Increased cytoplasmic level of migfilin is associated with higher grades of human leiomyosarcoma.

AIMS: Leiomyosarcomas (LMS) are malignant neoplasms composed of cells that exhibit distinct smooth muscle differentiation. The molecular and cytogenetic features of LMS are complex and no consistent aberrations have been reported to date. Mitogen inducible gene-2 (Mig-2), kindlin and migfilin are recently identified cell-matrix adhesion proteins. The aim was to determine the expression and distribution of these proteins in human smooth muscle tumours of somatic soft tissue. METHODS AND RESULTS: Immunohistochemistry was performed on a human LMS tissue microarray and on sections of human leiomyomas (LM) and normal smooth muscle. Migfilin was barely detectable in normal smooth muscle cells, whereas increased levels of migfilin were observed in the majority of LM and LMS. Furthermore, the cytoplasmic level of migfilin was strongly associated with higher tumour grades. Additionally, the cytoplasmic levels of migfilin and Mig-2 were correlated with each other, suggesting an association between the two in the cytoplasm. Kindlin was expressed in normal smooth muscle, LM and LMS, and its level did not correlate with tumour grade. CONCLUSIONS: Our results suggest a role for cytoplasmic migfilin in the progression of LMS and identify cytoplasmic migfilin as a potentially important biological marker for human LMS progression.

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.

Implications of microscopic satellites of the primary and extracapsular lymph node spread in patients with high-risk melanoma: pathologic corollary of Eastern Cooperative Oncology Group Trial E1690.

PURPOSE: To correlate the presence of extracapsular spread (ECS) of regional nodal metastases, and micrometastasis near the primary tumor, with disease outcome in the intergroup study E1690 in relation to the impact of recombinant interferon-alfa (rIFN alpha)-2b. PATIENTS AND METHODS: E1690 included 642 patients with American Joint Committee on Cancer stage IIB or III cutaneous melanoma. Patients were randomized into high- and low-dose rIFN alpha-2b treatment arms and an observation arm. Pathologic slides were reviewed for selected parameters from at least half of the subjects in all three arms. Evaluation of the primary tumor included notations regarding ulceration, mitotic activity, thickness, microscopic satellites (MS), and nodal ECS on a standardized pathology form. These data were collated in relation to relapse-free survival (RFS) and overall survival (OS) at 50 months' follow-up and studied using Cox regression analysis. RESULTS: Ulceration, mitotic activity, thickness, and size of tumor-bearing lymph nodes did not show a statistically significant correlation with either OS or RFS across all treatment arms. The presence of MS was correlated with RFS (P =.0008) and OS (P =.05). ECS correlated with RFS (hazard ratio = 1.44, P =.032) but not OS (P =.11). CONCLUSION: The presence of MS (in 6% [18 of 308 patients]) had a significant adverse impact on both RFS (P =.0008) and OS (P =.053). Ulceration, mitotic activity, thickness, and number of positive lymph nodes had no significant effect on OS in this subset study (univariate or multivariate Cox analysis). The presence of ECS in lymph nodes had a significant adverse effect on RFS (P =.032) but not on OS.

Cytokeratin immunophenotyping of an unusual cervical vertebral chordoma with extensive chondroid foci and perilaryngeal recurrence: a case report with review of the literature.

Chordomas are midline, slowly growing, and locally destructive tumors derived from vestigial remnants of the notochord. We present an unusual case of a cervical vertebral chordoma with extensive chondroid change that aggressively recurred in the anterior larynx and surrounding neck structures, and subsequently in the mediastinum, resulting in the death of the patient. Recent literature has investigated and debated the significance of chondroid elements in chordomas as a differential diagnostic and a prognostic indicator. In particular, the use of immunohistochemical stains for cytokeratin and mesenchymal markers in these areas as a means of distinguishing true from pseudocartilage has received much attention. In this study, we used a spectrum of cytokeratin subtypes (CK 7, 20, 5/6, AE1/3) to further characterize these chondroid areas, and observed that they were positive for the majority of the cytokeratin subtypes, suggesting pseudo, rather than true, cartilaginous change. Clinicopathologic features of this lesion and the recent literature are reviewed.

Comparative genomic hybridization of hepatocellular carcinoma: correlation with fluorescence in situ hybridization in paraffin-embedded tissue.

