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Molecular Pathology
Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)
Revised: 22 September 2012, last major update April 2012
Copyright: (c) 2008-2011, PathologyOutlines.com, Inc.
Table of contents - Molecular Pathology
DNA purification: introduction, analyzing purity, basic protocol, anion exchange chromatography, cesium chloride density gradient centrifugation, commercial DNA extraction machines, ethanol precipitation, organic extraction, PCR inhibitors, RNA purification, silica adsorption, tissue preparation
DNA sequencing: history, Maxam-Gilbert sequencing, Sanger sequencing, capillary electrophoresis, other innovations, real time, pyroseqencing, nanotechnology, Roche 454 FLX pyrosequencer, Illumina Genome Analyzer, HeliScope Sequencer
FISH: general, probes, protocol, probe patterns, images
Microarray: introduction, history, basics, considerations, errors
variations: antibody, bead based, cellular, CGH, solid phase, tissue (TMA)
PCR: definition, history, basics, Taq polymerase, reaction stages, thermocyclers applications
variations: general, methylation specific, multiplex, nested, real-time, reverse transcriptase
Fluorescent in situ Hybridization (FISH)
FISH-general - Molecular Pathology chapter
Used to visualize chromosomal deletions, amplifications, structural rearrangements and to identify whole chromosomes
Advantages: relative ease of sample preparation and analysis, high specificity, no requirement that the cells analyzed be activity dividing, can be performed on formalin-fixed, paraffin-embedded samples (allowing comparison of probe hybridization patterns to histology)
Applications: prenatal diagnosis and genetic counseling, oncology (especially chromosomal translocations), basic research, gene mapping, species identification of pathogens
Specimen types: solid tumors, bone marrow aspirates, peripheral blood (lymphocytes), amniotic fluid (amniocytes), skin (fibroblasts), chorionic villi
Uses interphase cells (non-cultured) or cultured cells in metaphase (culture + colcemid to block cells in metaphase)
History: FISH development followed development of cytogenetics
1882 - Walter Flemming published the first illustrations of human chromosomes
1956 - Identification of 46 human chromosomes
1959 - Identification of trisomy 21 in Down’s syndrome
1959 - Identification of XXY in Klinefelter's Syndrome (Nature 1959;183:302)
1959 - Identification of XO in Turner’s syndrome (Lancet 1959;1(7075):711)
1960 - Identification of Philadelphia Chromosome in chronic myelogenous leukemia (J Clin Invest 2007;117:2033)
1960’s - karyotype analysis now common, particularly with advent of dyes that reveal banding patterns specific to each chromosome pair
Later - radiolabeled probes used to bind to specific chromosomal sequences, leading to first molecular cytogenetic analyses of human tissue
FISH became common after development of fluorescent labeled nucleic acid probes
Currently 1 million+ cytogenetic analyses performed worldwide every year
FISH can be performed on tissue imprints, cytopreps, or bone marrow aspirate smears (J Clin Pathol 2005;58:629)
Ultrasound decalcification may allow more successful FISH, PCR and RT-PCR (AJSP 2006;30:892)
FISH can establish dizygotic origin of twin pregnancies if there are gender differences (Hum Path 1995;26:1175)
Sources for FISH test (advertisements): Propath
FISH probes - Molecular Pathology chapter
Like most assays involving nucleic acid binding and detection, the specificity of FISH depends on the nucleotide sequence of the probe and the stringency of the annealing and washing conditions used
Nucleic acid types
Double stranded DNA probes: more common; require denaturation prior to application and often reanneal to each other, lowering their effectiveness
Single stranded DNA probes
Oligodeoxyribonucleotide probes: usually short, up to 100 base pairs
RNA probes: usually single stranded complementary to target nucleic acid sequence; can be very specific but are easily degraded by mild alkaline conditions or Rnases, as are RNA target hybridization sequences
