Basic Concepts of Molecular Pathology

October 11, 2008

By Allen, Timothy Craig Cagle, Philip T; Popper, Helmut H

This month’s issue of the Archives of Pathology & Laboratory Medicine includes a unique special section on “Molecular Signatures of Lung and Pleural Tumors” that represents the proceedings of a special Joint Symposium of the European Working Groups for Molecular Pathology and Pulmonary Pathology at the 21st European Congress of Pathology in Istanbul, Turkey, in September 2007. This symposium and the subsequent special section were organized by Dr Helmut Popper of the Institute of Pathology at the Medical University of Graz, Graz, Austria, president elect of the Austrian Society of Pathologists and immediate past president of the European Working Group for Pulmonary Pathology. In this brief review, we have provided some definitions of terms and concepts used in the proceedings of the “Molecular Signatures of Lung and Pleural Tumors” special section for those readers who are not already familiar with molecular pathology. Molecular pathology may have once been a specialized component of the research laboratory or the clinical laboratory, but today molecular diagnostic and prognostic techniques are in common use within the anatomic pathology laboratory, especially in the realm of infectious organism diagnosis and cancer diagnosis. Molecular testing continues to expand as more easily obtainable archival paraffin-embedded tissue replaces fresh and frozen tissues as the source of DNA and RNA needed for molecular analysis, and as newer technologies allow for more streamlined methods of testing. This review addresses the increased application of molecular testing in lung pathology, specifically how the current state of molecular pathology may be applied to practical, everyday lung pathology diagnosis.


Genes, made up of nucleic acids, contain the information necessary for the construction of proteins from amino acids within a cell. Genes code for proteins required for metabolic reactions and cellular structure. DNA makes up genes, and RNA transcribes the genetic code held within the DNA into proteins. The genetic code within the genes is composed of nucleic acids, for which nucleotides are the building blocks. Nucleotides, made up of a sugar-phosphate backbone with a nitrogenous base, are either purines-adenine (A) and guanine (G) in DNA and RNA- or pyrimidines-thymine (T) and cytosine (C) in DNA (uracil [U] replaces T in RNA). The nucleotides that make up the genes are arranged in a double-stranded righthanded helix. Nucleotides in DNA are arranged sequentially so that a gene will code for a matching protein. Within a double helix pattern, A, a purine, always binds with T, a pyrimidine, and G always binds with C, giving a nucleotide sequence for which 1 strand is a “mirror image” of the other strand.1-6

There are 46 chromosomes (23 pairs) in a human diploid cell, on which all genes are located. Chromosomes are paired, and as such a gene is found on a locus on each of the 2 paired chromosomes, giving 2 copies, or alleles, of genes. Gametes are haploid rather than diploid and therefore contain only 1 allele for each gene. Diploid status is reestablished when the nuclear material from an egg and sperm combine during fertilization.1-6

Transcription, the synthesis of messenger RNA (mRNA) from a DNA strand, is a key step in the formation of protein coded by DNA. During transcription, enzymes called topoisomerases break a DNA strand and allow the DNA double-helix to uncoil, giving 2 DNA strands, 1 of which is the template for mRNA, called the DNA template. Base pairs are matched with the DNA template to produce a mirror image of the DNA template, except with the substitution of U for T, forming a strand of mRNA. A series of 3 base pairs in a gene, called a codon, code for a specific amino acid, so that a series of codons code for a particular sequence of amino acids resulting in the synthesis of a specific protein. Translation, the assembly of the protein molecule from the mRNA template, occurs with the addition of amino acids in a particular sequence based on the specificity of the mRNA. Transfer RNA assists in translation.1-6

After translation, modifications to the newly formed protein occur in order for it to function, to move within the cell, or to fold properly. Methylation, acetylation, phosphorylation, glycosylation, posttranslational cleavage, and the addition of lipid groups are examples of posttranslational modifications. End regions of chromosomes are made up of telomeres, hundreds of repeats of the nucleotide sequence TTAGGG. Some of these telomere sequences are lost each time a cell divides, until they are lost and the cell can no longer divide. This process is called senescence. A polymerase called telomerase is able to replace the DNA sequences at the end regions, allowing for continuing cell division-a significant feature in some cancers. 1-6

