Application of Molecular Techniques in Diagnostic Pathology
Moderators: Dr. Ricardo Lloyd and Dr. George Kontogeorgos
Section 2 -
In Situ Hybridization in Hematopathology
In situ hybridization (ISH) methods allow detection of nucleic acid sequences (DNA, RNA, host, or
foreign such as viral origin) in tissue sections or cell smear preparations. These powerful methods have
the distinct advantage of direct visualization of the target. These properties may increase the
specificity of the assay and may also provide insight into the biology of the disease. With regard to
specificity, knowledge of whether particular cells are expected to express a target can help in assay
development. With regard to biology, specific cell types within the tissue of interest can be identified
and examined for their staining properties. For example, detection of viral DNA or RNA sequences in
tissues will show which cell types are vulnerable to infection. This information would be lost in whole
tissue extract PCR methods. Another advantage is the ability to synthesize or clone probes against
almost any target. Thus detection of targets is not dependent on production of a suitable biologic
reagent with unpredictable properties as may be the case with antibody production for
ISH applications in hematopathology revolve around translocation detection, gene deletion, and
chromosome copy number for DNA. RNA applications are less common then DNA applications but may increase
in the future. We will briefly review some important steps in assay performance such as tissue
processing, cell conditioning, hybridization, and detection. We will then discuss some important uses of
ISH that have diagnostic, prognostic, or therapeutic importance. As metaphase applications usually fall
under the purview of cytogeneticists, we will focus primarily on interphase DNA ISH applications rather
than metaphase ISH.
DNA is relatively stable and a good target for ISH. Currently used crosslinking fixatives such as
formalin or metal containing fixatives such as acid zinc-formalin and B5 are acceptable.
Precipitative fixatives such as ethanol, methanol, or Carnoy's fixative are also suitable for
applications such as DNA fluorescent ISH (FISH). RNA is much less stable and this is a potential
drawback of ISH methods when RNA is the target. As anatomic pathologists, we primarily deal with fixed
tissues. Unfortunately, RNA is notoriously unstable and degrades quickly over time. Thus delay in
fixation should be avoided. Even delays of 5-10 minutes can result in substantial or complete loss of
low abundance targets. RNA is also sensitive to the presence of ubiquitous RNAases. Use of RNAase free
reagents, glassware, and instruments may be necessary for low abundance targets. Furthermore elevated
temperatures that are present even in routine tissue processing procedures (embedding in hot wax, heating
slides for tissue adherence) have a deleterious effect. Alternate tissue processing such as
microwave-assisted and ultrasound associated rapid fixation have been shown to result in superior
preservation of RNA with acceptable preservation of proteins and morphologic detail.
low temperature plastic or paraffin also has been shown to preserve RNA.
As with proteins, fixatives may also have marked effects on nucleic acid preservation. Neutral
buffered formalin does preserve RNA and DNA for ISH and from a practical standpoint is still the most
commonly used fixative. However, particularly for RNA preservation, it is not the most effective
fixative. Some commercially available alternate fixatives such as Prefer are unsuitable FISH
applications.  While standard neutral buffered formalin fixation certainly is adequate for
many common applications, few careful studies have been performed to understand optimal nucleic acid
preservation for RNA ISH that is applicable in a routine community-based surgical pathology laboratory.
In a study comparing multiple fixatives for RNA integrity and morphologic detail (including 70% formalin,
70% ethanol, Carnoy's, methacarn, Bouin's, and UMFIX) modified methacarn was the fixative of
choice.  Despite the inadequacies of formalin for optimally preserving RNA, the fact of the
matter is that formalin fixed tissues are commonly used diagnostically and archival tissues has been
shown to be suitable for ISH. 
ISH protocols can be divided into the following general steps: tissue pre-treatment, hybridization,
posthybridization washing, and probe detection. It is beyond the scope of this presentation to describe
detailed methods; however, general descriptions follow. The reader may refer to one of several good
methodologic reviews and websites of commercial probe vendors for details
(http://www.vysis.com/).  ISH can be done on whole cells (smears, cytospins, cell pellets)
or tissue sections (frozen or paraffin-embedded). Intact cells avoid problems such as analysis of
partial nuclei that occur in tissue sections; however, tissue section ISH allows architectural context to
be considered. Tissue sections must deparaffinized (if applicable), washed in buffer, and subjected to
cell conditioning. This occurs by controlled protease digestion (such as with proteinase K), which may
be combined with heat-pretreatment using microwave heating as is done for immunohistochemistry.
