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Antioxidative effects of sesamol and tocopherols at various concentrations in oils during microwave heating

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749
Molecular Identification of Metastatic Cancer to the
Skin Using Laser Capture Microdissection
A Case Report
Sara Milchgrub, M.D.1
Ignacio I. Wistuba, M.D.1,2
Bong K. Kim, M.D.3
Cynthia Rutherford, M.D.4
Jill Urban, M.D.1
Ponciano D. Cruz, Jr., M.D.3
Adi F. Gazdar, M.D.1,2
1
Department of Pathology, The University of Texas
Southwestern Medical Center, Dallas, Texas.
2
Hamon Center for Therapeutic Oncology Research, The University of Texas Southwestern
Medical Center, Dallas, Texas.
3
Department of Dermatology, The University of
Texas Southwestern Medical Center, Dallas,
Texas.
4
Division of Hematology and Oncology, Department of Internal Medicine, The University of Texas
Southwestern Medical Center, Dallas, Texas.
BACKGROUND. In the current study the authors report a 57-year-old woman with a
scalp tumor and cervical lymphadenopathy who had a previously resected duodenal carcinoid. Histologic and immunophenotypic characteristics of the duodenal carcinoid differed from those of the scalp and cervical lymph node tumors,
prompting the use of molecular methodologies to make the diagnosis.
METHODS. Paraffin embedded tissues from the duodenal carcinoid, scalp, and
lymph node tumors were dissected using microscopic visualization and laser
capture microdissection. DNA was extracted and polymerase chain reaction (PCR)
was performed to evaluate loss of heterozygosity and microsatellite alterations
using primers flanking 22 polymorphic microsatellite markers from 9 chromosomal regions, including genes associated with MEN-1 (11q), CDKN2 (9p), p53
(17p), and bronchial carcinoid (3p). Microdissected lymphocytes from the three
tissues were used as source of constitutional DNA (controls).
RESULTS. Fourteen of the 22 markers were informative (heterozygous in control
lymphocytes). A marker on 3p12 showed loss of the same parental allele in the
three tumors. A different marker on 3p14.2 showed an identical shifted band in the
three tumors indicative of a common microsatellite alteration.
CONCLUSIONS. The shared molecular abnormalities among the three tumors indicated a common clonal origin, leading to a diagnosis of primary duodenal carcinoid
with clear cell metastases to the scalp and cervical lymph nodes. These findings led to
radiation therapy and immunotherapy rather than chemotherapy. This case illustrates
the novel application of laser capture microdissection combined with PCR-based
analyses of genomic markers for the identification of the origin of metastatic disease.
Cancer 2000;88:749 –54. © 2000 American Cancer Society.
KEYWORDS: carcinoid, chromosome 3p, clear cell tumor, laser capture microdissection.
R
outine clinicopathologic workup often will reveal the primary
source of metastatic disease. However, in situations in which
metastasis is the only evidence of malignancy, the search for the
primary source may require collaboration across different specialties.
In this report, we present a case illustrating the novel use of laser
capture microdissection combined with polymerase chain reaction
(PCR)-based analysis of genomic markers to confirm the primary
origin of the malignancy.
Address for reprints: Sara Milchgrub, M.D., Department of Pathology, UT Southwestern Med. Ctr.,
5323 Harry Hines Blvd., Dalles, TX 75235-9073.
Received April 26, 1999; revision received October
18, 1999; accepted November 15, 1999.
© 2000 American Cancer Society
Case Report
A 57-year-old white woman presented with a rapidly expanding lesion
on the scalp that first was noted 8 months previously as a pea-sized
nodule. Her medical history was unremarkable except for an explor-
750
CANCER February 15, 2000 / Volume 88 / Number 4
FIGURE 1. (A) Frontal view of a disfiguring nodular plaque of the scalp extending to the forehead and ears. (B) Top
view showing telangiectasias, serous
crusting, and scarring alopecia.
atory laparotomy 4 years earlier for an upper gastrointestinal hemorrhage. The patient denied a history of
cancer, fever, loss of appetite, or weight loss. On examination, she had an erythematous nodular plaque
that spanned the frontal and temporal regions of the
scalp, with ill-defined margins extending into her forehead and ears (Fig. 1a). In certain portions, the plaque
was slightly yellow, telangiectatic, and boggy, with
serous crusting and loss of hair and hair follicles (Fig.