The polypeptide chain formed by the specific amino acid sequence causes the newly formed protein to fold into a tertiary arrangement giving it a 3-dimensional structure. Often, the newly formed protein is inert until made functional by a posttranslational modification such as phosphorylation or proteolytic cleavage. Phosphorylation, the addition to the protein of a phosphate group catalyzed by enzymes called kinases, may cause, for example, translocation of the protein from the cytosol into the nucleus. Dephosphorylation is the removal of a phosphate group catalyzed by enzymes called phosphatases. Phosphorylation and dephosphorylation of proteins are often important in the activation and deactivation of cell cycle proteins, signaling pathway proteins, and transcription factor proteins.1-6

Protein degradation is necessary to remove damaged proteins and limit signaling proteins such as those involved in cell survival and cell death. This degradation often needs to proceed quickly. Reversible cross-linkage to a polypeptide, termed ubiquitin, leads to the rapid degradation of proteins and is called ubiquinylation or polyubiquinylation.7

The control of gene expression is for the most part controlled by regulation of transcription initiation. Proteins called transcription factors, also termed transactivators or trans-acting factors, bind to DNA and regulate RNA polymerase activity, affecting gene expression either by inducing or activating the gene or by inhibiting the gene by reducing transcription levels.8-13 Transcription factors are necessary for RNA polymerase to initiate transcription, and transcription factors called transcriptional activators stimulate transcription of an RNA molecule from its DNA template.14,15


A variety of techniques exist for molecular pathology diagnosis, including nucleic acid extraction, Southern blotting, restriction fragment length polymorphism, sequencing, liquid bead microarrays, mass spectrometry, and comparative genomic hybridization, among others. Nucleic acid extraction historically has used organic techniques using chloroform and phenol; however, automated nucleic acid extraction exists today and is frequently used to purify nucleic acids for their use with other molecular methods. For practical laboratory-based molecular diagnosis of lung disease, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) are 2 of the most important techniques commonly used.

Fluorescence In Situ Hybridization

In situ hybridization uses DNA or RNA probes to evaluate intact cells for genetic changes. Probes visualized with a chromogen that produces a colored chemical at the reaction site is called chromogenic in situ hybridization and probes using fluorescent labels are called FISH. Evaluation of genetic alterations within intact cells allows for the detection of genetic alterations occurring in a specific group of cells or within a small number of examined cells and is a major benefit of the use of in situ hybridization in anatomic pathology. It is commonly used with cytologic and surgical specimens to detect tumor cells and certain microorganisms, and with surgical specimens of tumors for its prognostic utility and to determine treatment response. 16-22 Chromogenic in situ hybridization uses a probe that can be seen as a chromogenic reaction under light microscopy, whereas FISH is available as probe sets and multiprobe FISH cocktails. Peripheral blood, urine, sputum, endoscopic brushings and washings, and paraffin-embedded, formalin-fixed tissue are all suitable for FISH. However, fixation of tissue with formalin for longer than 48 hours may yield poorer FISH results.23