Prehybridization with hybridization buffer containing 50% formamide helps maintain stringency and is
followed by denaturation of probe and target using heat and formamide containing SSC (standard saline
citrate) buffer, depending on the particular application. Hybridization of the probe to the tissue takes
place at controlled temperature in a sealed chamber for varying times. Conditions important to the
hybridization include temperature, time of hybridization (30 minutes to overnight), salt concentration,
buffer pH, and probe type. Large probes (200-500 bp) are more sensitive in general than smaller
synthesized oligonucleotide probes (20-50 bp). RNA-RNA interactions using riboprobes may be more stable
(10-15 degrees C) than DNA-DNA or DNA-RNA interactions from DNA probes. Post hybridization washing in
successively higher stringency SSC-containing buffers to remove non-specific probe binding is then done.
Probe detection depends on design of the probe but can include directly labeled probes such as many
commercial FISH probes or chromogenic detection from secondary reagents in much the same way as
antibodies are detected in IHC. Amplification chemistries such as tyramide based systems can be used to
increase sensitivity. Radioactive methods have disappeared from clinical laboratories.
For detection of translocations, probe design strategies vary. Formats include single fusion, dual
fusion, and break-apart probes.  In single fusion formats, probes are designed near the
breakpoints and a single fusion signal results when a translocation is present. Greater sensitivity can
be gained with dual fusion probes in which the probes span the breakpoints and thus 2 fusion signals
result when the translocation is present. Break apart probes have different color probes on either side
of the breakpoint of a gene of interest. Normally, two fusion signals are seen (one from each allele).
When translocation occurs, the probes are split and individual signals are seen. The advantage of this
format is that a translocation can be detected in the gene of interest; however, the partner gene is
unknown. An example of where this might be useful is in the case of Burkitt lymphoma. CMYC translocation can involve IGH, kappa, or lambda
genes. A break apart format allows one to assess for CMYC translocation with a single assay; however, it
would not be known which immunoglobulin gene was involved. Interpretation of FISH signals depends on
probe format. Laboratory validation should be performed for each probe set to determine false positive
fusion signal rate and to gain experience with variant patterns. For probes used to detect copy number
changes such as deletion, cutoffs need to be determined and to understand false positive rates from
truncation artifact (examining a sectioned nucleus). 
III. Major Applications in Neoplastic Hematopathology
In routine diagnostic pathology, the major applications involve DNA detection, interrogating cells for
the presence of translocations or numerical abnormalities (gene/chromosomal gain or deletion). There are
relatively few RNA applications and most currently involve immunoglobulin light chain mRNA detection or
viral detection (EBV). We will briefly review current applications of ISH in myeloid and lymphoid
A. Chronic myeloproliferative disorders (CMPDs): Detection of the BCR-ABL1 fusion was one of their first major applications in hematopathology.
Detection of this translocation essentially defines chronic myelogenous leukemia (CML). It is present in
>95% of cases. Thus detection by FISH in blood or bone marrow confirms the diagnosis. The
translocation is also present in approximately 3-4% of precursor B ALL.  Interphase FISH is
useful to confirm primary diagnosis but it not useful in monitoring minimal residual disease, for which
QPCR methods have been shown to be predictive of outcome.  Other molecular abnormalities
are present in non-CML CMPDs such as JAK2 V617F mutation. However, ISH
methods are not generally useful in non-CML CMPD,other than to exclude the presence of BCR-ABL1.
B. Acute myeloid leukemias: With the WHO classification, demonstration of molecular abnormalities
became necessary for the correct diagnosis of AML. AML with recurrent cytogenetic abnormalities (AML
with AML1/ETO, CBFb /MYH11, PML/RARa and variants, and 11q23 abnormalities) required knowledge of the cytogenetic
result or specific molecular test designed to detect these abnormalities. All of these aforementioned
translocations or abnormalities can be detected by FISH methods using commercially available probes. The
first three abnormalities are associated with favorable prognosis while 11q23 AMLs have an intermediate
or poor prognosis.
