KeywordsSynovial Sarcoma Desmoplastic Small Round Cell Tumor Reverse Line Blot Assay Retinoic Acid Receptor Alpha Gene Preinvasive Cervical Lesion
Molecular pathology is the study of the molecular genetic causes of abnormal cell and tissue functioning with the goal of improved disease diagnosis and treatment. As a medical discipline, molecular pathology is a specialty training that incorporates the subject matter of genetics, inherited cancers, solid tumors, neoplastic hematopathology, infectious diseases, identity testing, HLA typing, and laboratory management and impacts both anatomic and clinical pathology practice.
Historical and Clinical Background
Pathology, the study of the origin, nature, and courses of diseases, has its foundation in the studies of Giovanni Battista Morgagni of Padua (1682–1771) (the “father of anatomic pathology”) and Rudolf Virchow of Berlin (1821–1902) (the “father of cellular pathology”). Dr. Morgagni documented the relationship between diseases and the gross changes observed in autopsy specimens, and Dr. Virchow established the correlation of cellular changes with disease having formulated the cellular theory of life, omnis cellula e cellula (all cells come from cells). Modern anatomic pathology retains gross and microscopic tissue examination as the basis of clinical diagnostics via standardized classifications; tumor specimens are described in terms of organ of origin (e.g., breast, colon, etc.), cell type (e.g., carcinoma, describing cells of epithelial type; adenocarcinoma, referring to a cancer of glandular origin), and tumor staging and tumor grade. The “TNM staging” characterizes solid tumors by the extent to which a tumor (T) has spread locally, the amount of regional lymph node (N) involvement, and whether or not there are metastases (M) to distant organs or lymph nodes. Tumor grade refers to the degree of cellular divergence from the normal condition. These criteria are increasingly supplemented by tests for additional biomarkers to improve disease diagnosis. The discoveries of molecular cell biology and the human genome project together with technological advancements resulting in methodologies that are reliable, rapid, and cost-effective have set the stage for molecular pathology as an essential and routine discipline for improved patient care. Potentially, molecular pathology will yield a comprehensive molecular classification of cancer improving early tumor diagnosis, resolution of equivocal diagnoses including tumors of uncertain grade or unknown organ of origin, tumor chemoresistance/drug therapy investigation, and individualized medicine. Numerous putative markers are reported in the scientific literature; the challenge is to identify those individual or sets of biomarkers that have high sensitivity, specificity, and predictive value (negative predictive value, positive predictive value) for vital clinical parameters. The following illustrates by way of current common techniques how molecular pathology can support cancer diagnosis.
Immunohistochemistry (IHC) allows by microscopic inspection the correlation of protein staining patterns with cytology or histology and is the most routinely used adjunct technique of anatomic pathology. Aberrant molecular genetic changes frequently result in an altered immunophenotype (nuclear, cytoplasmic, or membrane protein expression staining patterns). Typically, IHC involves application of a primary antibody to the antigen of investigation to tissue sections, followed by secondary antibodies labeled to allow chromogenic or fluorescent detection. IHC has been a routine part of pathology practice since the mid-1970s, often to identify cell or tissue origin. For example, the lineage of an undifferentiated tumor or the organ of origin of a metastasis can be identified after IHC with panel of antibodies for cytokeratins as the expression fingerprint of these cytoskeletal markers is not compromised by malignant transformation. IHC is also used to differentiate lymphomas and sarcomas. Panels of markers (e.g., CD5, CD23, CD43, cyclin D1, and bcl-6) are used to help distinguish different categories of lymphomas such as follicular, mantle cell, marginal zone, and chronic lymphocytic leukemias (leukemia diagnostics). Similarly, IHC markers including desmin, myogenin, CD99, CD43, and FLI-1 may be used to distinguish rhabdomyosarcomas, Ewing’s sarcoma, desmoplastic small round cell tumor (DSRCT), neuroblastoma, and lymphoblastic lymphoma.
“Genogenic” IHC supports the demonstration of genomic translocations that result in novel chimeric proteins; for example, ~90 % of Ewing sarcoma cases harbor a t(11;22)(q24;q12) translocation resulting in the EWS-FLI-1 chimeric product; overexpression of the FLI-1 protein can be used to distinguish Ewing’s sarcoma from histologically similar tumors. IHC can also be used as a marker of gene mutations that result in protein under- or overexpression; hereditary nonpolyposis colon cancer (HNPCC) is characterized by microsatellite instability (MSI) due to mutations in genes such as hMLH1 and hMSH2. These mutations translate as under-expression of MLH1 and MSH2 proteins detected by IHC.
