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Short Communications |
From the Departments of Pathology,*
Oncology,
and
Surgery,
The Johns Hopkins Medical
Institutions, Baltimore, Maryland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unfortunately, searching primary tumor tissue genetically for deletions can be difficult, especially in a neoplasm-like pancreatic adenocarcinoma. Pancreatic adenocarcinomas evoke intense desmoplastic responses, and, as a result, most of the DNA contained in a tumor is actually non-neoplastic DNA. This intimate admixture of normal and neoplastic tissues can mask the loss of a DPC4 allele in a cancer. This problem can be overcome by expanding the tumor in culture or by xenografting the cancer into mice, but such research techniques are clearly not practical in most cases, and because they require fresh, sterile tissue, they cannot be done retrospectively.13
Immunohistochemical analysis can overcome the problem of low neoplastic cellularity, because it is performed in situ. Immunohistochemical analysis has three additional advantages over genetic analysis: 1) because tissue morphology is maintained, it allows a direct correlation between gene expression and morphology; 2) it is less labor intensive than molecular analyses, and, therefore, larger numbers of lesions can be examined; and 3) it can be applied to archived, formalin-fixed tissues processed in a routine fashion.
We therefore labeled 46 previously genetically well-characterized periampullary adenocarcinomas with an antibody to the DPC4 gene product. By comparing Western blotting or immunohistochemical labeling of the DPC4 gene product with molecular genetic analyses of these same cancers, we could determine the specificity and sensitivity of this assay for the DPC4 gene product. If immunohistochemistry for the Dpc4 protein were a sensitive and specific marker for DPC4 genetic inactivation, then immunohistochemical analyses would be valuable in both investigative and clinical settings.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Included in this study were 5 cancer cell lines and 41 cancer xenografts. The cancer lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Of the cell lines, two were pancreatic (BxPC3, PANC1), two were colorectal (HCT116, RKO), and one was mammary (MDA-MB-468) in origin. Of the xenografts, 36 were infiltrating ductal adenocarcinomas of the pancreas, two were adenocarcinomas arising in association with intraductal papillary mucinous neoplasms of the pancreas, two were adenocarcinomas of the distal common bile duct, and one was an (ampullary) adenocarcinoma of the region of the papilla of Vater. Each of these xenografted tumors was originally obtained from a patient undergoing either Whipple resection or distal pancreatectomy at The Johns Hopkins Hospital between May 4, 1992, and March 27, 1995, as previously described.13 The presence and sequence of exons 1–11 of the DPC4 gene in each of these 46 cases had been determined in a previous study.1,2
Clinical and pathological data for the xenografted cancers were obtained from patients’ medical records, The Johns Hopkins Oncology Center information system database, and The Johns Hopkins Hospital Surgical Pathology files. Clinical and pathological characteristics included were gender, race, age, family history of cancer, alcohol use, tobacco use, symptoms, comorbidities, tumor differentiation, tumor size, tumor location, presence of nodal metastases at surgery, K-ras gene status, and microsatellite instability. Symptoms included jaundice, abdominal pain, weight loss, nausea/vomiting, and fevers/chills. Comorbidities included myocardial infarction, hypertension, peripheral vascular disease, diabetes mellitus, chronic obstructive pulmonary disease, peptic ulcer disease, acute pancreatitis, chronic pancreatitis, and inflammatory bowel disease.
Western Blot Analysis
Total cellular protein was harvested in Laemmli buffer without ß-mercaptoethanol, and protein concentrations were determined by use of bicinchoninic acid reagents (Pierce, Rockford, IL). Fifty µg of total protein were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by monoclonal antibody against Dpc4 (clone B8; working dilution, 4 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA). Blots were then incubated with a horseradish peroxidase-conjugated secondary antibody (Pierce). Detection was afforded by SuperSignal substrates (Pierce).
Immunohistochemistry
Multiple hematoxylin and eosin (H&E)-stained slides from each of the 41 primary cancers corresponding to the xenografted tumors were screened by light microscopy for sections having both infiltrating adenocarcinoma and non-neoplastic pancreas. Unstained 5-µm sections were then cut from the paraffin block selected for each case and deparaffinized by routine techniques. The slides were treated with sodium citrate buffer (diluted to 1x from 10x heat-induced epitope retrieval buffer, Ventana-Bio Tek Solutions, Tucson, AZ) and then steamed for 20 minutes at 80°C. After cooling for 5 minutes, the slides were labeled with monoclonal antibody to Dpc4 (clone B8, Santa Cruz), using the Bio Tek-Mate 1000 automated stainer (Ventana). Each slide was labeled with a 1:100 dilution of the antibody. The anti-Dpc4 antibody was detected by adding biotinylated secondary antibodies, avidin-biotin complex, and 3,3'-diaminobenzidine. Sections were counterstained with hematoxylin.