DNA and RNA probes hybridize to a specific target sequence that is of interest, for example, genes implicated in a specific type of cancer or an inherited disease, or to a certain microorganism.16-22 Probes are made using DNA fragments cloned from yeast or bacterial artificial chromosomes and, with FISH, are directly or indirectly fluorophore labeled, most often using Texas Red, fluorescing red, and fluorescein isothiocyanate, fluorescing green.24 Directly labeled probes have a fluorophore-labeled nucleotide inserted into the probe, so binding of the probe to its target in 1 hybridization step is all that is required to visualize the probe. Indirectly labeled probes have a reporter molecule such as biotin or digoxigenin attached covalently, requiring the additional step of the application of a fluorophore-labeled avidin or fluorophore- labeled antidigoxigenin. The additional step required with indirectly labeled probes is a disadvantage; however, indirectly labeled probes generally allow for stronger signals because of greater signal amplification.24 There are 4 general types of probes: chromosome enumeration probes, locus-specific indicator probes, telomeric probes, and chromosome paints. Chromosome enumeration probes hybridize to repetitive DNA sequences located near chromosome centromeres, and because the loss of a centromere is generally indicative of the loss of an entire chromosome, they are used to enumerate the number of copies of a certain chromosome within a cell.25,26 Locus-specific probes are probes to unique sequences and are most frequently used to determine whether specific genes are amplified, translocated, or deleted. Telomeric probes hybridize to unique DNA sequences located very close to telomeres. The probes do not hybridize to telomeric sequences. Chromosomal paints are a mix of probes that probe to the entire length of 1 or more chromosomes. A FISH specimen must undergo prehybridization to allow a probe to efficiently hybridize to cellular DNA targeted by the probe while protecting the cell from morphologic disruption. Following prehybridization, the probe and cellular DNA are denatured so that the probe can hybridize to the cellular DNA it is targeting. Hybridization usually takes between 4 and 12 hours. A type of DNA called Cot DNA is added during hybridization to hybridize highly repetitive DNA sequences that are located within the genome so that the probe DNA does not nonspecifically bind these repetitive sequences and yield a multitude of nonspecific signals rather than the appropriately specific signal.24,25,27,28 The nonbound probe is then removed by washing, a nuclear counterstain that weakly fluoresces is added so that the nucleus can be identified, and an antifade is added to retard fluorophore photobleaching. The signal produced by the FISH fluorophore is then examined with fluorescence microscopy.

Polymerase Chain Reaction

Since its introduction in 1985,29 PCR has been refined to be an efficient and sensitive method of studying the molecular pathology of primary and metastatic neoplasms, inflammatory mechanisms, and infectious diseases.30-37 Today, automated instruments designed for the laboratory tabletop are available. Polymerase chain reaction amplifies DNA via a repeated 3-step process of denaturation, annealing, and extension. Double-stranded DNA that is the target is denatured at high temperature to yield 2 intact single strands of complementary DNA. Then at a lower temperature, specially designed single-stranded DNA primers anneal or bind to a specific targeted area of the single-stranded DNA. Because there are very large numbers of DNA primers relative to the full-length complementary DNA strand, the target DNA anneals with the primer DNA much more frequently than with the complementary DNA when cooling occurs. In the third step, extension, Taq polymerase identifies the now partially double-stranded DNA and extends the primers by polymerization, to yield, at the end of the first cycle, 2 doublestranded copies of a portion of the target DNA generated from 1 copy. Because the DNA primers only recognize the DNA for which they have been specifically designed, only that specific segment of DNA, making up a small part of the entire DNA present in the original DNA, is preferentially amplified. Subsequent cycles produce numerous shorter double-stranded DNA PCR products, so that more than a billion copies of the original double-stranded DNA are produced after 30 cycles, and more than a trillion are produced after 40 cycles. Following amplification, post-PCR analysis of the markedly increased amount of targeted DNA sequence product can be performed. The target sequence can be shown to be present in a specimen by the use of amplicons run on polyacrylamide or agarose electrophoresis, or via Southern blotting with probe hybridization, comparing the lengths of the targeted DNA sequence with DNA “ladder” markers. DNA sequencing can be performed on the amplicons, or studies to identify mutations may also be performed.38,39 Because PCR enormously amplifies DNA, great caution must be used in performing PCR to avoid cross-contamination of a specimen with even very small amounts of DNA.

