Small-cell neuroendocrine carcinoma displays unique profiles of tumor-suppressor gene loss in relationship to the primary site of formation

Small-cell neuroendocrine carcinoma (SCNC) is a well characterized malignancy with distinctive cellular morphology and aggressive biologic behavior most frequently encountered in the lung but also noted for origin from other sites. The basis for this difference in incidence and the impact of primary site location on the molecular pathogenesis of the neoplasm is not well understood. To address this issue and to identify reliable molecular markers of potential diagnostic value for primary site localization of this tumor, we have compared the genetic profile of cancer-related gene damage of SCNC arising from a variety of organ sites. The analysis involved microdissected paraffin-embedded formalin fixed specimens of SCNC. Tumors were organized into 3 groups: lung (n = 18), head and neck region (n = 5), and gastrointestinal tract (n = 5). Genotyping evaluated allelic imbalance (loss of heterozygosity) involving genomic regions containing p53 (17p13), L-myc (1p34), OGG1 (3p26), MCC/APC (5q21), p16 (9p21), PTEN (10q23), and point mutational change in K-ras-2 (12p12) using polymerase chain reaction-based microsatellite analysis and DNA sequencing. Distinct genotypic profiles of allelic imbalance using this panel was seen for each group of SCNC enabling primary site determination to be suggested based on genotypic profiling of microdissected tissue samples. Despite similarity in histologic appearance, our study suggests that SCNC have a unique pattern of acquired allelic damage that is determined in part by primary site of tumor development. These attributes can be effectively used for primary localization of metastatic SCNC, thereby assisting in the diagnosis and classification of this neoplasm.     

Molecular analysis of clonality of sporadic angiomyolipoma

Angiomyolipoma, which consists of three intimately intermixed components, smooth muscle, blood vessels, and adipose tissue, is variably considered a hamartoma, a choristoma or a true neoplasm. This study has investigated the clonality of sporadic angiomyolipomas in seven women, each with a single lesion, by determining the pattern of X-chromosome inactivation. Polymerase chain reaction (PCR) amplification of the highly polymorphic human androgen receptor gene (HUMARA) was performed on the DNA extracted from the paraffin-embedded lesional tissue microdissected to sample the admixed smooth muscle and blood vessel component (SMC/BV) and the adipose tissue component. All seven patients were heterozygous for HUMARA polymorphism upon amplification of undigested DNA from non-lesional tissue and were therefore informative for further analysis. In all patients, lesional DNA, representative of the components, was predigested with HpaII restriction enzyme for amplification of the methylated allele. In six patients, the lesions were clonal, while in one, polyclonal. The polyclonal lesion was small and had less than 20 per cent SMC/BV component. Microdissected SMC/BV component was clonal in 6/7 lesions; the scanty SMC/BV in the remaining lesion did not yield amplifiable DNA. Microdissected adipose tissue was polyclonal in all seven lesions. Angiomyolipomas are three clonal lesions due to a clonal smooth muscle cell and blood vessel component, while the polyclonal adipose tissue is probably metaplastic or reactive. Copyright © 1999 John Wiley & Sons, Ltd.     

Genetic evidence for a clonal link between low and high-grade components in gastric MALT B-cell lymphoma

High-grade MALT lymphomas often contain low-grade tumour components; both cell populations have been shown to express the same immunoglobulin light chain previously. However, the clonal link between the low and high-grade components has not been established at the genetic level. To investigate this link, we have examined low- and high-grade components microdissected from tissue sections of four high-grade gastric MALT lymphomas. PCR and sequence analyses were performed to identify clone-specific rearranged immunoglobulin heavy chain gene sequences. In each of these cases, the PCR products from the two components were identical in size by electrophoresis. Direct sequencing revealed common clone-specific immunoglobulin heavy chain gene rearrangements in both lesions of each case, providing genetic evidence for a clonal link. These results support the proposal that high-grade MALT lymphomas generally evolve from low-grade clones.     

Metaplastic carcinoma of the breast: p53 analysis identified the same point mutation in the three histologic components.