Two of the authors of this study (R. E. W. and R. H. H.) independently evaluated the immunohistochemical labeling of the carcinomas. Each author was unaware of the DPC4 gene status of each case. The labeling in each case was scored as "diffusely positive," "focally positive," or "negative." "Diffusely positive" labeling was defined as strong and uniform expression of Dpc4 in the cytoplasm of cells, with focal expression of Dpc4 in nuclei. "Focally positive" carcinomas contained two distinct populations of cells, those that labeled with the antibody to Dpc4 and those that did not. Cases were regarded as "negative" only when no expression of Dpc4 was seen in the cytoplasmic or nuclear compartments of cells.
The interpretation of immunohistochemical labeling of the cancers was highly robust, with agreement between the observers in all cases but one (PX68). One author scored the adenocarcinoma in this case as "focally positive," whereas the other scored it as "negative." After re-examination of this case by both authors, this case was considered to be "focally positive."
Cases known to show either homozygous deletion of the DPC4 gene or a mutation combined with loss of heterozygosity in the DPC4 gene were expected to show no expression of Dpc4. Cases known to have at least one wild-type DPC4 gene were expected to show diffusely positive expression of Dpc4. When unexpected results were obtained, the immunohistochemical labeling was repeated with the same and/or new blocks. This included one DPC4 homozygously deleted case designated as "diffusely positive," two DPC4 wild-type cases designated as "focally positive," and one DPC4 wild-type case designated as "negative." In each of these four cases, repeat analysis confirmed the original immunohistochemical findings.
Normal pancreatic ducts, islets of Langerhans, acini, lymphocytes, and stromal fibroblasts, which all show moderate to strong expression of the DPC4 gene product, served as positive internal controls in each of the sections.
Genetic Reanalysis of Cases Showing Unexpected Results by Immunohistochemistry
Each of the four cases showing unexpected results by immunohistochemistry was rescreened for homozygous deletions of exons 1 to 11 of the DPC4 gene, as previously described.1-3 In addition, a newly identified exon (exon 0) of the gene was also examined (B Vogelstein and AB Sparks, unpublished data). Exon 0 had not yet been identified when the original homozygous deletion screening of the 46 cancers was performed. Primers used for amplification of exon 0 by polymerase chain reaction were 5'-ACCTTCTCCCCAGAGCTGTCG-3' and 5'-GAGCTCGGCGTAGAGTGGGCG-3'.
Statistical Analysis
Cross tabulations were analyzed with 
2or
Fisher exact tests when appropriate. Means were compared with the
Student’s t-test or the nonparametric Mann-Whitney rank sum
test. The latter was used to compare distributions when the assumption
of normality was not valid. All of these tests were two-tailed.
Survival times between groups were calculated using the method of
Kaplan and Meier14
and were compared using the log-rank
statistic. Tests were performed using Statistica for Windows (StatSoft,
Tulsa, OK).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The monoclonal antibody to Dpc4 was first tested by Western
blotting. Dpc4 is a 64-kd protein. As seen in Figure 1
, antibody to Dpc4 identifies a 64-kd
product in the lysates of the HCT116, PANC1, and RKO cell lines.
In contrast, no product is labeled within the lysates of
BxPC3 and MDA-MB-468. These results match the previously known genetic
characterizations of these cell lines.1
HCT116, PANC1, and
RKO all have wild-type DPC4 genes, whereas the
DPC4 gene is homozygously deleted in BxPC3 and
MDA-MB-468.1,3,9
Therefore, detection of Dpc4 by Western
blot using the B8 monoclonal antibody directly mirrored the gene
status.
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In all cases, normal acinar, ductular, stromal, and islet cells
were strongly labeled with the antibody (Figure 2)
. Both cytoplasmic and nuclear labeling
was seen.
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shows examples of
immunohistochemical labeling of the cancers with antibody to the Dpc4
protein. Of the 41 periampullary adenocarcinomas, 17 (41%) were
labeled diffusely positively; two (4.9%) were labeled focally
positively; and 22 (54%) were not labeled. Sixteen (94%) of the 17
adenocarcinomas that were labeled diffusely positively had at least one
wild-type DPC4 allele (Figure 3A)
. The remaining carcinoma
that was labeled diffusely positively in archival tissues had a
homozygously deleted DPC4 gene detected in xenografted
tumor. The two adenocarcinomas that were labeled focally positively had
at least one wild-type DPC4 allele on genetic analysis. Of
the 22 carcinomas that were not labeled with the antibody to the Dpc4
protein, 18 (82%) had homozygously deleted DPC4 genes
(Figure 3B)
, three (14%) had DPC4 genes exhibiting an
intragenic mutation of one allele and loss of heterozygosity in the
other, and one (4.5%) had a wild-type DPC4 allele. The
three mutations included two nonsense mutations, TAC to TAG (aa 412)
and AGA to TGA (aa 515), and one deletion causing a frameshift,
CAGAGCTCC to C (aa 516).