Because of the urgency with which PML/RARa must be recognized, this is arguably the
most important translocation to demonstrate at diagnosis by molecular methods such as FISH, as opposed to
waiting for the results of standard cytogenetics. Interphase FISH testing in freshly isolated blood or
bone marrow cells can be done in 24-36 hours, giving clinicians confirmatory evidence of AML with PML/RAR a and allowing confident use of all trans
retinoic acid during induction chemotherapy. Likewise, failure to confirm the presence of this specific
abnormality or presence of a variant such as t(11;17)(q23;q21) PLZF/RAR a will stop unnecessary therapy with this agent.
C. Myelodysplastic syndrome (MDS): As opposed to AML in which specific translocations define some
subtypes, MDS is still classified largely by morphologic criteria – by presence of dysplastic morphology,
ringed sideroblasts, and blast percentages in blood and bone marrow. Cytogenetic abnormalities are
common, however, and have prognostic importance. The international prognostic scoring system (IPSS)
stratifies karyotypic abnormalities into good, intermediate, and poor risk (Table 1).  The
mere presence of an abnormal karyotype also has diagnostic importance in confirming the presence of a
clonal process. –7/del(7q), del(5q), del(20q), and –Y are among the most common abnormalities.
These can be detected by FISH with appropriate probes. Since FISH can yield results without
metaphases, the sensitivity of detecting abnormalities by FISH is potentially higher than standard
karyotyping. However, some studies have suggested that the additional yield is not large.
Some abnormalities such as trisomy 7 and +8 are missed by cytogenetics. 
The two methods should be considered complementary. Despite the commercial availability of "FISH MDS
Panels" it is still uncertain whether abnormalities detected by FISH but not by routine karyotyping have
the same prognostic relevance. Some data is becoming available suggesting the patients with cryptic
abnormalities do have a worse prognosis than FISH normal cases.  Although not routine,
many clinicians are now requesting FISH studies for abnormalities such as 5q-. This is being driven in
large part by the new therapeutic indication of lenalidomide in MDS with 5q-. 
Table 1: Cytogenetic Risk Groups of the International Prognostic Scoring
|Risk ||Abnormalities ||Median Survival|
|Good ||Del(5q) only, del(20q) only, -Y only, normal ||3.8 yrs|
|Intermediate ||+8, single miscellaneous, double abnormalities ||2.4 years|
|Poor ||Complex (>3 abnormalities), chromosome 7 abnormalities ||0.8 years|
A. B-cell non-Hodgkin lymphoma: Several non-Hodgkin lymphomas have characteristic translocations
that aid in diagnosis. BCL2/IGH fusion leads to overexpression of BCL2 and
is found in 80-90% of follicular lymphoma (FL). The forced expression of this anti-apoptotic protein
leads to prolonged cell survival and is important in lymphogenesis.  Because of
variability in breakpoints, PCR-based assays can not detect a significant minority of cases, particularly
in fixed tissues.  FISH assays span the different breakpoints and thus will detect nearly
all translocations and can be applied in fixed tissues (either disaggregated cells or in tissue
sections). IGH/CCND1 fusion is characteristic of mantle cell lymphoma
(MCL). It leads to overexpression of cyclin D1, plays a central role in pathogenesis of MCL, and is
found in greater than 95% of cases. FISH is a superior method than amplification-based assays for
reasons similar to IGH/BCL2. Extranodal marginal zone B-cell lymphomas of
mucosa associated lymphoid tissue type (MALT lymphomas) have several recurrent translocations.
Translocations such as API2/MALT1, IGH/MALT1, and IGH/BCL10 appear to be pathogenic and ultimately lead to activation of NFk B.
Presence of one of these characteristic translocations would support a diagnosis of
MALT lymphoma; however, the percentage of cases that harbor the translocations varies by anatomic site.
Besides being useful in confirming diagnosis, there is some clinical information to be gained since it
has been demonstrated that patients with API2/MALT1+ gastric MALT lymphoma
usually do not respond to anti-helicobacter therapy.