1b). Her anterior and posterior cervical lymph nodes
were enlarged, firm, and nontender. The rest of the
physical examination was unremarkable except for a
well healed abdominal scar from her previous surgery.
Laboratory values showed normal complete blood
count, liver enzymes, and renal function; plasma levels of cholesterol and triglycerides were moderately
elevated, and chest X-ray, plasma, and urine protein
electrophoresis were normal. At this point, the differential diagnosis included cutaneous lymphoma, a histocytic neoplasm, metastatic disease, and, less likely,
amelanotic malignant melanoma.
A skin biopsy was performed and hematoxylin
and eosin (H & E) staining of the specimen showed a
thick dermal infiltrate of pale-staining cells with
foamy cytoplasm arranged in nest-like aggregates with
a patchy infiltrate of lymphocytes and plasma cells
(Fig. 2C and D). Staining for periodic acid–Schiff (PAS)
with and without diastase was negative. An excisional
cervical lymph node biopsy also was performed and H
& E staining of the specimen showed obliteration of
normal architecture by sinusoidal expansion of similarly pale-staining cells traversed by vascularized fibrous tissue septa (Fig. 2E). These cells had abundant
vacuolated cytoplasm, large vesicular nuclei, and eosinophilic nucleoli (Fig. 2F). They also were PAS negative. Because of the clinical consideration of lym-
phoma,1 flow cytometry and T-cell receptor gene rearrangement analyses of skin and lymph node specimens were conducted; these studies did not document monoclonality. Because fresh tissue was not
available, ultrastructural studies were not performed.
At this point, the most likely diagnosis was metastatic
carcinoma containing clear cells.
Given the sustained rapid growth of the scalp
tumor, there was an impending need to begin treatment, the nature of which depended on the specific
diagnosis. Thus, the patient again was questioned and
past records from another medical center were requested for review. This process revealed that her
intestinal hemorrhage was due to a 1-cm duodenal
carcinoid that was excised locally without bowel resection. Her postoperative course was unremarkable;
annual evaluations including abdominal computed
tomography (CT) scans and urine 5-hydroxyindoleacetic acid levels were normal and she had been
asymptomatic until the scalp nodule developed. Archival tissue from the duodenal tumor showed replacement of the submucosa and muscularis propria
by large aggregated polygonal cells arranged in a trabecular pattern with an organoid architecture typical
of neuroendocrine tumors (Fig. 2A). The tumor cells
had finely granular eosinophilic cytoplasm, distinct
nucleoli, and stippled chromatin (Fig. 2B); focal aggregates showed cells with clear cytoplasm. Cellular pleomorphism was minimal.
Immunohistochemical stains were performed on
the duodenal, scalp, and cervical lymph node tumors
using horseradish-peroxidase-conjugated streptavidin-biotin technique with diaminobenzidine as the
color read-out. None of the tumors showed S-100 protein and HMB-45 immunoreactivity, excluding the
possibility of Langerhans’ cell histiocytoses and mel-
Molecular Identification of Metastasis/Milchgrub et al.