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. This
"cancerization of ducts" highlights the strong contrast
between the labeling seen in the cancers and normal tissues.
One of the two carcinomas that labeled focally positively for Dpc4 had
an interesting morphological pattern. The carcinoma from this case had
two distinct morphologies. One was that of a moderately differentiated
adenocarcinoma, and the other was a more poorly differentiated
carcinoma that did not form glands. Remarkably, the gland-forming
component was labeled with the anti-Dpc4 antibody, but the poorly
differentiated component was not (Figure 3D)
. The other carcinoma that
was labeled only focally for Dpc4 showed only the pattern of a
moderately differentiated adenocarcinoma.
Genetic Reanalysis of Tumors Showing Unexpected Results by Immunohistochemistry
Four carcinomas showed unexpected results by immunohistochemistry. These included one DPC4 homozygously deleted case designated as diffusely positive, two DPC4 wild-type cases designated as focally positive, and one DPC4 wild-type case designated as negative. Xenografts from each of these carcinomas were again screened for homozygous deletions, including a screen of a newly described DPC4 exon (exon 0) (B Vogelstein and AB Sparks, unpublished data).
One of the two carcinomas that originally were thought to have a
wild-type gene but that expressed Dpc4 only focally was found to
have a deletion in exon 0. The homozygous deletion was confirmed by
using duplex polymerase chain reaction with a positive internal
control primer set. Presumably the xenografted tissue for
molecular analysis which showed the exon 0 deletion came from the
poorly differentiated component of this neoplasm that did not label
with the Dpc4 antibody (Figure 3D)
.
The DPC4 gene status of the xenografts of the three other carcinomas (two wild type and one homozygously deleted) was verified with the repeat molecular assays. In particular, multiple parallel xenografts from one case were examined at the molecular level, and all parallel xenografts showed the same homozygous deletion, indicating origin of the homozygous deletion in the patient’s primary carcinoma rather than during the establishment of the xenografts.
Summary of Results
Including the newly discovered exon 0 homozygous deletion,
immunohistochemical labeling matched the genetic data in 38 (93%) of
the 41 cases. Of 18 cases with wild-type DPC4 genes, 17
(94%) were labeled diffusely or focally positively for the protein,
and 21 (91%) of 23 cases with inactivated DPC4 genes were
not labeled with the antibody. (See Table 1
for results.)
Thus, immunohistochemistry for the DPC4 gene product is a
highly sensitive (91%) and specific (94%) marker for genetic
alterations. Immunohistochemistry did not differentiate between a
mutant DPC4 allele combined with loss of heterozygosity and
a homozygously deleted DPC4 gene: Both types of cases showed
complete absence of Dpc4 labeling in the nucleus and the cytoplasm. We
did not have missense mutations available in our tumor panel, but if
missense mutations were to occur in regions necessary for nuclear
translocation of Dpc4, the mutations might result in intact cytoplasmic
but absent nuclear labeling.
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Clinical and Pathological Data
Clinical, pathological, and genetic data on the 41 xenografted primary periampullary adenocarcinomas were collected and analyzed. There were no significant differences in any clinical, pathological, or genetic characteristic between patients whose tumors expressed (focally or diffusely) or did not express the Dpc4 protein. These characteristics included gender, race, age, family history of cancer, alcohol use, tobacco use, symptoms, comorbidities (see Materials and Methods), tumor differentiation, tumor size, tumor location, presence of nodal metastases at surgery, K-ras gene status, and microsatellitein stability. In addition, although there was a definite trend that patients whose tumors were labeled positively for Dpc4 had longer survival (mean/median survivals of 17.5/16.5 months) than those whose tumors were not labeled (mean/median survivals of 14.9/9.0 months), this difference did not reach statistical significance in this group of 41 patients (P = 0.06).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The DPC4 gene produces a 64-kd protein that influences gene transcription and growth arrest.16-22 In fact, the Dpc4 protein has three distinguishable domains (for DNA binding, transcriptional activation, and nuclear translocation), and mutations in each these domains can lead to a loss of Dpc4 function.23 Dpc4 mediates a transforming growth factor-ß (TGF-ß)–induced signaling pathway.23-33 Although it is generally held that Dpc4 is required for TGF-ß–induced growth arrest, recent evidence shows that TGF-ß sometimes may act in a Dpc4-independent manner.34,35
The genetic analysis of an infiltrating cancer for DPC4 alterations can be greatly limited by low neoplastic cellularity in a tumor. Therefore, we undertook this study to determine whether immunohistochemical analysis for the DPC4 gene product could serve as a surrogate marker for DPC4 gene alterations. Immunohistochemical analysis is technically less cumbersome and more widely available than are genetic assays, and it provides a way to correlate specific morphological and genetic changes in a large number of cases. If sensitive and specific, immunohistochemical analysis for DPC4 gene expression could be used in both the clinical and investigative settings.