Detection types
Fluorescent probes: most common, includes rhodamine, Texas red and fluorescein; used in most current assays; useful because require no secondary detection reagents, can be stored for relatively long periods, have strong signals with fluorescent excitation
Enzymatic probes: includes digoxygenin-antidigoxygenin, biotin-streptavidin; works well but often has high background signals; analysis is limited by short half lives of enzymatic activities
Radioactive probes: used in early studies, includes 3H and 32P; cumbersome to use, take days-weeks to complete, require extra safety precautions and are limited by radioactive decay of probes
Target types
Bind specific sequences (or locus-specific FISH): bind one nucleotide sequence found at one chromosomal region or specific to a few diseases; also used to identify deletions (with another probe used as a positive control) or amplifications of specific gene sequences; examples - BCR/ABL fusion transcript due to t(9;22)(q43;q11.2) in CML
Bind unique sequences: bind unique sequences found on different chromosomes, such as centromeric and subtelomeric regions; used to identify chromosomal changes in malignancy, birth defects or developmental delay
Whole chromosome probes: collections of many probes, each specific for different regions of each chromosome and each labeled with a different fluorophore, which together gives each chromosome a unique color; used to locate translocations difficult to otherwise identify
Probe Length: ranges from short oligonucleotides of 100 base pairs to nucleic acid sequences over 1 million base pairs; probe must be large enough to bind specifically to target, but not so large that its size interferes with efficient hybridization; optimal probe size depends on its target sequence - specific unique target sequences require shorter probe sequences but large target sequences (chromosomal deletions over large areas) often need larger probes
Probe Sequence and Target Binding: based on sequence from human genome project, grown in bacteria containing plasmids with specific sequences
FISH protocol - Molecular Pathology chapter
Goal is to identify targets with high specificity, although research projects seeking related sequences intentionally use lower specificity
FISH employs three sequential steps: (a) sample fixation, (b) probe denaturation and hybridization, (c) several washing steps
Fixation: requires that sample is fixed to microscope slide so it does not wash off, but loose enough to allow probe-target hybridization
Probe denaturation and hybridization: usually marked excess of probe to target sequence is used to insure complete target binding; stringency of binding is manipulated using salt and detergent concentrations, temperature, and hybridization time; hybridization volumes are relatively low (10 to 20 ul), to minimize volume of expensive probes
Hybridization temperatures are typically 37° to 60° C in buffers containing 50% formamide and SSC buffer
Tissue samples are usually pretreated with proteases, appropriate nucleases, and other reagents to remove sample constituents that might interfere with FISH (examples - RNase pretreatment to remove RNA from hybridization to DNA, proteolysis of DNases or RNases to avoid probe degradation, DNase pretreatment to remove DNA from reactions hybridizing probe to sample RNA)
FISH Probe patterns - Molecular Pathology chapter
Following hybridization and washing, most probes are visualized by fluorescence when subjected to the proper excitation light wavelengths
Absence of signal: indicates deletion of the probe target (but need proper controls)
Extra signals: may indicate trisomy or other aneuploidy
Fusion signal: two different fluorophores in close proximity often emit a third color (example red and green together emit yellow)
