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Consultations in Molecular Diagnostics |
From the Division of Molecular Diagnostics, Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
From the Editor
The Journal of Molecular Diagnostics would like to announce the establishment of a new feature in the Journal, "Consultations in Molecular Diagnostics." This manuscript category will include short, case-directed articles meant to illustrate a classic principle, a difficult interpretation, or a new approach or application in molecular diagnostics. In keeping with the multifaceted mission of the Association for Molecular Pathology, we anticipate that this feature will be useful in education of our residents and fellows, technologists, medical and pathology colleagues, and ourselves. We welcome submissions for "Consultations in Molecular Diagnostics." Suitable topics are expected to span the spectrum of molecular diagnostics, including infectious disease, molecular studies of hematological and solid tumors, genetics, identity testing, and new technologies. Submissions will be peer reviewed and should include a case report, along with discussion of the molecular approach as it led to resolution of the case. Manuscript submissions from AMP members and nonmembers, technologists, and trainees are encouraged. Questions regarding "Consultations in Molecular Diagnostics" may be directed to the Senior Editor.
Karen L. Kaul Senior Editor
Patient History
A 19-year-old woman presented with a 3-week history of fatigue, easy bruisability, petechiae, and recent gingival bleeding. Past history was unremarkable. Several bruises were noted on her trunk and extremities; there was no organomegaly. Coagulation studies were consistent with disseminated intravascular coagulation (DIC): prothrombin time (PT) was 19.9 seconds (10.0–12.8), activated partial thromboplastin time (APTT) 50.2 seconds (24.4–33.2), and fibrinogen <50 mg/dl (50–300). Other laboratory results included: WBC, 1.8 x 109/l; Hgb, 10.8 g/dl; MCV, 99.5 fl; and platelets, 31,000/µl. The leukocyte differential showed 56% neutrophils, 2% bands, 3% blasts; 34% lymphocytes (rare atypical cells), 2% monocytes, and 3% eosinophils. The peripheral smear showed occasional macrocytes, rare poikilocytes, and diminished platelets.
The bone marrow aspirate and biopsy were normocellular with focal hypercellular areas. There were 25% blasts and 29% atypical promyelocytes, some with multiple Auer rods. Megakaryocytes were not seen; stainable iron was present. Cytochemical stains demonstrated blasts positive for myeloperoxidase and Sudan Black B. Flow cytometric studies on the marrow showed a large number of CD33, CD117, and myeloperoxidase-positive cells, which were negative for CD34 and HLA-DR, a phenotype consistent with, but not diagnostic for, acute promyelocytic leukemia (APL). Routine cytogenetics confirmed the diagnosis of APL (FAB M3); a translocation involving chromosomes 15 and 17, characteristic of APL,1 was identified.
Molecular Studies
Molecular analysis for the t(15;17) PML-RAR
breakpoint
was requested for follow-up study of residual disease. Figure
1depicts the two common breakpoint cluster regions described in the PML
gene on chromosome 15 and the consistent breakpoint region in the
RAR gene on chromosome 17.2
bcr1 refers to breakpoints
within PML intron 6; bcr3 to breakpoints within PML intron 3. A third,
less common, breakpoint region within exon 6 of PML (bcr2) has also
been described. All breakpoints fuse the PML gene to a consistent
breakpoint cluster region in intron 2 of the RAR gene on chromosome
17. This results in a t(15;17) PML-RAR fusion gene and a reciprocal
t(17;15) RAR-PML gene. Evidence suggests that t(15;17) PML-RAR is
the transforming moiety and is transcribed in APL. Alternatively
spliced products3
will be discussed below. Reports that
different breakpoints confer different prognoses have not been
consistent.4, 5, 6, 7, 8
|
, all breakpoint regions can be encompassed in a single
reverse transcription (RT)-PCR assay. Additional sensitivity may be
gained by a second round of PCR amplification using nested primers.
Chimeric mRNAs resulting from different breakpoints vary in size.