Real-time PCR is becoming a more and more popular method of molecular pathology research and diagnosis that eliminates the need for post-PCR analysis and allows for relatively quick detection of DNA targets, including specific mutations.40-44

The best way to examine specific genes present in a certain cell type, such as in tumor cells, is to examine those cells’ mRNA. As RNA is not stable enough to work with easily in a laboratory, reverse transcription can be used to convert mRNA into its complementary DNA.With reverse transcription, mRNA is the template used for the production of a strand of DNA, opposite or reverse of typical cellular transcription. The complementary DNA can be used as the template for PCR in a process called reverse transcriptase-PCR (RT-PCR). Reverse transcriptase-PCR can be used to examine genes that are expressed, overexpressed, underexpressed, or not expressed in a specific cell type by the isolation of specific mRNA.44-47 Altered gene expression is a characteristic of malignant transformation, and those alterations allow for the identification of the presence of cancer cells via the detection of mRNA transcripts specific to those tumor cells. Tumor markers have been identified that are specific to solid organ cancers, and RT-PCR is highly sensitive in detecting differentially expressed tumor- related mRNAs. Some studies have indicated that RT-PCR can detect as few as 1 cancer cell in a million normal cells.44,48 Real-time quantitative RTPCR has become popular for detecting and quantifying RNA targets in a variety of cancers. It requires no post-PCR analysis and is efficient and automated. Several studies have used real-time quantitative RT-PCR to evaluate lymph nodes for micrometastases, including for non-small cell lung carcinoma (NSCLC), and to examine peripheral blood for potential dissemination of lung cancer cells during lobectomy.49-52


Normal human DNA contains 2 alleles for every genetic locus, the majority of which are identical, or homozygous, and the loss of 1 allele results in no pathologic change. Some genetic loci have 2 differing copies of alleles and are heterozygous. The majority of these heterozygous alleles allow for normal variance and do not result in pathologic changes; however, some of these heterozygous loci have the potential to cause pathologic genetic variations, with resultant disease. If there is a loss of the normal, or “wildtype,” allele at a locus, with resultant “loss of heterozygosity,” the remaining aberrant allele can cause cellular damage. At homozygous loci, the loss of an allele can occur and be followed by gene silencing or point mutation, causing loss of tumor suppressor genes.53,54 Detection of loss of heterozygosity using older methods such as Southern blot analysis and restriction fragment length polymorphism analysis are low-throughput, tedious, and inefficient; however, newer, high-throughput single nucleotide polymorphism arrays have allowed for more efficient examination of loss of heterozygosity.55-57

Cancers, including lung cancers, commonly exhibit loss of heterozygosity, causing the inactivation or silencing of genes critical for growth regulation and homeostasis. Cigarette smoking has been associated with loss of heterogeneity of sites on chromosome 3, and the association is greater in patients who began smoking at a young age.58-61 More than 90% of small cell carcinomas and more than 70% of NSCLCs contain loss of heterozygosity.57,62,63 Among NSCLCs, squamous cell carcinomas exhibit loss of heterozygosity in more than 90% of cases, compared with adenocarcinomas, showing loss of heterozygosity in approximately 70% of cases. In NSCLC, loss of heterozygosity generally involves genetic foci on chromosomes 1p, 3p, 8p, 9p, 13q, 17p, 19p, Xp, and Xq. In small cell carcinomas, loss of heterozygosity generally involves chromosomes 3p, 4q, 5q, 4q, 10q, 13q, 15q, and 17p.57,62- 66 Losses found in both small cell carcinomas and NSCLCs, involving chromosomes 3p, 13q, and 17p, are probably related to inactivation of critical tumor suppressor genes including retinoblastoma, p53, and fragile histidine triad (FHIT).57,62,63 Loss of heterozygosity in premalignant conditions and malignant diseases of the lung represents both early- and late-stage changes in the progression of disease; however, the continuum of losses makes it hard to evaluate the specific contribution of each loss. Loss of heterozygosity has also been identified in some benign lung diseases, including asthma and chronic obstructive pulmonary disease, probably reflecting the genetic predisposition identified in these diseases.67-70 Loss of heterozygosity has also been identified in cases of usual interstitial pneumonia (idiopathic pulmonary fibrosis) and suggests premalignant potential in those cases.71