examples: mantle cell lymphoma has diagnostic t(11;14)(q13;q32), which juxtaposes the 11q13 BCL1 gene locus next to the 14q32 immunoglobin sequence; if one locus is labeled green and the other red, a yellow signal indicates the translocation, but separate green and red signals means no translocation
Split Signal Fluorescence: differently labeled probes flank a region broken in a specific translocation; with red and green probes but no translocation, two yellow signals will be seen (unaffected chromosomes), but with the translocation, separate green and red signals will be seen
Sub-Deletion Signal Fluorescence: two differently labeled probes are used; red and green probes may be located flanking different possibly deleted areas; no deletion - 2 yellow signals; 1 deletion - one yellow signal and one red or green signal; examples include
Prader-Willi/Angelman, Cri-du-Chat, Williams, and Steroid Sulfatase Deficiency syndrome
FISH images - Molecular Pathology chapter
Breast cancer:
Breast carcinoma and HER2 amplification - IHC, CISH and FISH; FISH amplification #1; #2; #3; #4; IHC and FISH #1; #2; #3; not amplified-FISH; not amplified-CISH and FISH; HER2 and TOP2A (see Breast-malignant chapter)
Breast medullary carcinoma - FISH and IHC show HER2 amplification in high grade invasive ductal carcinoma (figures A/B), but not in medullary carcinoma (figures C/D)
Breast secretory carcinoma - t(12;15)
Breast cancer during pregnancy - FISH shows cells from male fetuses within tumor
Kidney tumors:
Congenital mesoblastic nephroma - cellular subtype has t(12;15)(p13;q25) translocation, results in ETV6-NTRK3 fusion gene, figures E and F
Renal papillary adenoma - trisomy 12
Leukemia/lymphoma:
Acute myelogenous leukemia with inv(16)(p13;q22) or t(16;16)(p13;q22) - H&E, cytogenetics and FISH; H&E, RT-PCR and FISH
Acute promyelocytic leukemia with t(15;17)(q22;q12) - skin infiltration-various H&E and FISH; negative and positive control of t(15;17)
Anaplastic large cell lymphoma - t(2;5)(p23;q35), fusion of ALK and NPM - rib tumor
Burkitt’s lymphoma - t(8;14)(q24;q32.3), fusion of c-myc and IgH or t(8;22)(q24;q11), fusion of c-myc and lambda light chain - figures
CLL / SLL - trisomy 12, FISH shows three green signals in 2 cells on right side; trisomy 12 (figures 5a, 6)
Mantle cell lymphoma - t(11;14)(q13;q32), fusion of BCL1/cyclin D1 and IgH - FISH; H&E, stains, FISH;
Mantle cell lymphoma - t(8;14)(q24;q32.3), fusion of c-myc and IgH (rare), figure 4A
Myeloid neoplasm associated with PDGFRB rearrangement - rearrangement of PDGFRB - #1; #2
Myeloid neoplasm associated with FGFR1 rearrangement - FISH assay of FIM (also called ZNF198) and FGFR1
PreB ALL with t(9;22)(q34;q11) - contributed by Dr. Julia Braza, Beth Israel Deaconess Medical Center, Boston, Massachusetts (USA) - bcr-abl FISH probe
FISH images - Molecular Pathology chapter (continued)
Soft tissue and bone tumors:
Chondroma - FISH with HMGA2 rearrangement
DFSP related sarcomas - COL1A1-PDGFB fusion gene - H&E and FISH
Infantile (congenital) fibrosarcoma - t(12;15)(p13;q25), fusion of ETV6 and NTRK3 - ideogram and FISH
Inflammatory myofibroblastic tumor - ALK staining, FISH and karyotype; FISH for ALK
Pigmented villonodular synovitis - trisomy 5 and 7
Synovial sarcoma - t(X;18)(p11.2;q11.2), fusion of SYT and SSX1 or SSX2 - FISH; FISH and karyotype; heart tumor - COBRA-FISH karyogram with t(X;18) as part of complex karyotype
Other tumors:
Glioblastoma (pediatric) - molecular changes - various
Glioblastoma multiforme - EGFR amplification by FISH
Intravenous leiomyomatosis - t(12;14)(q14-15;q23-24), fusion of HMGA2/HMGIC and various - intravenous leiomyomatosis
Lung carcinoma and Kras mutation - H&E and FISH of Kras mutation
Ovarian granulosa cell tumor - trisomy 12, FISH of ovarian granuloma cell tumor: trisomy 12, monosomy 17
Pleomorphic adenoma of salivary gland - t(3;8)(p21;q12), fusion of CTNNB1 and PLAG1 genes - karyotype, FISH and CISH
Other:
Tetraploidy in neonate who died shortly after death - FISH and karyotype
Presence of female cells in penile swabs after recent vaginal intercourse - image (Archives 2000;124:1080)
End of Molecular Pathology chapter
