Download Positive Immunoglobulin Gene Rearrangement Study by the

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Single Case Report
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Positive Immunoglobulin Gene
Rearrangement Study by the Polymerase
Chain Reaction in a Colonic
Adenocarcinoma
FRANKLIN C. LING, M.B.B.S., COLLEEN E. CLARKE, R.T.,
AND DAVID LILLICRAP, M.D., F.R.C.P.C.
The detection of clonal rearrangements of the immunoglobulin
heavy chain gene by the polymerase chain reaction provides a
rapid method to differentiate monoclonal from polyclonal Blymphocyte proliferations. It has been shown to be highly specific
and so far, no false-positive results have been described. A case
of a poorly differentiated colonic adenocarcinoma that showed
a "false positive" clonal immunoglobulin heavy chain gene rearrangement by the polymerase chain reaction technique is reported. DNA contamination was unlikely because of the strict
adherence to the laboratory polymerase chain reaction protocol
and also the repeated demonstration of the same amplified band
in a separate experiment using DNA extracted from another
piece of tumor tissue. The apparent monoclonal immunoglobulin
heavy chain gene rearrangement in the first polymerase chain
reaction may be related to a combination of the paucity of lymphoid cells in the tissue sample and the presence within this
small number of lymphocytes of a clonal reactive cell population.
It is, therefore, important to correlate the routine microscopic
and immunohistochemical findings in the interpretation of polymerase chain reaction results, especially when working with
nonlymphoid tumors and lymphocyte-poor lesions. (Key words:
Immunoglobulin heavy chain gene rearrangement; Polymerase
chain reaction; Adenocarcinoma; Clonal proliferation) Am J Clin
Pathol 1992; 98:116-119
The recent development of the polymerase chain reaction
From the Department of Pathology. Queen's University and Kingston
(PCR) to detect clonal rearrangements of the immunoGeneral Hospital, Kingston, Ontario, Canada.
Supported by the Clare Nelson Bequest Research Fund from the
Kingston General Hospital.
Dr. Lillicrap was supported by an Ontario Ministry of Health Career
Scientist Award.
Received August 28, 1991; received revised manuscript and accepted
for publication November 11, 1991.
Address reprint requests to Dr. Lillicrap: Department of Pathology,
Kingston General Hospital, Kingston, Ontario, Canada, K7L 2V7.
globulin heavy chain (IgH) gene has provided a rapid and
practical method to distinguish monoclonal from polyclonal B-cell proliferations.1,2 This method shows great
promise in routine diagnostic surgical pathology because
of its simplicity and short turnaround time, and because
it is relatively inexpensive compared to previous studies
using Southern blot analysis.
A.J.C.P.-July 1992
LING, CLARKE, AND LILLICRAP
117
IgH Gene Rearrangement Study i' PCR in Colonic Adenocarcinoma
The primary problem with the method is that monoclonality is only detected in about 80% of B-cell lymphoproliferative disorders.1,2 In a recent study performed at
our institution, only 19 of the 34 cases of B-cell nonHodgkin's lymphomas showed monoclonal rearrangements by the PCR technique (sensitivity rate, 56%). (Ling
F, Lillicrop D, unpublished observations). Possible reasons
for the failure of amplification in some cases include suboptimal priming by available consensus primers, mutation
or translocation involving the IgH gene, and the presence
of PCR inhibitors in the tissue.
The PCR technique is very specific for clonal B-cell
lymphoproliferative lesions and no false-positive reactions
have been reported. We describe a case of a poorly differentiated adenocarcinoma that showed a monoclonal
rearrangement of the IgH gene by PCR. The possible
causes for false-positive results and practical implications
of this result are discussed.
CASE REPORT
A 71-year-old man came to the Kingston General Hospital with a
right lower quadrant mass and weight loss. Computed tomographic scan
showed a large tumor in the cecum and the ascending colon with metastatic deposits in the liver. A palliative right hemicolectomy with ileotransverse anastomosis was performed. The entire surgical specimen was
immediately submitted to the laboratory. In addition to routine histologic
examination, fresh tissue also was taken from the tumor for PCR and
Southern blot analysis of IgH gene rearrangement as part of an ongoing
study of the sensitivity and specificity of these methods to detect clonal
B-cell proliferations.
