastic risk. Microarray-based expression profiling applications are discussed in a following section.
Although useful applications of nucleic acid-based markers for diagnosis and management of patients with solid tumors have lagged behind their use for hematolymphoid neoplasia, there is recent significant growth. A range of molecular abnormalities can be detected, including chromosomal translocations, point mutations, allelic loss, gene amplification, microsatellite instability, epigenetic abnormalities such as methylation, and altered expression of single or multiple genes. Translocation, deletion, and amplification often can be detected by FISH as well as PCR or Southern blot methods for tumors with molecular abnormalities detectable by cytogenetic or molecular methods (see Table 1-6 in Chapter 18 , Conventional and Molecular Cytogenetics of Neoplasia). A rich array of molecular diagnostic applications relating to sarcomas is discussed separately (see Chapter 97 ).
Hereditary Cancer Syndromes
Molecular genetic testing is important for a range of autosomal dominant familial cancer syndromes in patients with appropriate family history and/or clinical phenotypes. Testing usually is done on adults, but it may include children. For common cancers, the nature of family history is crucial to decide whether testing a single gene makes sense. Patients undergoing gene testing should receive pre- and post-test counseling and give written informed consent; they also should consider consulting a genetic counselor or geneticist.
Hereditary cancers with mendelian inheritance are a small subset (<5%) of solid tumors, but the detection of a disease-causing mutation is enormously beneficial to these patients and family members. Major syndromes include hereditary breast and ovarian cancer (BRCA1, BRCA2, TP53, and CHEK2 genes), hereditary colorectal cancer with polyposis (APC gene), and hereditary nonpolyposis colorectal carcinoma (MSH2, MLH1 and other genes). Familial cases testing negative for mutations likely arise from undetected mutations (discussed in the following paragraphs) or complex inheritance due to abnormalities in several genes impacted by hormonal or environmental factors.
Hereditary cancers present a testing challenge because typically many mutations are spread over large areas of causative genes. This necessitates sequencing many or all exons to include exon–intron junctions or performing “mutation scanning” assays followed by sequencing regions with abnormalities. Scanning techniques such as single-strand conformation polymorphism (SSCP) analysis and denaturing high pressure liquid chromatography (DHPLC) reveal subtle physical differences caused by nucleotide changes and typically detect more than 95% of simple mutations. Guidelines exist for the functional interpretation of sequence variants, which is crucial for counseling patients and family members. Previously described mutations and neutral polymorphisms (i.e., nucleotide changes notassociated with disease) often are available in disease-specific databases. Previously undescribed sequence variants that produce prematurely truncated proteins via nonsense, frameshift, and splice site mutations are likely to cause disease. New missense mutations that change single amino acids have uncertain functional consequences and often are reported as “variant of uncertain significance (VUS).” Segregation studies of a variant among family members may be helpful, and conversion analysis, a technology that separates and allows study in isolation of mutant patient alleles, facilitates functional resolution for many alleles that fall into the VUS category. Major structural changes such as partial loss or duplication within a gene can be the basis for mutation in 15% to 20% of cases in hereditary cancers such as breast cancer due to BRCA1. Sequencing and screening methods rarely detect major structural changes, because no unique DNA nucleotide changes result. Newer methods such as MLPA permit comprehensive screening for duplications and deletions focused on changes in copy number of exons.    
Many other hereditary cancer syndromes can be sought clinically or on a research basis. A selected group with genes that can be analyzed focusing on fewer than 10 exons includes MEN-2; type 2 multiple endocrine neoplasia (RET)  ; hereditary paraganglioma/pheochromocytoma (SDHD, SDHB, SDHC)  ; Li-Fraumeni syndrome (TP53); von Hippel-Lindau syndrome (VHL)  ; type 1 multiple endocrine neoplasia (MEN1)   ; and Cowden syndrome (PTEN).  
Allele Imbalance and/or Copy Number Variation
The development of solid tumors commonly is accompanied by genetic instability leading to loss and, less commonly, amplification of small or larger regions of DNA from various chromosomes.  Such loss of heterozygosity (LOH) can be measured in two ways: (1) via PCR assays directed at microsatellite length polymorphisms that occur throughout genome, or (2) using single-nucleotide polymorphism (SNP) microarrays.   In the first, the ratio of different-sized alleles in the sample of interest can be compared to the ratio obtained from a normal (often adjacent) sample of the same individual. These ratios normally should be ∼1.0. Because of connective tissue and vascular and inflammatory cells within a tumor, a marker may not be lost entirely, and conservative ratios (e.g., <0.66 or >1.50) typically are used to determine whether there is LOH for a marker. In a SNP array, hybridization levels of labeled sample DNA are compared to normal patterns at thousands of known normal and variant genomic sequences represented on the array.