Burkitt lymphoma is essentially
defined by the presence of a CMYC translocation involving one of the
immunoglobulin genes, most commonly IGH. FISH testing for this is very
useful in confirming a diagnosis of Burkitt lymphoma (BL). It should be noted that CMYC translocations can be seen in non-Burkitt lymphomas (diffuse large B-cell
lymphoma) and can be a secondary event as opposed to the primary oncogenic event in BL.
B. In the area of T-cell lymphomas, 60-80% of anaplastic large cell lymphomas contain a translocation
harboring an ALK translocation (usually NPM/ALK, although other uncommon variants exist)
of the ALK protein tyrosine kinase likely plays an important pathogenic role in lymphomagenesis. ALK+
lymphomas appear to comprise a distinct group of ALCL with a favorable prognosis compared to ALK-negative
ALCL. It remains to be seen if these ALK- ALCL cases are truly a form of ALCL or whether they should be
considered a type of peripheral T-cell lymphoma, unspecified. FISH assays may be useful for diagnosis of
ALCL; however, it should be noted that IHC is an excellent surrogate for the translocation since it is
not expressed in normal lymphoid cells.
C. Chronic lymphocytic leukemia: Much has been learned about the molecular genetic abnormalities in
CLL in the past decade. With regard to chromosomal abnormalities, FISH played a central role in defining
these abnormalities and their clinical relevance. Because prior data on chromosomal abnormalities was
based on standard cytogenetics it was thought that trisomy 12 was the most common abnormality; however,
with the use of interphase FISH, del 13q has been shown to be the most common abnormality. In a landmark
paper, Dohner and colleagues described the frequency of chromosomal abnormalities by FISH in CLL and
importantly showed the association with outcome.  In descending order the abnormalities
included del 13q (55%), del 11q (18%), trisomy 12 (16%), del 17p (7%), and del 6q (6%). Some cases may
have more than one abnormality. Del 17p was associated with treatment resistance and particularly poor
outcome (median time to first treatment and survival of 9 and 32 months, respectively). FISH studies
have become important in the routine evaluation of CLL patients.
D. Multiple myeloma (MM): As with CLL, much has been learned about the genetic abnormalities in MM
in recent years. Both numerical abnormalities and translocations are common and have clinical
importance. Hyperdiploidy (48-75 chromosomes), often with multiple trisomies of odd chromosomes
(3,5,7,9,11,15,19,21) has been associated with good prognosis. It is seen in approximately 50% of
cases. Primary translocations of IGH with one of several partner genes
(FGFR3, Cyclin D3, Cyclin D1, c-MAF, and MAFB) are seen in 40% of MM. These are usually seen in the non-hyperdiploid
tumors. Patients with FGFR3 and c-MAF/mafB
translocations have poor prognosis. Likewise, deletion of chromosome 13 has been reported to have a poor
outcome independent of other factors.  Techniques of interphase FISH are being developed,
such as combined immunofluorescence and FISH, to specifically assess plasma cells in mixed samples such
as bone marrow aspirate smears.
Studies are beginning to validate the importance of
interphase FISH; however, the results are not straightforward. Schmidt-Wolf recently reported in a
series of 130 patients that IGH translocation and del 13q14 were associated
with adverse outcome. Dewald and colleagues reported that t(14;16), t(4;14), del 17p13 and monosomy 13
were associated with poor outcome by metaphase FISH. However, interphase FISH showed t(14;16) and
t(4;14) were associated with poor outcome but chromosome 13 anomalies were of intermediate prognosis.
While there is certainly information to be gained by FISH studies in MM, the role of
routine FISH in the diagnosis and prognosis of MM is yet to be defined.
E. Acute lymphoblastic leukemia (ALL): Translocation detection and cytogenetics are extremely
important in diagnosis and prognosis of ALL. Treatment risk groups are defined in large part due to
cytogenetic features. While FISH studies detect these abnormalities in ploidy and also translocations.
Cytogenetics remains the standard. One abnormality that deserves specific mention is the TEL-AML1 fusion. This occurs in approximately 25% of pediatric ALL (precursor B)
and cannot be reliably detected by standard cytogenetics. It has been associated with a favorable
prognosis in conventionally treated patients.
FISH is ideal for detecting this
translocation. FISH is also useful in cases that fail standard cytogenetics. As expected, interphase
FISH results in a much higher rate of abnormalities than cytogenetics alone. Once again, they are
complimentary techniques. 
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