RT-PCR studies for the PML-RAR fusion transcript in the subject’s
bone marrow are shown in Figure 2
. Total cellular RNA was reverse-transcribed using MMLV reverse
transcriptase, followed by two separate rounds of PCR amplification for
35 cycles. The nested PCR product was separated by 2% agarose gel
electrophoresis.
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, lanes 1 and 2, are 
X174/HaeIII and
pBR322/MspI digests as molecular size markers. The upper
three X174 markers are 1353, 1078, and 872 bp, and the upper four
pBR322 markers are 622, 527, 404, and 307 bp. Lanes 3 and 4 are derived
from starting volumes of 5 and 1 µl of resuspended total cellular RNA
isolated from bone marrow of the subject. Positive controls in lanes
5–10 are serial 10-fold dilutions of RNA from an APL patient with a
bcr3 breakpoint diluted in normal peripheral blood RNA, beginning in
lane 5 at 1:100 positive to normal RNA. Lanes 11 and 12 are nested
RT-PCR reactions from normal peripheral blood RNA; as expected, no
product is seen. Lane 13 is a minus-RNA (water) sample carried through
the RT and nested PCR steps; lane 14 is a second aliquot of water
placed in the second (nested) PCR reaction. Both are intended as
controls for reagent contamination.
In lanes 3 and 4 (the patient’s sample), several bands in the 300–900
bp range are seen; two are fairly prominent and differ in size from the
350-bp band in the bcr3 breakpoint positive control. Identical
patterns were seen when the assay was repeated; RT-PCR studies using a
primer in PML exon 6 to specifically detect the bcr-1 breakpoint in PML
was also positive. The multiple bands in lanes 3 and 4 represent PCR
products from full-length and alternatively spliced fusion transcripts
of a bcr1 fusion PML-RAR gene in which one or more of exons 4, 5,
and 6 have been excluded from the spliced fusion
transcript.3
Discussion
Because several RT-PCR bands from the patient are less
than the full length expected for a bcr1 breakpoint, these are
potentially misinterpretable as nonspecific amplification products.
Interpretation of individual samples on the same run or samples on
different assay runs may be further complicated because transcripts can
amplify differentially at varying concentrations of input RNA (Figure 2
, lanes 3 and 4); note, however, that products at 450 bp, and
several others faintly, are present at both RNA concentrations
examined. Confirmation of bands as specific alternatively spliced
transcripts using Southern transfer and hybridization of
oligonucleotide probes is complicated by a variety of potential splice
patterns. With experience, alternative splicing is readily recognized
in a given assay and must be kept in mind when interpreting unexpected
products in any RT-PCR assay in which one or more additional exons
intervene between the exons containing the PCR oligonucleotide primers.
In part to remove this issue and minimize chances for a false-negative
result for t(15;17) analyses in our laboratory, we currently perform
separate amplification reactions using PML exon 3 and exon 6 primers
with a common exon 3 RAR primer. Bcr1 samples produce a single band
with the exon 6 primer and an alternatively spliced pattern with the
exon 3 primer.
Footnotes
Address reprint requests to Jeffrey A. Kant, Division of Molecular Diagnostics, Department of Pathology, University of Pittsburgh Medical Center, S701 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15213. E-mail: kantja{at}msx.upmc.edu
Accepted for publication May 24, 2001.
References
transcripts that encode aberrant PML proteins and PML/RAR isoforms in acute promyelocytic leukaemia. EMBO J 1992, 11:1397-1407[Medline]
isoforms and detection of minimal residual disease by retrotranscriptase/polymerase chain reaction to predict relapse. Blood 1993, 82:1264-1269 isoform and CD13 expression in patients treated with all-trans retinoic acid. Blood 1994, 84:3843-3849 break/fusion sites in PML exon 6: identification of a subgroup with decreased in vitro responsiveness to all-trans retinoic acid. Blood 1995, 86:1540-1547 isoform at presentation and detection of PML/RARa by reverse transcription polymerase chain reaction (RT-PCR) in remission of acute promyelocytic leukemia (APL). Blood 1994, 84:379a
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