A rare case of metaplastic carcinoma of the breast with both squamous metaplasia and cartilaginous metaplasia was reported. Histologically, the neoplasm revealed complex features, which were consisting of invasive ductal carcinoma, squamous carcinomatous component and chondrosarcomatoid component. Gradual transition of each component was recognized microscopically. p53 mutation analysis disclosed the same point mutation in three histologically different components, but not in the normal epithelium. Based on the morphologic findings, immunohistochemical findings and the p53 mutation analysis, we concluded that these three components in the tumor originated from the same duct progenitor cells.     

Tumor-to-tumor metastasis: lung adenocarcinoma metastasizing to a follicular variant of papillary thyroid carcinoma

Cancer-to-cancer metastasis into a thyroid neoplasm is an uncommon phenomenon with possible diagnostic difficulties. Here, we describe a case of lung adenocarcinoma metastatic into a follicular variant of papillary thyroid carcinoma (FVPTC). A 60-year-old woman with no prior history of malignant neoplasm presented with a nodule in the right lobe of the thyroid gland, some masses in the left lung were found by radiological examination. Histopathological examination of the thyroidectomy specimen demonstrated two different components of carcinoma in a single thyroid nodule; one was FVPTC and the other was high-grade adenocarcinoma. Although both components shared the TTF-1+/CK7+/CK19+/CK20-/SP-A- immunoprofile, only the former was positive for thyroglobulin, and only the latter was positive for CEA. The epidermal growth factor receptor (EGFR) gene mutation at exon21 (L858R) was present only in the latter. The lung biopsy specimen showed cytological, immunohistochemical, and EGFR genotypic features similar to those of the high-grade adenocarcinoma component of the thyroid nodule. These findings resulted in a reliable diagnosis of lung adenocarcinoma metastasizing into an FVPCT and treatment with EGFR-targeted therapy. These results demonstrate that a panel of immunohistochemical staining and molecular analysis is helpful for both diagnosis and appropriate postoperative treatment for a patient with cancer-to-cancer metastasis.     

Teratomatous genotype detected in malignancies of a non-germ cell phenotype

Originating from post-meiotic germ cells, mature ovarian teratomas (MOT) are genetically homozygous tumors within heterozygous hosts. MOT may be associated with malignant tumors of a non-germ cell phenotype (so-called malignant transformation). Based on the presence of in situ changes, some cases have been hypothesized to arise from teratomatous tissue. However, other malignancies associated with mature teratomas, such as sarcomas, may originate from either teratomatous elements or preexisting somatic ovarian tissue. Eight cases of MOT containing various histologic types of malignancy, including four squamous cell carcinomas, two sarcomas, one thyroid carcinoma, and one carcinoid tumor, were selected for study. Using selective tissue microdissection and PCR-based analysis of the extracted DNA, we compared the genotypic pattern of the mature teratomatous components to the associated malignant neoplasm with a random panel of highly informative genetic markers for different chromosomes. In all eight cases, genetic analysis of the malignant component revealed a homozygous genotype. In seven cases, the genetic profiles of mature teratomas and the associated malignant tumors were identical, suggesting a direct pathogenetic relationship between these lesions. In one case, the malignant component revealed homozygosity of different alleles compared with mature teratoma, suggesting independent teratomatous growth processes. This finding indicates that some ovarian malignancies of the non-germ cell phenotype arise in teratoma and fall into the spectrum of germ cell tumors.     