751
FIGURE 2. Histology of tumor tissues. (a) Duodenal tumor infiltrating the intestinal muscular wall (H & E, ⫻10). (B) Magnification of the duodenal tumor showing
a rare focus comprised of polygonal cells with clear cell cytoplasm, distinct nucleoli, and stippled chromatin (H & E, ⫻40). (C) Scalp tumor infiltrating the dermis
(H & E, ⫻10). (D) Scalp tumor comprised of pale-staining cells with granular cytoplasm, vesicular nuclei, and prominent nucleoli (H & E, ⫻40). (E) Cervical lymph
node infiltrated by clear cells (H & E, ⫻10). (F) Cervical lymph node with cells containing vacuolated cytoplasm, large vesicular nuclei, and eosinophilic nucleoli
(H & E ⫻40).
anoma. The duodenal tumor showed diffuse keratin
expression and focal strong reactivity for chromogranin, synaptophysin, and somatostatin. Only chromogranin was expressed focally in the lymph nodes;
the skin tumor was negative for the three neuroendocrine markers. The clear cells in the skin were strongly
immunoreactive for lysozyme and ␣-l-antitrypsin and
weakly positive for CD68, which are nonspecific markers for cells of histiocytic derivation. Finally, keratin
was not expressed in the skin and lymph node tumors,
thereby excluding the possibility of metastatic carcinoma containing clear cells, such as renal cell carcinoma.
An expanded laboratory workup revealed a normal skeletal survey and an abdominal CT scan showed
simple cysts in the liver and kidneys that had been
noted previously and were unchanged. At this point, a
diagnosis of a metastatic clear cell carcinoid was favored. To confirm this diagnosis, the three tumors
were studied by molecular analyses to identify the
same clonal origin.
MATERIALS AND METHODS
Paraffin embedded specimens from the duodenal,
scalp, and cervical lymph node tumors were subjected
to dissection using a PixCell laser capture microdis-
752
CANCER February 15, 2000 / Volume 88 / Number 4
FIGURE 3. Loss of heterozygosity (LOH) and a microsatellite alteration (MA) in microdissected DNA of duodenal carcinoid cells (D) and of clear cell tumor cells
from the scalp (S) and cervical lymph nodes (LN). Nonmalignant lymphocytes (L) from these tissues served as controls. Microsatellite marker D3S1274 at
chromosomal region 3p12 showed LOH of the same parental allele in the three tumor tissues. D3S4103 at 3pl4.2 showed identical shifted bands indicative of the
same MA in the three tumor tissues. By contrast, D3SI612 at 3p22-24 showed retention of heterozygosity in the three tumor tissues. Bars indicate the positions
of the major allelic bands for LOH.
section apparatus (Arcturus Engineering, Mountain
View, CA).2 In this technique, a transparent thermoplastic film (ethylene vinyl acetate polymer) is applied
to the surface of the tissue section on a standard glass
histopathology slide; a carbon dioxide laser pulse then
specifically activates the film above the cells of interest. Strong focal adhesion allows selective procurement of the targeted cells. Carcinoid tumor cells from
the duodenal tumor and the clear cells within the
scalp and lymph node tumors were microdissected.
Lymphocytes from the tumors were similarly microdissected and served as sources of constitutional DNA
(controls). DNA extraction was performed from sequential sections (microslides) of each sample.2 Multiplex PCR was performed on 5 ␮L of proteinase Kdigested samples, each containing DNA from at least
100 cells. Loss of heterozygosity and microsatellite
alterations were evaluated using primers flanking 22
dinucleotide and multinucleotide microsatellite repeat polymorphisms located at 9 chromosomal regions: 3p12 (D3S1274), 3p14.2 (D3S13OO, D3S4103,
and D3S1234 at the FHIT gene), 3p14.1-21.3
(D3S1613), 3p2l (D3S1029), 3p22-24.2 (D3S1612,
D3S1351, D3S2432, and D3S1537), 3p25 (D3S1293),
9p21 (IFNA and D9S1748), 11q13 at the multiple
endocrine neoplasia type 1 (MEN-1) gene locus
(D11S4097, D11S4908, D11S599, D11S480, D11S4936,
INT2, and PYGAL), and 17p13 (TP53 dinucleotide and
pentanucleotide repeats). All primer sequences were
obtained from the Genome Database except for those
in the TP53 gene.3,4 For all samples, multiplex PCR (up
to eight markers) was performed in the first amplification, followed by uniplex PCR for individual micro-
satellite markers.3,4 Loss of heterozygosity was recognized by the complete absence of one parental allele
in tumor tissues. Microsatellite alterations were identified by a shift in size of one or both parental alleles.