Routinely, IHC markers are employed to assess tumor aggressiveness and the appropriate course of treatment; breast tumor diagnostics requires IHC for estrogen receptor (ER), progesterone receptor (PR), and HER-2 status. ER and PR positive tumors are generally less aggressive and are more responsive to hormone suppression treatments such as tamoxifen; HER-2 positive tumors tend to be aggressive but are candidates for treatment with herceptin.
IHC can also be used in the direct demonstration of oncogenic viruses. Detection of Epstein-Barr virus (EBV) latent membrane proteins (LMPs) and/or nuclear antigens can be used in the diagnosis of nasopharyngeal carcinoma, Burkitt lymphoma, and Hodgkin’s lymphoma. Detection of human herpesvirus 8 (HHV-8) may assist in the diagnosis of Kaposi sarcoma, and detection of the HPV L1 capsid protein of infective virions may be a marker of low-grade cervical lesions.
In Situ Hybridization
Fluorescent in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) allows the demonstration of DNA or RNA in tissue samples by hybridization with labeled nucleic acid probes or synthetic analogs; like IHC, in situ hybridization (ISH) supports the direct correlation of test data with specimen morphology. FISH techniques represent an efficient and easier option than classical metaphase cytogenetic techniques for the detection of chromosomal rearrangements; additionally, ISH supports interphase cytogenetic applications.
FISH is utilized in the diagnosis of hematological malignancies. For example, chronic myelogenous leukemia (CML) is characterized by the BCR-ABL reciprocal translocation involving fusion of the BCR region of chromosome 22 with the ABL region of chromosome 9. The fusion results in a shortened chromosome 22 (the Philadelphia chromosome). Predictable FISH signal patterns can be observed in interphase cells depending on whether the probes span or flank the translocation breakpoint. Other hematological diagnoses by FISH include acute promyelocytic leukemia (APL) (t(15;17)(q22;q12)) and follicular lymphoma (t(14;18)(q32;q21)). Soft tissue tumors such as synovial sarcomas can also be diagnosed by FISH; the t(X;18)(p11;q11) translocation is present in ~90 % of these tumors and results in the juxtaposition of the SYT gene (18q11) and the SSX gene (Xp11).
Other ISH applications include investigation of chromosome instability/aneusomy in tumors or cell lines using panels of centromeric probes. Locus-specific probes can be used to investigate the association of defined abnormalities with tissue morphology. Centromeric and locus-specific probes can also be used to examine intra-tumoral heterogeneity and to investigate the relationship between tumors and putative precursor lesions. Combining ISH with IHC allows observation of the correlation between nucleic acid detection and protein expression patterns. Specialized FISH techniques such as spectral karyotyping (SKY) or multiplex (M) FISH utilize chromosome paints specific for each of the 24 human chromosomes and detectable by fluorescent microscopy and computer imaging. Application of the paints to metaphase spreads from tumors allows the identification of chromosomal rearrangements with a resolution down to ∼2–3 Mb.
Polymerase Chain Reaction
Pathology specimens are frequently highly limiting with respect to the amounts of nucleic acids that can be recovered for diagnostic or research applications. The polymerase chain reaction (PCR) utilizes a thermostable DNA polymerase, DNA primers, and deoxyribonucleic acid building blocks to amplify a DNA template; theoretically, after 30 cycles of denaturation, annealing, and extension, the starting template DNA sequences are amplified one billion-fold. Consequently, PCR techniques have been at the vanguard of molecular pathology investigations. Using fluorescent-labeled primers, PCR can be adapted for real-time quantitative (Q) PCR to allow an estimation of relative DNA load or mRNA expression. By combining PCR with microdissection of specific tissues or cells off a slide, it is possible to correlate PCR data directly with tissue morphology.