Useful clinical applications of LOH analysis include evaluation of oligodendrogliomas, where LOH for markers on the short arm of chromosome 1 and the long arm of chromosome 19 are both diagnostic and prognostic  ; to detect recurrence in bladder cancer; and to discriminate, by comparing LOH patterns, de novo second primary tumors from metastatic spread of a single primary tumor.Potential utility also has been demonstrated with cytologic bile duct brushing samples for pancreatic neoplasia, which can be combined with direct analysis for KRAS mutations.   Because STR analysis is a form of genetic identity testing, these sorts of sample screens also are useful to resolve the vexing question of whether a tissue “floater” from one specimen has somehow become included in the paraffin block of another. High-resolution SNP arrays produce complex data that must be correlated clinically but show promise for revealing patterns in neoplastic or preneoplastic lesions.  
In addition to full gene or targeted sequencing in hereditary cancer syndromes, specific somatic sequence alterations have been described in various tumor specimens. Given the variability among studies, well-designed clinical trials are essential to establish the utility of individual biomarkers or panels in diagnosis, prognosis, selection of therapy, and population screening.
One useful application not directed at a specific gene or mutation is microsatellite instability (MSI) analysis.   In some tumors, additional, often minor, bands of different size are noted in addition to the bands corresponding to germline alleles seen in normal cells. MSI bands reflect genetic instability leading to insertions or deletions of nucleotides within an STR or single nucleotide repeat sequence; the additional alleles are believed to result from defects in mismatch repair (MMR) systems under control of genes such as MSH2 and MLH1. MSI is useful as a screening assay for colon ( Fig. 19-5 ) or other tumor tissue from patients suspected of the HNPCC syndrome. A positive MSI result typically triggers immunohistochemical testing of MMR gene expression and, based on those results, direct sequencing to identify potential germline mutations. Whether MSI contributes to improved survival in colon cancer is controversial; disease-free but not overall survival may be improved. A small percentage (∼15%) of sporadic colorectal cancers also demonstrate MSI, and screening for the common BRAF mutation may allow their identification, thus saving workup for HNPCC.
Figure 19-5 Microsatellite instability (MSI) assessment of ascending colon carcinoma specimen. Capillary electrophoresis tracing shows normal (upper) and tumor (lower) analysis by STR (BAT26) analysis. The jagged peak in the upper tracing represents apparent homozygosity for a single allele. The jagged appearance arises from “stutter” during PCR amplification. Note the novel, smaller (left-shifted) allele in the tumor. This sample showed additional MSI using other markers; immunohistochemistry demonstrated MSH2 protein expression in normal colonic eithelium but not in tumor cells. (Courtesy of Antonia Sepulveda, MD.)
Mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene identified in 10% to 20% of North American, and a higher percentage of Asian, non-small-cell lung cancers initially were thought to correlate with higher response rates to the EGFR-targeted agents gefitinib and erlotinib, although a clear survival benefit has been difficult to demonstrate and results have varied among studies. EGFR mutations are more common in patients with adenocarcinomas, women, and individuals who have never smoked. Targeted sequencing of BRAF, RET/PTC and RAS genes for specific mutations may offer potentially useful information for prognosis and management in patients with papillary thyroid carcinoma, although the frequency of their detection appears to be method-dependent. Oncogenic mutations of KIT or platelet-derived growth factor receptor alpha (PDGFRa) are common and serve as useful therapeutic targets for tyrosine kinase inhibitors in gastrointestinal stromal tumors (GIST); as in CML, development of secondary mutations leads to resistance. Mutations in the p53 (TP53) gene, a prominent feature of the hereditary Li-Fraumeni syndrome, occur in somatic form in half of all human cancers, with lower levels in leukemia and higher levels in colorectal and ovarian cancer. A number of studies suggest a role for this central mediator of cell death in tumor behavior and response to treatment, but its interactions are complex, and diagnostic, prognostic, or therapeutic utility requires much study.  