Extracellular messenger molecules such as hormones, inflammatory cytokines, and growth factors, called ligands, bind to specific cell surface receptors and activate messengers within the cytosol leading eventually to activation of nuclear transcription factors that, due to the extracellular message, direct the transcription of a specific gene product, such as the transcription of a protein involved in cell growth. This cascade of events is termed signal transduction, and the series of steps within the cascade is termed signal transduction pathway or signaling pathway. Growth factor receptors are a common cell surface receptor, on which polypeptide growth factors such as epidermal growth factor are ligands attaching to those receptor protein-tyrosine kinases, activating the receptor and causing it to bind with intracellular proteins, which in turn continue the signaling pathway. Epidermal growth factor receptor is a member of the type I growth factor receptor tyrosine kinase family. Epidermal growth factor receptor has other ligands that bind to it other than epidermal growth factor, including transforming growth factor alpha, and these ligands, receptors, and signaling pathways play a central role in many lung cancers as well as some nonneoplastic pulmonary diseases.72-77 The extracellular ligands’ “messages” are transmitted via a signaling pathway. Cell differentiation and proliferation, cell survival, and cell death and apoptosis are regulated by signaling pathways, the majority of which “cross-talk” with other signaling pathways in a complex manner. Several important signaling pathways have been well studied. For example, the Wnt/B/catenin pathway, termed the canonical Wnt signaling pathway, involves Wnt binding to Frizzled cell surface receptors, which in turn activate Disheveled, causing the inhibition of protein kinase glycogen synthase kinase 3, which in turn releases dephosphorylated beta-catenin from the adenomatous polyposis coli- axin complex. beta-Catenin associates with T-cell factor/lymphoid enhancer-binding factor transcription factors causing the induction of Myc.78-84 Other important signaling pathways include the JAK/ STAT pathway, involving signal transducers and activators of transcription (STAT) proteins and Janus kinase (JAK) nonreceptor protein tyrosine kinases; the Ras/Raf-1/MAPK pathway, a significant pathway in carcinogenesis, including epithelial cell proliferation; the nuclear factor-kappaB transcription factor and nuclear factor- kappaB signaling pathways that regulate immune system proteins, cell survival and proliferation proteins, and apoptosis proteins; and the PI3K/Akt/mTOR pathway important in regulating cell survival; among many others.85-93


Errors in replication, extracellular influences such as UV light, radiation, and chemicals, and endogenous influences such as oxygen radicals routinely cause DNA damage, generally depurination, deamination, nonenzymatic methylation, and hydrolysis, sometimes with the attachment of a chemical group to DNA, the combination of which is termed an adduct. Several DNA repair pathways exist to excise the damaged DNA and replace it with newly synthesized DNA based on its undamaged complementary DNA strand. These DNA repair pathways are important in an individual’s susceptibility to lung cancer and response to lung cancer therapy. The base excision repair pathway repairs small isolated foci of DNA damage including reduced or oxidized single bases or fragments and small, nonbulky adducts. The nucleotide excision repair pathway repairs DNA damage involving both strands. The defects cannot be simply replaced because no matrix for the DNA segment is available. These defects can cause DNA helical structure deformity, such as cross-links, bulky chemical adducts, and pyrimidine dimers. The DNA damage response pathway, also termed the doublestrand break repair pathway, involves a cascade of events and repairs damage to double-stranded breaks. The direct damage reversal pathway repairs DNA damage caused by alkylating compounds in cigarette smoke.94-102