BACKGROUND: Proto-oncogene MYC, mapped to chromosomal band 8q24 and the genes for hepatocyte growth factor (HGF at 7q21) and its receptor, MET, at chromosomal band 7q31, have an important role in the biology and growth of normal and neoplastic liver. Comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) studies have reported frequent abnormalities of chromosomes 1 and 8 in hepatocellular carcinomas (HCCs) of various clinical and pathological stages. Chromosome 7 involvement is reported to be less frequent. MATERIALS AND METHODS: Frozen tissue from 17 HCCs was used for CGH analysis and sections of corresponding formalin-fixed, paraffin-embedded HCC tissue were used for dual-color FISH with locus-specific (LSI-cMYC for chromosome 8q24 and LSI D7S486 for chromosome 7q31) and centromeric probes, CEP8 (8p11.1-q11.2) and CEP7 (7p11.1-q11.2) (Vysis, Inc, Downers Grove, IL). This study intended to determine the pattern of chromosomal aberrations in early-stage (incidental) HCC and large surgically resected HCC, and also compared the efficiency and usefulness of the two cytogenetic methods. RESULTS: CGH showed abnormalities on chromosomes 1q, 5q, 7q, 8q, 9, 10, 13q, 15, 16, 17p, 18q, 19, 20, 21, 22, and X. Gains of 8q were noted in 50% of the HCCs, including five cases of incidental HCCs by CGH. Increase in copy numbers of MYC detected by FISH was noted in 25% of tumors that had shown 8q gains by CGH and in five cases with no chromosome abnormalities noted by CGH. Three cases with 7q31 copy number abnormalities were found by FISH in addition to those detected by CGH. CONCLUSION: Combined use of CGH and FISH may provide important information about early and/or primary genetic changes in the development of HCC.

Comparative immunohistochemical and molecular analysis of uterine and extrauterine leiomyosarcomas.

Histologic criteria defining malignancy in smooth muscle tumors are currently site specific. This study was undertaken to determine whether, in leiomyosarcomas (LMS) occurring in different anatomic locations, there were differences in patterns of expression of molecules that have been demonstrated to be associated with biologically aggressive behavior in malignant neoplasms, and also to determine their diagnostic utility. Formalin-fixed paraffin-embedded tissue blocks were selected from 16 extrauterine leiomyosarcomas (EULMS), 14 cases of uterine leiomyosarcomas (ULMS) and from five cases each of uterine and extrauterine leiomyomas (LM). Utilizing immunohistochemical (ABC) techniques with antigen retrieval, we assessed serial sections of each tumor for immunoreactivity with Glut1, CD44s, bcl2, cyclin D1, and estrogen receptor. Molecular genotyping for detecting k-ras-2 point mutation, p53 gene loss, and mdm2 gene amplification was performed on microdissected tumor samples from the same histologic sections. All of the uterine and extrauterine LM were diffusely positive for CD44s, bcl2, and cyclin D1, and uniformly negative for Glut1. In contrast, 50% of the ULMS and 25% of EULMS were Glutl positive. Moreover, Glut1 positivity closely correlated with aggressive biologic behavior reflected by distant metastatic spread. Eighty-percent of LM and 70% of the ULMS were estrogen receptor positive, whereas only one retroperitoneal tumor had focal weak positivity. Over 80% of the extrauterine and 50% of the uterine sarcomas showed absence of CD44s immunoreactivity. Percentage of cyclin D1 immunoreactivity was independent of tumor grade and inversely proportional to the percent of bcl2 positivity. An LMS of the male breast contained k-ras-2 exon 1 point mutation (codon 12 aspartate substitution of glycine). P53 allelic imbalance was present in 29% of ULMS and 57% EULMS. Mdm2 amplification was present in three of six EULMS but not in ULMS. In addition to clinical staging, Glut1 positivity together with patterns of immunoreactivity of CD44 and bcl2 may be helpful in identifying aggressive smooth muscle tumors of the uterus and some EULMS. The presence of estrogen receptor staining may be helpful in identifying uterine versus nonuterine LMS. Although sample numbers are too small for definite conclusions, this study suggests that there are differences in glucose transport, expression of adhesion molecules, and estrogen receptors in ULMS and EULMS, which in part may be due to the estrogen dependency of the ULMS. P53 mutations and mdm2 amplifications appear to be more frequent in EULMS.

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.

Distant skin and soft tissue metastases from sarcomas.

BACKGROUND AND OBJECTIVES: This small series documents the clinical and pathological features and the rarity of distant skin and soft tissue metastases from sarcomas. MATERIALS AND METHODS: Five cases of sarcomas from different anatomical locations that had metastasized to skin and subcutaneous soft tissue were identified in three women and two men. The age range was 41-77 years. The primary tumors had wide excisions, followed by either radiation or chemotherapy, or both. The histological types were epithelioid sarcoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, and leiomyosarcoma. Metastases occurred to the skin and soft tissue of the chest wall, leg, breast, and abdominal wall. The diagnosis was established by excision biopsies for three cases and by needle biopsy and fine-needle aspiration for two cases. RESULTS: Three patients died within 7 months of the diagnosis of soft tissue metastases that were always histologically high grade and never solitary. One patient is alive with lung metastasis discovered 17 months after excision of primary. Lung metastases occurred either simultaneously or within a short period after soft tissue metastases. CONCLUSION: Distant skin and soft tissue metastases from sarcomas are very rare and often occur as a terminal event.