The potential to look for mutations from desquamated adenoma or carcinoma cells in stool as a screening approach for colonic cancer has been entertained for some time. DNA seems to be stable for reasonable amounts of time as it traverses the colon and in collected stool. Studies targeting stool in large numbers of patients using multitarget assays that detect 15 to 21 mutations have performed better than fecal occult blood detection in detecting invasive cancers or adenomas with high-grade dysplasia; however, these assays still are not as good as colonoscopy.  
Extremely-high-throughput picoliter DNA sequencing platforms offer the potential to assess the full spectrum of simple nucleotide sequence mutations in cancers at very low levels and to follow changes over time.   Early studies indicate enormous breadth and complexity for investigators to probe and validate for clinical significance.
NEWER DIAGNOSTIC TECHNIQUES AND POTENTIAL APPLICATIONS
Another approach to detection of cancer-related alterations in a wide range of tumors is the study of cytosine methylation status in upstream CpG islands; these show promise as early screening markers for cancer in relatively noninvasively obtained specimens such as saliva, sputum, pleural fluid, pancreatic juice, or even peripheral blood.      Silencing/inactivation has been associated with hypermethylation of tumor suppressor genes, DNA repair genes, or proto-oncogenes believed to be causal or linked to mutational changes in the development of cancer. Evidence also has been presented that as neoplasia develops, hypermethylation affects different gene groups progressively. Methylation markers may be useful for tumor diagnosis or subclassification. The most extensively studied genes and tumors include MSH2, MLH1, and CDKN2A-p14 (colorectal, including patients with ulcerative colitis), GSTP1 and RASSF1A (prostate), and CDKN2A-p16 (lung, esophagus, and pancreas). A number of other hypermethylated genes also have been described in lung and other cancers. For some genes (KRAS, HRAS, MYC, FOS, BCL2) hypomethylated status has been associated with neoplasia. Methylation status in some tumors correlates with their response to therapy, and prognostic differences in leukemia or myeloma suggest possible therapeutic use of hypomethylating agents.
The pharmacogenetic paradigm for personalized medicine in cancer is thiopurine-S-methyl transferase, the homozygous absence of which in 1 of 300 individuals can lead to marrow aplasia following standard doses of 6-mercaptopurine or azathioprine in patients with acute leukemia or inflammatory bowel disease or post transplantation. An understanding of increasing numbers of common and rare gene polymorphisms associated with metabolism of anticancer drugs such as glucuronidation of irinotecan by UGT1A1   holds promise of developing a better dose schedule for effective treatment and fewer adverse reactions. Many pharmacogenetic relationships are complex, and clinicians understandably hesitate to use assays in the absence of validated algorithms. Genotyping by molecular methods offers distinct advantages, but may produce some false negative results.  
Microarray expression profiling for diagnosis, classification, and therapeutic selection in an assortment of leukemias and lymphomas has demonstrated impressive power in investigational studies.         Expression profiling also has been a powerful tool for biomarker discovery in the diagnosis and management of patients with solid tumors, and profiling studies of solid tumors have provided exciting results despite the seemingly greater challenge of analyzing samples with varying numbers of nonneoplastic vascular, stromal, and inflammatory cells. Initially dogged by reproducibility issues among laboratories, recent consortium studies indicate that commercially manufactured arrays are promising for clinical use.    The first cancer-directed microarray assay was approved by U.S. regulatory agencies in February 2007 for breast cancer assessment, and other applications are in the pipeline, including those for assessment of metastatic tumor of unknown origin, leukemia, lymphoma, and colorectal cancer. Although clinical trials data are lacking, arrays theoretically offer platforms to predict response and aid selection of individualized therapies. Because a modest number of targets (<100, or possibly even <10) may contain the bulk of useful information, targeted assays using quantitative RT-PCR or low-density-array platforms may be viable clinical platforms.       Prior studies and recent data suggest there may be overlapping or even distinct sets of genes that give comparable predictions. Some assays may be done on paraffin-embedded tumor samples, but if array-based assays become more prevalent, pathologists and surgeons accustomed to placing biopsy specimens in fixative for morphologic and immunohistochemical analysis may need to adapt, because RNA from fresh tissues is likely to provide the highest-quality results. The use of microdissection (manual and laser-capture aided) to enrich with relevant regions for analysis is likely to increase.