The cell cycle is a sequential series of very tightly regulated events governing cell proliferation, including entry into DNA replication, replication, entry into cell division, cell division, and cell rest. The cell cycle is divided into 5 phases: G0 (cell at rest), G1 (preparation for DNA synthesis), S (DNA synthesis or replication), G2 (integrity check of replicated DNA), and M (mitosis with nuclear and cellular division), which provide orderly control of DNA replication and cell division in response to external and internal stimuli. Cyclin-dependent kinases form complexes with proteins called cyclins that tightly regulate the progression of the series of steps in the cell cycle by activating and inactivating proteins by phosphorylation, including proteins that act as “brakes” on cell cycle progression and cell proliferation. The cell cycle is controlled by many interacting pathways and positive and negative feedback loops, and it may be stimulated appropriately or inappropriately in various inflammatory diseases. Cell cycle regulation loss is a very important step in uncontrolled cell proliferation during carcinogenesis.

Cell cycle checkpoints prevent damaged DNA to be passed on to daughter cells by temporarily arresting the cell cycle at specific steps. It allows damaged DNA to be repaired, or, if the damage is too severe, to send cells into apoptosis (programmed cell death). The primary checkpoint in the cell cycle is the restriction point in G1 where “commitment” to the cell cycle occurs. Other checkpoints include an S-phase checkpoint and a G2-M checkpoint.103-106 A complex of damage sensor proteins, the Rad9-Rad1-Hus1 heterotrimer complex and the Rad17-RFC complex, detect DNA damage at these checkpoints. After DNA damage is repaired, the DNA damage checkpoint is silenced and the cell cycle restarts.

Growth factor signaling initiates the cell cycle and maintains the transition through the G1 phase; however, once the cell passes through the cell cycle’s restriction point, it no longer requires growth factor signaling, and the cell is “committed” to the cell cycle. The retinoblastoma (Rb) gene product, pRb, governs progression past the restriction point of the cell cycle and governs the expression of genes involved in DNA synthesis. Progression of the cell cycle also depends on activation of cyclin D.107,108 One of the main functions of p53, the TP53 gene product, is to “protect” the DNA through the arrest of the cell cycle at checkpoints in response to DNA damage or to promote the induction of apoptosis when damage is too severe.109,110 As such, p53 has been referred to as the guardian of the genome. Both Rb and p53 have critical roles in the management of the cell cycle, and abnormalities of Rb and p53 are the most common abnormalities associated with the cell cycle dysregulation of malignancy; however, due to the large number of redundancies, feedback loops, and interacting pathways, many abnormalities can produce effects mimicking direct f Rb or p53 loss. The CDKN2A gene encodes for 2 unrelated protein products, p16INK4A, a cyclin-dependent kinase inhibitor, and p14ARF. Abnormalities of either of these can produce effects mimicking abnormalities of the Rb or p53 genes.111,112 Indeed, abnormalities of CDKN2A, p16, p14, and MDM2, as well as other genes and their products upstream or downstream of Rb and p53, can produce loss of cell cycle control mimicking the direct loss of Rb and p53.


Diagnostic and prognostic techniques using molecular technology are increasingly being used in anatomic pathology laboratories. Understanding genes responsible for proliferation, differentiation, and apoptosis, the basic tools used for evaluation of molecular changes, and factors involved in genetic susceptibility will help the pathologist better understand current and future molecular testing methodologies and their relevance to the diagnosis and therapy of lung diseases.


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Timothy Craig Allen, MD, JD; Philip T. Cagle, MD; Helmut H. Popper, MD

Accepted for publication June 17, 2008.

From the Department of Pathology, The University of Texas Health Science Center at Tyler (Dr Allen); the Department of Pulmonary Pathology, The Methodist Hospital, Houston, Tex (Dr Cagle); and the Institute of Pathology, Medical University of Graz, Graz, Austria (Dr Popper).

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Timothy Craig Allen, MD, JD, Department of Pathology, University of Texas Health Science Center at Tyler, 11937 US Hwy 271, Tyler, TX 75708-3154 (e-mail: timothy.allen@uthct.edu).

Copyright College of American Pathologists Oct 2008

(c) 2008 Archives of Pathology & Laboratory Medicine. Provided by ProQuest LLC. All rights Reserved.

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