Oligoclonality of a "composite" gastric diffuse large B-cell lymphoma with areas of marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue type

We analyzed a case of oligoclonal Helicobacter pylori-associated, primary gastric diffuse large B-cell lymphoma (DLBCL) with areas of extranodal marginal zone B-cell lymphoma (MZBL) of mucosa-associated lymphoid tissue (MALT) type in a 61-year-old male patient. The patient had two separate tumors, in the corpus and in the antrum, each showing two morphologically distinct components (DLBCL and MZBL of MALT type). To determine the clonal relationship between the large- and small-cell components, we microdissected four samples from morphologically distinct tumor components at both tumor sites. PCR analysis revealed rearranged immunoglobulin heavy chain (IgH) genes of different sizes in three of the four microdissected samples. Cloning and sequencing of the PCR products displayed different IgH gene rearrangements in the two small cell components of the antrum and the corpus. A third genuinely differently rearranged IgH gene was found in the large-cell components of both antrum and corpus. Our results suggest that in primary gastric DLBCL with areas of MZBL of MALT type, the small-cell component may comprise more than one tumor clone. Furthermore, the large-cell component may evolve independently from coexisting MZBL.     

The value of polymerase chain reaction detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection

AIMS: The aim of this study was to investigate the usefulness of polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection (LCM). METHODS: The PCR DNA amplification method was used to detect M. tuberculosis in granulomas microdissected from one section stained by haematoxylin and eosin (H&E) from a formalin-fixed paraffin-embedded specimen. The results were compared to those obtained from PCR performed from 10 whole paraffin sections of 5 micro m each, and with the histology, culture and the patient's clinical findings. RESULTS: Forty-nine formalin-fixed and paraffin-embedded samples from 49 patients with a histological suspicion of a mycobacterial infection were investigated. Using culture as the reference method, the sensitivity for the detection of M. tuberculosis was 92% and the specificity was 100% using PCR from microdissected granulomas and were similar to those obtained by using PCR from 10 whole sections. CONCLUSIONS: The PCR method of examination of microdissected granulomas from deparaffinised sections is a sensitive, specific and rapid method for the detection of M. tuberculosis in formalin-fixed and paraffin-embedded samples. The method is as sensitive as that using PCR on 10 whole tissue sections, thus making it suitable for small biopsies. However, although these methods reduce the delay in diagnosis, culture remains the gold standard for identification of mycobacteria in tissue. Culture also allows for the testing of antibiotic sensitivity of any isolated species, in this way determining appropriate treatment.     

Cytogenetic findings in a breast stromal sarcoma. Application of fluorescence in situ hybridization to characterize the breakpoint regions in an 11;19 translocation.

Cytogenetic analysis of a stromal breast sarcoma revealed a complex karyotype that included a reciprocal 11;19 translocation, along with multiple numerical changes, deletions, and other unbalanced structural rearrangements. Karyotypic abnormalities have not been reported previously in this rare neoplasm that arises from mesenchymal breast tissue, and the t(11;19) is of interest because various types of sarcoma are characterized by specific reciprocal translocations. Because of the pericentric nature of the breakpoints on chromosomes 11 and 19 in the t(11;19), classical cytogenetic banding could not reveal the centromeric origin of the translocation derivatives. Using nonisotopic in situ hybridization with chromosome 11 and 19 alpha-satellite probes, the centromere of each derivative chromosome was determined, and the rearrangement was interpreted as a balanced translocation, t(11;19)(q12 or q13.1;p12 or p13.1). This abnormality has not been described previously in any breast tumor.     

Loss of Heterozygosity on Chromosome 11p15 during Histological Progression in Microdissected Ductal Carcinoma of the Breast

Microdissection of histologically identifiable components from formalin-fixed, paraffin-embedded tissue sections allows molecular genetic analyses to be correlated directly with pathological findings. In this study, we have characterized loss of heterozygosity (LOH) at chromosome 11p15 at different stages of progression in microdissected tumor components from 115 ductal carcinomas of the breast. Microdissected foci of intraductal, infiltrating, and metastatic tumors were analyzed to determine the stage of progression at which LOH at 11p15 occurs. LOH was detected in 43 (37%) of 115 cases. Foci of intraductal carcinoma could be microdissected from 85 cases, of which 30 (35%) showed LOH at some stage of progression. LOH was detected in the intraductal component in 26 of these 30 cases. Interstitial deletions were characterized by using a panel of 10 highly polymorphic markers. The smallest region of overlap (SRO) for LOH at 11p15 was bounded by the markers D11S4046 and D11S1758. LOH at 11p15.5 showed no correlation with estrogen receptor status, the presence of positive lymph nodes, tumor size, histological grade, or long-term survival. We conclude that 11p15 LOH usually occurs early in breast cancer development but less frequently does not develop until the infiltrating or metastatic stages of tumor progression.     

