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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. REFERENCES 1. 2. 3. 4. Jaffe ES, Costa J, Fauci AS, Cossman J, Tsokos M. Malignant lymphoma and erythrophagocytosis simulating malignant histiocytosis. Am J Med 1983;75:741–9. Emmert-Buck MR, Bonner RF, Smith PD, Cuaqui RF, Zhuang A, Goldstein SR, et al. Laser capture microdissection. Science 1996;274:998 –1001. Hung J, Kishimoto Y, Sugio K, Virmani A, McIntire DD, Minna JD. Allele-specific chromosome 3p deletions occur at an early stage in the pathogenesis of lung carcinoma. JAMA 1995;273:558 – 63. 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