MATERIALS AND METHODS
DNA Extraction
DNA was extracted from fresh tumor tissue by treatment with sodium dodecyl sulfate and Proteinase K followed by phenol chloroform extraction, as previously described.3 The extracted DNA was precipitated with
ethanol and redissolved in TRIS-EDTA buffer.
Southern Blot Analysis
The extracted DNA was digested with the restriction
endonucleases EcoRl, Hindlll, and BamHl, size fractionated by agarose gel electrophoresis, and transferred to a
nylon membrane (Gene Screen Plus, Dupont Canada Inc,
Lachine, Quebec) by the Southern blotting method.4 The
membrane was hybridized with an alpha - 32 P-dCTP-labeled probe from the J region of the IgH gene.5 After
washing to a stringency of 0.1 X standard saline citrate,
0.1% sodium dodecyl sulfate at 65 °C for 30 minutes, the
blot was subjected to autoradiography for 1 to 7 days.
Polymerase Chain Reaction
The PCR was performed according to the method of
Wan and colleagues6 with some modifications. The prim-
ers used were as follows. For the third framework portion of the V region: 5' ACACGGC(QT) (G/
QTGTATTACTGT 3' (Fr3A); for the J region: 5' TGAG
GAGACGGTGACC 3' (LJH); or 5' GTGACCAGGGTNCCTTGGCCCCAG 3' (VLJH). A nested PCR procedure was performed in a Perkin-Elmer Cetus Thermal
Cycler using Thermus aquaticus (Taq) polymerase (Perkin-Elmer Cetus, Norwalk, CT). The PCR reaction mixture (100 JUL) contained 0.2 jtmol/L of each primer, 5
units of Taq polymerase, 0.8 mmol/L of a dNTP mix, 3
mmol/L magnesium sulphate, 16.6 mmol/L ammonium
sulphate, 10 mmol/L beta-mercaptoethanol, and 67
mmol/L TRIS-HCl (pH 8.0). A first round of 25 PCR
cycles was performed using primers Fr3A and LJH. In
the second round of 20 PCR cycles, Fr3A and the internal
nested primer VLJH were used. Each PCR cycle consisted
of denaturation at 94 °C for 2 minutes, annealing at 60
°C for 2 minutes, and extension at 72 °C for 2 minutes.
The final amplified product was electrophoresed and visualized under ultraviolet light after staining with ethidium
bromide.
Special precautions were taken to avoid DNA crosscontamination and PCR carryover. Stringent laboratory
guidelines were followed during the entire DNA extraction
and PCR procedures, as suggested by Kwok and associates.7 These included extraction of DNA in a class II biological containment cabinet separate from our PCR area,
dedicated sets of supplies and positive-displacement pipetting devices for PCR, use of disposable gloves, and
inclusion of "no DNA template" tubes as negative controls in every run.
RESULTS
Histologic sections of the tumor showed a poorly differentiated adenocarcinoma extending through the muscularis propria into the serosal fat. There was only a mild
host inflammatory response that was present primarily at
the margins of the tumor and consisted predominently
of small lymphocytes. The tumor cells showed strong
positive staining for low-molecular-weight keratin and
negative staining for leukocyte common antigen.
Southern blot analysis showed a normal germline band
with no evidence of clonal IgH gene rearrangement. The
PCR, however, yielded a discrete amplified band (Fig. 1,
T l ) suggesting a clonal B-cell proliferation. Because the
PCR finding was completely unexpected for what had
now been identified morphologically as an adenocarcinoma, DNA was extracted from a separate piece of fresh
frozen tumor tissue and processed for a second PCR. The
result of the second PCR is also shown in Figure 1 (T2).
The amplified products from the second piece of tumor
tissue formed a diffuse band on the gel, but in addition
the same amplified band seen in the first PCR was
Vol. 98 • No. 1
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ANATOMIC PATHOLOGY
Single Case Report
T1 T2
L
126—• : " 1 ^
s*4
FIG. 1. Polymerase chain reaction analysis of IgH gene rearrangement.