Clonal patterns in phaeochromocytomas and MEN-2A adrenal medullary hyperplasias: histological and kinetic correlates

The relationship among histological features, cell kinetics, and clonality has not been studied in adrenal medullary hyperplasias (AMHs) and phaeochromocytomas (PCCs). Thirty-four PCCs (23 sporadic and 11 MEN-2A (multiple endocrine neoplasia type 2A)-related tumours, the latter associated with AMH) from females were included in this study. Representative samples were histologically evaluated and microdissected to extract DNA and evaluate the methylation pattern of the androgen receptor alleles. At least two tissue samples (from the peripheral and internal zones in each tumour) were analysed with appropriate tissue controls run in every case. The same areas were selected for MIB-1 staining and in situ end labelling (ISEL). Malignant PCCs were defined by histologically confirmed distant metastases. All monoclonal AMH nodules from the same patient showed the same X-chromosome inactivated. Six sporadic PCCs revealed liver metastases (malignant PCC) and eight additional sporadic PCCs showed periadrenal infiltration (locally invasive PCC). All informative PCCs were monoclonal, except for five locally invasive PCCs and one benign PCC that revealed polyclonal patterns. Those cases also showed a fibroblastic stromal reaction with prominent blood vessels, focal smooth muscle differentiation, and significantly higher MIB-1 (126.8+/-29.9) and ISEL (50.9+/-12.8) indices. Concordant X-chromosome inactivation in nodules from a given patient suggests that MEN-2A AMH is a multifocal monoclonal condition. A subgroup of PCCs characterized by balanced methylation of androgen receptor alleles, high cellular turnover, and stromal proliferation also shows locally invasive features.     

Loss of heterozygosity analysis shows monoclonal evolution with frequent genetic progression and divergence in esophageal carcinosarcoma

Carcinosarcoma (spindle cell carcinoma) of the esophagus is a rare neoplasm that shows squamous cell carcinoma (SCC) with a variable component of spindle cell sarcoma. Clinical and pathologic features of this neoplasm have been well documented, but the histogenesis has long been a matter of speculation and dispute. In an attempt to clarify the clonality and genetic relationships in the evolution of this neoplasm, we microdissected a total of 36 carcinomatous and sarcomatous foci from six esophageal carcinosarcoma (CS) and analyzed the allelic status with 25 microsatellite markers on chromosomal arms 3p, 5q, 6q, 8p, 9p, 11q, 13q, 17p, and 18q. In all cases, we found multiple and homogenous allelic losses in both the carcinomatous and sarcomatous components, strongly supporting the concept of monoclonal origin for this neoplasm. Homogeneous allelic losses were detected most frequently on 17p (5 cases), a chromosomal arm that included the p53 locus, followed by 3p, 11q, and 13q (3 cases); 9p (2 cases); and 8p and 18q (1 case). Moreover, five of the six cases showed additional or divergent allelic losses at more than one chromosomal locus at some of the microdissected foci, indicating genetic progression (2 cases) or genetic progression and divergence (3 cases). In four cases, the genetic changes indicated that an original clone of a pure SCC apparently acquired carcinosarcomatous or sarcomatous phenotype by successive genetic changes. On the other hand, we saw no evidence for tumors in which a sarcoma appeared to give rise to a carcinosarcomatous or carcinomatous subclone in the examined cases. In conclusion, our data support the concept that esophageal CS is derived from a single clone originating from a SCC. Furthermore, we showed genetic heterogeneity to accompany the phenotypic divergence, with patterns of genetic alterations that are consistent with both progression and divergence within individual tumors.     