RESULTS
Fourteen of the 22 microsatellite markers were informative (i.e., heterozygous in control lymphocytes) and
at least 1 of these markers was informative at each
chromosomal region analyzed. Molecular changes (allelic loss or microsatellite alteration) involved two of
these markers, and the results from all three tumor
specimens were identical. A loss of the same allele was
detected in a single marker in 3p12 in all three tumor
specimens (Fig. 3). Furthermore, an identical microsatellite alteration in the 3p14.2/FHIT gene was noted
in the three tumor specimens (Fig. 3). These molecular
results indicate a common origin of the skin, lymph
node, and duodenal tumors. In tandem with the morphologic features, these results support the diagnosis
of scalp and cervical lymph node metastases from a
primary duodenal carcinoid.
DISCUSSION
The skin is an unusual site for carcinoid metastasis.5-8
In fact, to our knowledge the patient reported herein is
the first reported case of a clear cell variant of carcinoid metastatic to the skin.9,10 The most common
clear cell metastasis to skin is from renal cell carcinoma. Other metastases with clear cells include balloon cell melanoma and clear cell variants of germ cell
tumors; chondrosarcoma; non-Hodgkin lymphoma;
Molecular Identification of Metastasis/Milchgrub et al.
and neuroendocrine malignancies of pituitary, thyroid, parathyroid, pancreatic, or adrenocortical origin.
The differential diagnosis in this case of a rapidly
growing tumor on the scalp in association with scarring alopecia and cervical lymphadenopathy histologically proven to contain clear cells included primary
skin neoplasms such as melanotic balloon cell melanoma or T-cell or B-cell lymphoma, which may be
comprised of clear cells.11 Leukemia, multiple myeloma, and Langerhans’ cell (X) or non-Langerhans’
cell (non-X) histiocytosis also may be associated with
an exuberant xanthomatous reaction, albeit rarely.12
Finally, there was the possibility of metastatic carcinoma.
Although the diffuse growth pattern in the skin
and lymph nodes was not suggestive of a typical metastatic tumor of epithelial origin, individual neoplastic
cells from all three tumor sites had similar histopathologic features, suggesting that the duodenal carcinoid
may have given rise to less differentiated clear cell
metastasis. Although the majority of carcinoid tumors
may be recognized by their characteristic pathologic
features, by the immunohistochemical demonstration
of neuroendocrine cell properties, or by the electron
microscopic demonstration of the characteristic intracytoplasmic dense core granules, in the current case
pathologic and immunohistochemical studies provided negative or conflicting data and ultrastructural
studies were not performed because of the absence of
fresh tissue. To confirm the possibility of metastatic
clear cell carcinoid, molecular methodologies were
utilized.
Because molecular analysis requires a high degree
of purity in the phenotype of tissues or cells to be
studied, and because of the limited amount and the
preserved condition of archival tissue available to us,
we employed the newly described technique of laser
capture microdissection under microscopic visualization.2 This powerful technique exploits the ability of a
laser beam to simultaneously microdissect specific tissue targets (even single cells) and adhere such targets
to thermoplastic film that then can transfer the otherwise unaltered microdissected tissue for analysis.2
Its capacity to procure selected cell populations from
a section of complex and heterogeneous tissue permitted the isolation and study of malignant (carcinoid
and clear cells) and nonmalignant cells (lymphocytes)
from the same tumor specimens for the presence of
loss of heterozygosity and microsatellite alterations.