Amplified DNA from the first and second PCR of the colonic tumour
are in tracks Tl and T2, respectively. Track P is marker DNA with
relevant size markers labelled in base pairs along the left hand side. Track
L is amplified DNA from a B-cell lymphoma. The track between P and
Tl is a negative control in which no DNA template was included in the
reaction.
still identifiable, although it was less intense than in the
initial PCR.
DISCUSSION
Immunoglobulin gene rearrangement analysis by the
PCR is very specific for B-cell lymphoproliferative disorders. Previous studies have shown no false-positive reactions. The case reported here showed a positive PCR
for IgH gene rearrangement although the morphologic
and immunohistochemical stains were diagnostic of adenocarcinoma. Furthermore, IgH gene rearrangement
study was negative on Southern blot analysis.
One of the primary problems with the PCR is contamination by DNA from extraneous sources. Because of the
exquisite sensitivity of this technique, even very low levels
of DNA contamination can result in erroneous test data.
The effect is especially problematic if contamination involves products of a completed PCR that provides high
concentrations of a specific DNA template.
DNA contamination is unlikely to have been the cause
of false-positive results in this case because of meticulous
specimen handling and strict adherence to laboratory PCR
protocol. In addition, there were no unexpected findings
in other PCR analyses at the time of this study. Furthermore, the finding of the same amplified band in a second
independent PCR using DNA extracted from another
piece of tumor tissue essentially precludes contamination
as the cause of false-positive results.
Nevertheless, there were significant differences between
the results of the first and the second PCR. Although the
same amplified band could still be recognized in the second PCR, it was much less distinct and was present with
a diffuse smear in the background. The difference in appearance of the amplified products in the first and second
PCR implies some important differences in the cellular
composition between the two tissue samples despite the
fact that they were both taken from the same tumor. Unfortunately, morphologic confirmation of this theory was
not possible in this case. Understanding the underlying
principle of PCR analysis of IgH gene rearrangement helps
to explain the observed differences and may suggest a possible cause for the false-positive reaction in this case.
In contrast to the use of the PCR to amplify a segment
of DNA unique to the neoplastic clone, such as t(14;18)
chromosome breakpoints in follicular lymphomas, 8 the
amplification of rearranged IgH genes is not specific for
the neoplastic population. The detection of monoclonality
by PCR is, in essence, a quantitative comparison of all
rearranged IgH gene segments to identify a predominant
rearrangement that will appear as a distinct band on electrophoresis. It follows that the specificity of the PCR in
predicting monoclonality will rely on the presence of a
sizeable and representative population of lymphoid cells.
If very few lymphoid cells are sampled and amplified, a
distinct "monoclonal" band may be produced, regardless
of whether the lymphocytic infiltrate is neoplastic or reactive. This "sampling artefact" will be more evident in
PCR than Southern blot analysis because of the inherent
sensitivity of the former procedure.
Adequate sampling of lymphoid cells is not a problem
in lesions rich in lymphocytes. However, it could be a
factor in lymphocyte-poor lesions, as in this colonic adenocarcinoma. It is possible that the first PCR reaction
was performed on tissue that contained sheets of carcinoma cells with very few lymphocytes and thus resulted
in a false-positive monoclonal reaction. The production
of a less distinct band against a stronger polyclonal background in the second PCR could be due to sampling of
more lymphoid cells in the second specimen, thereby
ameliorating the effects of the sampling artefact.
Another possible explanation for the false-positive PCR
was the presence of a clonal lymphocytic proliferation in
response to specific antigens on the carcinoma cells. Clonal
lymphoid rearrangements have been detected in a variety
of nonmalignant lymphoproliferative disorders.9 Although this might have been a contributing factor in this
case, it alone cannot explain the observed differences between the two PCR reactions and the negative Southern
blot result.