Genotypic analysis of primary and metastatic cutaneous melanoma

Microdissection genotyping was performed on 16 cases of melanoma, including two cutaneous and one lymph node metastases. Three benign nevi were used as controls. Where possible, tumor was microdissected at several sites. Genotyping involved assessment of loss of heterozygosity [LOH]), which was accomplished using a panel of nine polymorphic tetranucleotide microsatellites. Polymerase chain reaction was performed on the normal tissue sample to establish microsatellite heterozygous status. Informative markers were then tested on microdissected lesional tissue and scored for the presence and extent of allelic imbalance (AI). Microsatellite informativeness varied from 33% to 66%. Benign nevi were without AI. All invasive melanomas manifested acquired allelic loss, which involved 75% or 100% of the markers shown to be informative for each subject. Eleven of 13 (84%) primary melanomas demonstrated intratumoral heterogeneity of AI consistent with development of tumor subclones with differing genotypic profiles within thin as well as thick melanomas. Although a consistent pattern did not emerge among the markers, LOH of 9p21 (D9S254) occurred in 60% (9/15) of the cases followed by 40% of cases displaying LOH of 1p34, p53, 10q (MXI1), and 10q23 (D10S520) and 25% with 5q21 (D5S 592) abnormalities. A third of the cases including the metastatic foci demonstrated two different patterns of AI affecting alternative alleles of the same genomic marker within different parts of the melanoma. Two melanomas in situ did not display LOH of any markers in the informative cases although the in situ component in the invasive tumors had allelic losses that were in part similar to the invasive areas. The results of this study support the expanded use of microdissection genotyping and explore other markers to define the unique mutational profile for malignant melanoma that may complement other histologic characteristics of melanoma.     

Morphology of isolated colonic crypts

The traditional paradigm of colonic fluid and electrolyte transport includes a spatial separation of absorptive and secretory processes to surface and crypt cells, respectively. Recent studies of isolated microperfused colonic crypts revealed constitutive Na-dependent fluid absorption while secretion is regulated by one or more neurohumoral agonists. One obvious reason for the difference found in microdissected crypts is their separation from the lamina propria milieu. While it has been shown that isolated crypts are devoid of obvious lamina propria elements, including pericryptal fibroblasts, detailed morphologic information of the content of isolated crypts has been lacking. To characterize the morphology of the isolated crypt, we performed transmission electron microscopy (TEM) and immunofluorescence on microdissected and Ca2+ chelated crypts. Crypt cell type analysis was carried out separately on intact rat colon using light microscopy. TEM revealed a complete lack of either lamina propria cells or extracellular material in crypts isolated by either technique. TEM also revealed a subtle difference between the two isolation methods, with intact basal membranes in microdissected crypts but focal disruption of basal membranes in Ca2+- chelated crypts. Immunofluorescent stains for two basement membrane components (laminin and collagen type IV) revealed the presence of adherent basement membrane only on microdissected crypts; evidence that the plane of separation differs in these two preparations. Crypt cell type analysis on intact rat colon revealed an equal proportion of goblet cells in the right and left colon (approximately 50%) when measuring the middle 70% of the crypts - the area studied during crypt microperfusion. This morphologic analysis will increase our understanding of the observed physiology of isolated colonic crypts.     

Hamartomatous endocervical polyp with heterologous mesenchymal tissue

We present an endocervical polyp with heterologous elements. Although a few neoplastic cervical lesions with cartilaginous and adipocytic heterologous tissue have been reported, an endocervical polyp with heterologous cartilage and adipose tissue has not been reported before our case. The patient was a 33-year-old woman who presented with abnormal uterine bleeding. On physical examination, there were no remarkable findings other than a cervical polyp protruding into the cervical canal. The polyp was removed. Pathological examination revealed an endocervical polyp with typical epithelial features. The stroma of the polyp contained mature cartilage islands and adipose tissue. There were also many thick-walled vascular structures. Neither stromal periglandular condensation nor atypia was found. Mitotic figures were not observed. Arteriolar structures did not contain internal elastic lamina. In our opinion, these pathological findings are all consistent with a hamartomatous lesion rather than with a true neoplasm.     