Coupled with PCR amplification of selected molecular
markers, this methodology enabled us, in duodenal,
skin, and lymph node specimens of two identical molecular abnormalities, to show the presence of loss of
heterozygosity and microsatellite alteration involving
753
different polymorphic markers. These findings provided very strong evidence for a common clonal origin
for the three tumors, leading to the conclusion that
the patient in the current study had a carcinoid tumor
of the duodenum with clear cell metastases to the
scalp and cervical lymph nodes.
Although the majority of carcinoid tumors follow
a benign course, their clinical behavior cannot be
predicted reliably based on histology.13,14 However,
the presence of nuclear atypia, pleomorphism, and
mitotic activity may suggest an aggressive clinical
course. A retrospective study has shown a correlation
between extent of tumor invasion and the rate of
incidence of metastasis and death.14 Approximately
85% of carcinoids classified as deeply invasive (halfway expansion through the intestinal wall or measuring ⬎ 2.5 cm in greatest dimension) metastasized and
65% were fatal, whereas ⬍ 1% of superficially invasive
carcinoids metastasized and resulted in death.14 In the
patient presented herein the duodenal tumor measured ⬍ 2.5 cm but had expanded more than halfway
through the bowel wall, which was sufficient to cause
bleeding. This feature categorizes the current case as a
deeply invasive carcinoid tumor with a relatively poor
prognosis. Had this categorization been recognized at
the time of the initial diagnosis, an argument may
have been made for more extensive bowel resection.
To our knowledge only a few genetic changes have
been reported in association with carcinoid tumors,
and these include allelic losses and mutations at the
MEN-1 gene (chromosome 11q).15-17 More recently,
deletions in 3p have been reported with bronchial
carcinoids.18 Therefore, selected markers in both of
these chromosomal regions were studied in all three
tumors. Markers in 9p and 17p also were evaluated
because these chromosomal regions are sites of the
CDKN2 and TP53 genes, respectively, which are mutated or exhibit allelic loss in a wide variety of malignancies. Because of the technical difficulties of performing sequencing studies from the limited amounts
of paraffin embedded materials available, mutational
analysis of the MEN1 gene was not performed. Loss of
heterozygosity was demonstrated for a marker in the
3pl2 region, with absence of the same parental allele
detected in all three tumor specimens. Furthermore,
using a different marker, an identical microsatellite
alteration was shown in the 3p14.2/FHIT gene in all
three tumor specimens. Microsatellite alterations produce changes in the number of polymorphic DNA
repeat units and are detected as altered electrophoretic mobility of one or both alleles.19 The biologic
significance of these alterations is not known, although an association with genomic instability has
754
CANCER February 15, 2000 / Volume 88 / Number 4
been speculated because of their high rate of incidence in neoplastic conditions.20,21
Our molecular findings constituted very strong
supportive evidence for a common clonal origin for
the three tumors. These results, together with the
morphologic features, lead to the clinical conclusion
that the patient had a carcinoid tumor of the duodenum with clear cell metastases to the scalp and cervical lymph nodes.
Because carcinoid tumors respond poorly to conventional chemotherapy, the patient was treated with
radiation therapy to the scalp and cervical lymph
nodes.22,23 Initially, she appeared to respond well,
with regression of the tumors of the skin and lymph
nodes. However, on completion of radiation treatment (total dose of 60 grays), a chest CT scan showed
the development of several tumor nodules in lung
parenchyma. Despite treatment with systemic interferon-␣ (up to 8 million U 3 times a week),22,23 her
condition worsened with the emergence of new subcutaneous tumors of the head and neck, an increase in
and enlargement of the pulmonary lesions, and the
development of hilar adenopathy and pleural effusion.
This case reminds us that carcinoid tumors are
not all indolent, that these tumors can present in
protean ways including intestinal hemorrhage, and
that they can metastasize without discernible liver
involvement. A second lesson concerns the limitations
of routine pathologic examination and immunohistochemical stains for identifying the source of metastases that no longer resemble the parent tumor. Finally,
our experience illustrates the novel application of laser capture microdissection combined with PCRbased analysis of genomic markers for diagnosing the
primary origin of metastatic disease.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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