PCR is used diagnostically to screen for gene mutations (by PCR product sequencing or by techniques such as single-strand conformation polymorphism (SSCP)). Mutation screening of all 19 hMLH1 exons, all 16 hMSH2 exons, or all 10 exons of the hMSH6 is possible in the diagnosis of HNPCC; MSI can be investigated directly by PCR for a panel of 5–12 standardized microsatellite loci that includes mono- and dinucleotide repeat sites (colon cancer). PCR mutation screening is also applicable in the diagnosis of other familial tumors such as the APC gene in familial adenomatous polyposis, which can lead to an autosomal dominant condition resulting in the development of myriad colon polyps, some of which may have the potential to progress to colon carcinoma. Mutation screening is also performed for BRCA1/BRCA2 germ line mutations and breast cancer risk.
Numerous candidate genes associated with sporadic tumors have been described; however, uncertainty about the relationship of mutations in these genes to tumor diagnosis and prognosis has stalled translation to routine screening tests. For example, mutations leading to aberrant expression of the tumor suppressor TP53 protein (“guardian of the genome”) are estimated to be present in up to 50 % of sporadic tumors. Despite the undoubted contribution of p53 mutations to tumor pathology, p53 mutation screening is of uncertain clinical utility.
PCR is used in the diagnosis of oncogenic viruses; in particular, several PCR strategies are available for detecting HPV and determining which of more than 40 HPV types associated with cervical lesions are present in a patient (cervical smear) sample. In the reverse line blot assay, labeled PCR product is hybridized to a blot spotted with HPV type-specific probes. Patients positive for high-risk HPV types may be at an increased risk for high-grade lesions/cancer. An elevated HPV DNA load as determined by Q-PCR may also correlate with increased tumor risk.
Reverse-transcriptase (RT) PCR converts RNA to DNA for PCR amplification. RT-PCR allows the detection of RNA fusion transcripts and is widely used in soft tissue tumor diagnostics. For example, alveolar rhabdomyosarcoma diagnosis is aided by the detection of mRNA transcripts created after the t(2:13)(q35;q14) reciprocal translocation that juxtaposes part of the PAX3 gene on chromosome 2 with part of the FKHR gene on chromosome 13. The API2/MALT1 RT-PCR test can be applied to stomach or other gastrointestinal tissues for the diagnosis of B-cell tumors of mucosa-associated lymphoid tissue (MALT lymphoma). This condition commonly develops against a background of chronic inflammation caused by Helicobacter pylori infection. Diagnosis of DSRCT can be aided by RT-PCR for transcripts from the t(11:22)(p13;q12) translocation associated with DSRCT and involving the EWS and WT1 genes. RT-PCR is also used in the diagnosis of Ewing’s sarcoma, synovial sarcomas, and BCL-ABL/CML. Q-RT-PCR is used in the diagnosis of APL to detect transcripts of the fusion of the retinoic acid receptor alpha gene (RARA) with the promyelocytic leukemia (PML) gene; monitoring the tumor burden may be helpful in predicting disease relapse. The PCR-based methods have greater sensitivity than their FISH counterparts for detecting tumor-specific rearrangements; nevertheless, “false-negative” diagnoses may occur because of variant translocation points not detected by PCR primer sets. RT-PCR also supports the assessment of HPV integration. The amplified papillomavirus oncogenic transcript (APOT) assay confirms integration by the detection of HPV transcripts that are contiguous with human sequence transcripts.
Improved techniques and new findings are a constant in molecular pathology. For example, application of a sophisticated high-throughput PCR mutation screening strategy of more than 13,000 genes in 11 breast and 11 colorectal cancers has shown that individual tumors typically accumulate ~90 mutant genes and that only a subset of these contribute to the neoplastic process; an average of 11 genes per tumor were mutated at significant frequency. Follow-up of such studies in course of time may reveal (sets of) mutations that have high utility as sporadic tumor diagnostic markers. Similarly, microarray cDNA technology and the findings of proteomics studies are expected to greatly increase and refine knowledge of the molecular basis of cancer, translating into effective clinical tests including improved IHC, ISH, or PCR, or the development of cost-effective microarray or proteomic tests for limited sets of biomarkers. Additionally, there remains much to be learned about genome regulation; for example, the significance of microRNAs in the control of gene expression and contribution to tumor biology has only recently been recognized, and there is increasing awareness of the importance of epigenetics in tumor etiology. Advances in the conceptual frameworks within which cancer is understood will also direct the interpretation of biomarkers. While oncogenes and tumor suppressor genes remain key concepts, there is a growing appreciation of cancer as a process involving a multi-interaction of cellular systems.