Pancreatic mucinous cystic neoplasms with sarcomatous stroma: molecular evidence for monoclonal origin with subsequent divergence of the epithelial and sarcomatous components.

Neoplasms with mixed carcinomatous and sarcomatous growth patterns occur in many organs and tissues. The pathogenesis of these cancers is thought to be either the result of two independent neoplastic processes merging to form a single tumor, or a neoplasm of monoclonal origin that develops phenotypic diversity. To address this issue, we characterized molecular alterations in separately microdissected epithelial and sarcomatous areas in three cases of pancreatic mucinous cystic neoplasms with sarcomatous stroma. Using microsatellite markers for six chromosomal loci commonly deleted in infiltrating ductal adenocarcinomas of the pancreas, we found genetic alterations to be virtually identical between the sarcomatous and epithelial components of two of the three neoplasms. In the third neoplasm, we found allelic losses and retentions to be identical at five of the six chromosomal loci, but at a single locus, we noted allelic loss in the neoplastic epithelial component but not the sarcomatous component. The same neoplasms were also analyzed for activating point mutations in codon 12 of the K-ras gene by using mutant-enriched polymerase chain reaction and allele-specific oligonucleotide hybridization. A K-ras mutation was identified in the epithelial component of one of the three neoplasms (the same tumor with an additional allelic loss in the neoplastic epithelial cells), but the sarcomatous component of this tumor was wild-type at codon 12 of K-ras, as were both components of the other two neoplasms. Overall, these results suggest a monoclonal origin with subsequent divergence of the neoplastic epithelial and sarcomatous portions of these neoplasms.     

Aortic body tumour with adjacent ectopic thyroid tissue in a dog

A 12-year-old neutered male Husky dog had a neoplasm at the base of the heart which did not invade surrounding tissues. Microscopically, the neoplasm was composed of nests and sheets of polyhedral cells subdivided into lobules by trabeculae of fine fibrovascular stroma. Adjacent to the neoplasm was a rim of ectopic thyroid tissue that appeared histologically normal. The possible differential diagnoses for the neoplasm were aortic body tumour, ectopic thyroid tumour and ectopic parathyroid tumour; the ultrastructural characteristics revealed it to be an aortic body tumour.     

Max interacting protein 1: loss of heterozygosity is frequent in desmoplastic melanoma.

Max interacting protein 1 (MXI1), a negative regulator of myc oncoprotein with tumor suppressor properties, has been mapped to chromosome 10q24-25. MXI1 gene loss, demonstrated by loss of heterozygosity (LOH) analysis (allelic imbalance), is a frequent event in astrocytomas and other forms of glial neoplasia. Development and progression of malignant melanoma likely involves several cooperative oncogene/tumor suppressor gene alterations, many of which remain to be elucidated. We sought to discover whether desmoplastic melanoma (DM) exhibited MXI1 LOH. Archival fixative treated tissue was used for genotyping; this necessitated a microdissection-based molecular approach uniquely designed for minute tissue samples. Nineteen formalin-fixed tissue samples from 11 patients representing primary, locally recurrent, and metastatic DM were available for study. In each case, normal and neoplastic tissue was microdissected under stereomicroscopic observation as a basis for genetic analysis. We identified MXI1 LOH in neoplastic tissue by observing allelic imbalance for a microsatellite repeat polymorphism in the 3' nontranslated region of the MXI1 gene in subjects shown to be informative. Colorectal adenocarcinoma (n = 21) and astrocytomas (n = 19) were similarly analyzed, serving as negative and positive controls, respectively, for MXI1 LOH. Five of 11 DMs in subjects informative for the MXI1 microsatellite manifested MXI1 allelic imbalance consistent with tumor suppressor LOH. Genotype fidelity with respect to MXI1 status was present in two patients from whom primary and recurrent tumor was available for comparative analysis. We found that MXI1 LOH was independent of tumor stage and survival. MXI1 LOH seems to be a relatively frequent and early event in DM development and progression, consistent with neuroectodermal histogenesis of the neoplasm.