Immunohistochemistry for the DPC4 gene product indeed proved to be a highly specific and sensitive marker for DPC4 gene inactivation. Immunohistochemical labeling correctly identified the demonstrable DPC4 gene status in 38 of 41 cancers (93%). The three discordant cases included one with a wild-type DPC4 gene in the xenograft that did not express the Dpc4 protein in the primary carcinoma, one with a homozygously deleted DPC4 gene in the xenograft that labeled diffusely with the antibody in the primary carcinoma, and one with a homozygously deleted DPC4 gene in the xenograft that labeled focally with the antibody in the primary carcinoma. Another case with a wild-type DPC4 gene in the xenograft focally expressed the Dpc4 protein in the primary carcinoma, and the immunohistochemical labeling and genetic analysis were considered concordant in this case.
There may be explanations for these genetic-immunohistochemical mismatches. The case with the wild-type DPC4 gene that was negative for the Dpc4 protein may have involved inactivation of the DPC4 gene through mechanisms other than homozygous deletion or mutation coupled with loss of heterozygosity. For example, mutation in or methylation of the DPC4 promoter may turn off transcription of the gene.36 Alternatively, it is possible that there was a mutation in a portion of the gene that had not been examined. For example, exon 0 of DPC4 was discovered some years after the initial reports of DPC4 inactivation in pancreatic adenocarcinomas (B Vogelstein and AB Sparks, unpublished data).
The positive labeling seen in the cancers with homozygously deleted
DPC4 genes is more difficult to explain. One explanation for
positive labeling in these homozygously deleted cancers is
cross-reactivity of the anti-Dpc4 antibody with another protein. A more
intriguing explanation is that different areas of the neoplasms were
sampled for genetic and immunohistochemical analyses, and it is
possible that tumor heterogeneity, with DPC4 loss being a
late event in the portion of the tumor sampled for xenografting, may
account for this discrepancy. Indeed, one of the two carcinomas that
labeled only focally for Dpc4 had two distinct morphological patterns.
The better differentiated glandular component expressed Dpc4, whereas
the distinct, more poorly differentiated component did not (Figure 3D)
.
It is possible that this more poorly differentiated pattern represents
progression of the invasive cancer to a more aggressive phenotype,
evidenced by both histology and genetics, and that it was this portion
of the neoplasm that was sampled for xenografting. This progression
would be similar to that seen in adenomas and carcinomas of the
colon.37-40
It is also possible that two neighboring
sites of invasive cancer could form a "collision tumor."
Finally, this study includes the first description of a deletion in exon 0 of the DPC4 gene in a pancreatic cancer.
In summary, immunohistochemical labeling for the Dpc4 protein is an accurate and easy method to detect DPC4 gene alterations with high sensitivity and specificity. In addition, immunohistochemical labeling provides information not obtainable through genetic analysis. For example, because it is performed in situ, immunohistochemistry may provide evidence for the heterogeneity of Dpc4 expression in a given carcinoma.
Because of its simplicity and availability, immunohistochemical labeling for Dpc4 has direct clinical applications. For example, labeling for Dpc4 may help to distinguish trapped glands in chronic pancreatitis (which should express Dpc4) from infiltrating adenocarcinomas of the pancreas (slightly more than half of which will not express Dpc4). In addition, the immunohistochemical assay for Dpc4 may lead to answers in the investigative arena. For instance, the immunohistochemical study of pancreatic duct lesions, the putative precursors for pancreatic adenocarcinoma, may help determine the stage at which DPC4 inactivation contributes to neoplastic progression.
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Acknowledgements |
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Footnotes |
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Supported in part by the NIH Specialized Program of Research Excellence (SPORE) in gastrointestinal cancer grant CA62924, by NIH grant CA68228, by PHS grant CA67751–03, and by generous donations from the Helen S. Heller and Daniel Kim memorial funds for pancreatic cancer research.
Accepted for publication September 27, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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