Cancer-Specific Detection at Low Levels in Blood, Bone Marrow, and Tissue
For some time, investigators have probed the utility of tumor-specific molecular markers to identify metastatic carcinoma cells in sentinel or other lymph nodes or circulating in the blood with the hope of using such assays to determine patient prognosis and, in some cases, management. Work has continued to detect cells by both molecular and nonmolecular methods.   Some groups have developed rapid PCR methods that would allow such markers to be used intraoperatively, but these applications have remained resistant to clinical application in the absence of prospective trials. Difficulties have included false-positive results due to a lack of specific expression or expression from pseudogenes. False-positive results also have been noted in normal control samples. Advances in assays performed on cell-free circulating nucleic acids offer alternate possibilities.
It has become increasingly clear that microRNAs (miRNAs), which are small RNA molecules of 19 to 24 nucleotides, play important roles in the regulation of normal gene expression. Accumulating evidence indicates that miRNAs, individually or in groups, act as tumor suppressors or oncogenes in hematolymphoid and solid tumors.    Assays that target or profile miRNAs are very likely to become available in the near future.
BUSINESS, REGULATORY, AND ETHICAL ISSUES IN MOLECULAR DIAGNOSTICS
Business and regulatory trends continue to affect molecular diagnostic testing in oncology. Oncologists probably will see package inserts recommending pharmacogenetic testing for increasing numbers of pharmaceuticals. As noted earlier in this chapter, interpretation of genotypes in the context of other medications and patient status probably will be complex.
Intellectual property positions on tumor markers, nucleic acid variations, techniques, and clinical applications are significant.   The BRCA1 and BRCA2 genes for hereditary breast and ovarian cancers are a well-known example. Patents to both genes are held by a single entity, and full gene analysis in the United States is restricted to a single laboratory. Exclusive licensing of patents also leads to single-provider situations. Conflicts have not yet emerged, but clini cians and laboratories remain concerned that panels of molecular markers in microarrays or other platforms may be delayed or difficult to offer because of multiple and conflicting patents.
There is continued government and public interest in oversight of molecular testing, particularly laboratory-developed tests. The Food and Drug Administration (FDA) has circulated revised guidelines for analyte-specific reagents, the basis for laboratory-developed tests. It appears that the FDA will require review and clearance of “special controls” for a new class of assays—in vitro diagnostic multi-analyte index assays—which process multiple laboratory results and, sometimes, clinical information to provide treatment recommendations via proprietary interpretive algorithms. Both developments may affect the types of complex assays available. Standards and calibrators should produce improved comparability of results among laboratories and methods. Beyond hereditary cancer syndromes, it is unclear whether establishing a genetic specialty area for laboratory accreditation, proposed by some, might affect molecular testing in oncology.
Payment for increasingly complex molecular assays continues to be a challenge. Reimbursement for molecular testing is governed largely by Current Procedural Terminology (CPT) codes, whose levels were undervalued initially and have gone largely unrevised for 15 years. Reimbursement levels typically are not sufficiently attractive to justify investment in oncology assay development at biotechnology companies, a situation that has prompted developers of complex assays to approach third-party payers directly to request payment under “miscellaneous” billing codes. Proposals have been advanced regarding improved payment for new technologies, but with political pressures for budget neutrality, the possibility of better reimbursement remains uncertain.
Over the next two decades, molecular diagnostic assays will become increasingly important tools for the determination of tumor diagnosis, prognosis, and residual presence, as well as initial and follow-up therapy. Targeted individual molecular markers probably will remain useful, and may even be predominant for the near future. However, validated panels of markers or expression profiling of tumors using microarrays or proteomic methods show potential to dramatically supplement or replace single markers in determining diagnosis, prognosis, and whether available therapies will be beneficial. New understandings of molecular pathogenesis in cancer will develop from continuing research with microarrays and proteomic methods, leading, we hope, to targeted therapies and opportunities to monitor treatments with molecular assays. Intellectual property and regulatory actions may limit the rate at which clinical applications are developed because of licensing and cost considerations. There will be increasing demands on physicians to ensure that patients understand the impact molecular tests have on decisions for their management, and, with a plethora of possibilities, to choose wisely for cost-effectiveness.
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