Immunoglobulin heavy gene rearrangement study by
the PCR technique is a new and powerful method to detect
clonal B-lymphocyte proliferations. Although it is highly
specific, false-positive reactions can still occur, as illustrated by this case. We have examined 19 other control
cases, including T-cell lymphomas (two), Hodgkin's disease (three), reactive lymphadenopathy (five), reactive
marrow lymphocytosis (seven), and metastatic carcinoma
(two), and none of them have shown a monoclonal rear-
A.J.C.P. • July 1992
SCHOFIELD, AGOSTINI, AND YUNIS
119
Microvillus Inclusion Disease
rangement on PCR (Ling F, Lillicrop D, unpublished observations). The case reported here is the only case of
false-positive reaction we have encountered and this is
also the only primary colonic carcinoma included in our
study. The authors believe that the most probable explanation for the positive PCR in this instance is related to
the paucity of lymphoid cells in the tissue samples. This
report reemphasizes the importance of correlating the tumor morphologic and immunohistochemical findings in
the interpretation of PCR results.
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4.
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6.
7.
8.
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2. McCarthy KP, Sloane JP, Wiedemann LM. Rapid method for dis-
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Arnold A, Cossman J, Bakhshi A, Jaffe ES, et al. Immunoglobulin
gene rearrangements as unique clonal markers in human lymphoid neoplasms. N Engl J Med 1983;309:1593-1599.
Wan JH, Trainor KJ, Brisco MJ, Morley AA. Monclonality in B
cell lymphoma detected in paraffin wax embedded sections using
the polymerase chain reaction. J Clin Pathol 1990;43:888-890.
Kwok S, Higuchi R. Avoiding false positives with PCR. Nature
1989;339:237-238.
Crescenzi M, Seto M, Herzig GP, Weiss PD, et al. Thermostable
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William CL, Griffith BB, Whittaker M. Molecular genetic approaches
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Med 1990;10:119-149.
Gastrointestinal Microvillus
Inclusion Disease
DEBORAH E. SCHOFIELD, M.D., ROCCO M . AGOSTINI, J R . , B.S.,
AND EDUARDO J. YUNIS, M.D.
A 3-year-old girl of Navajo heritage had intractable diarrhea
beginning at 4 days of age and resulting in long-term hyperalimentation. Investigation before multivisceral transplantation included biopsies of the rectum, stomach, duodenum, and liver.
The diagnosis of microvillus inclusion disease was established
by documentation of microvillus inclusions in duodenal epithelial
cells. A trial of somatostatin therapy was ineffective in controlling
the diarrhea. Subsequently, a multivisceral organ transplant
provided a unique opportunity to establish the gastrointestinal
extent of involvement of this disease. Ultrastructural microvillus
inclusions were identified in the duodenum, jejunum, ileum, and
colon, but not in the gallbladder. A few inclusions also were
documented in gastric antral epithelial cells. Alkaline phosphatase stains performed on paraffin-embedded material showed a
few inclusions in the antrum of the stomach and many inclusions
throughout the small intestine, primarily in surface epithelial
cells but also in upper crypt cells. (Key words: Villus atrophy;
Microvillus inclusion; Chronic intractable diarrhea of infancy;
Familial enteropathy; Intestinal alkaline phosphatase) Am J Clin
Pathol 1992; 98:119-124
Microvillus inclusion disease was first characterized in
1978,' although cases probably had been included in earlier reports of protracted diarrhea occurring in infancy.2'3
Intractable diarrhea typically begins early in infancy and
the prognosis is poor. Diagnosis is based on demonstration
of mucosal atrophy with loss of villi accompanied by ultrastructural demonstration of internalized inclusions of
From the Department of Pathology, Children's Hospital of Pittsburghmicrovilli within enterocyte cytoplasm. Familial cases
and the University of Pittsburgh, Pittsburgh, Pennsylvania.
have been reported and occur in a pattern consistent with
autosomal recessive inheritance. We are unaware of reReceived August 20, 1991; received revised manuscript and accepted
for publication November 11, 1991.
ports of ultrastructural findings in organs other than the
Address reprint requests to Dr. Schofield: Department of Pathology,
small intestine, colon, rectum, and kidney. When a 3The Children's Hospital, 300 Longwood Avenue, Boston, Massachusetts
year-old girl afflicted with this unrelenting disease under02115.
Vol. 98 • No. I