Genotype- and gender-dependent hepatic alcohol dehydrogenase (ADH) activity in developing mice.

Ethanol is metabolized in human and rodent liver primarily by the enzyme alcohol dehydrogenase (ADH). Hepatic ADH activity in adult males and females of seven inbred strains of mice was determined to examine genotype- and sex-dependent variability among the strains and the level of sexual dimorphism in each of the strains. ADH activity varied considerably among the strains, which could be categorized into high-activity strains C57BL/6, C57Brcd, and Swiss, and relatively low-activity strains C3H, CBA, and DBA. Adult Swiss, AKR, C57BL/6, and DBA females exhibited significantly higher levels of hepatic ADH than their male counterparts, whereas no gender differences were seen in C3H, CBA, and C57Brcd. Young mice of high and/or low ADH activity strains viz. C57BL/6 and C3H did not exhibit gender differences in ADH activity at weanling but the enzyme levels increased by sixth week in females and remained higher thereafter. The progeny of a high-activity strain with sexual dimorphism (C57BL/6) and a low-activity strain lacking gender difference (C3H) exhibited intermediate levels of ADH and age-dependent sexual dimorphism.

Hepatocyte growth factor and c-MET in benign and malignant peripheral nerve sheath tumors.

Hepatocyte growth factor (HGF), secreted by mesenchymal cells, has pleiotropic biological activities on several cell types. HGF and its receptor, the c-met proto-oncogene product (c-MET) have been implicated in the genesis and progression of several carcinomas and sarcomas. It has been suggested that MET/HGF autocrine signaling may contribute to tumorigenesis in sarcomas. HGF has been recently found to be a mitogen for rat Schwann cells and to be present in neurofibromas in NF1 patients. In this investigation, we assessed the immunoreactive patterns of HGF and MET in benign and malignant peripheral nerve sheath tumors (PNST) using archival formalin-fixed tissue. The standard avidin-biotin-peroxidase method was used. All benign tumors were negative with HGF. Eight cases of MPNST were positive with both HGF and MET. In some malignant PNST, positivity with both ligand and the receptor may be indicative of an autocrine mediated signal transduction and may implicate HGF/MET in tumor progression. Immunoreactivity with MET was strikingly greater in MPNST in contrast to benign PNST; this finding may prove to be helpful in distinguishing some histologically low-grade MPNST from cellular and atypical benign PNST.

Cytogenetic and histologic correlation of peripheral nerve sheath tumors of soft tissue.

Cytogenetic analysis was performed on 11 peripheral nerve sheath tumors of soft tissue from 10 patients. They include 6 benign and 5 malignant schwannomas. Five cases which include two benign, one cellular and two malignant schwannomas had a known association with a nerve, but only one patient with malignant schwannoma has clinically documented neurofibromatosis type I. All the patients had a normal diploid constitutional karyotype. Two cases of cellular schwannoma were analyzed by routine cytogenetic analysis and fluorescence in situ hybridization (FISH). One tumor was karyotyped as 45, XX,-13,-22 +mar; and the other case had a 45,X,-Y,t(1;17) (p12;q11.2) karyotype. In the latter, the breakpoint in 17q occurred below the centromere and is at or in the region of the Neurofibromatosis Type 1 (NF1) gene. Four benign tumors had a normal diploid karyotype. One hypodiploid malignant schwannoma with myxoid features demonstrated monosomy of chromosomes 17 and 22 by FISH analysis. The rest of the malignant schwannomas showed a wide range of numerical and structural aberrations, with frequent loss of 22q and gains of chromosomes 2 and 7. Loss of a sex chromosome was observed in cellular as well as malignant schwannomas. Regional karyotypic evolution was noted in one malignant schwannoma. Cytogenetic analysis may prove to be useful in identifying tumors, such as cellular schwannomas, which, because of their histologic features may be inadvertently categorized as malignant. Simultaneous involvement of NF1 and NF2 genes, which are located on chromosomes 17q and 22q, respectively, should be investigated at a molecular level in both benign and malignant tumors of peripheral nerves.

Studies on the effect of ethanol on dominant lethal mutations in Swiss, C57BL6 and CBA mice.

Swiss, C57BL6 and CBA males were given 0.1 ml of 40% ethanol per mouse per day for three consecutive days, intraperitoneally. These males were mated with untreated virgin Swiss females employing a 4-day mating schedule and three consecutive matings were carried out. In another study, C57BL6 males were given an ascending gradient of 5% to 40% ethanol in drinking water for a total period of 11 weeks. These males were mated with C57BL6 females for 2 weeks. Females were dissected at mid-term pregnancy for the examination of uterine contents including total, live and dead implants. All the investigations comprised at least two or three independent experiments which were evaluated independently as well as after pooling the data. Swiss, C57BL6 and CBA males given 0.1 ml of 40% ethanol, intraperitoneally, gave no evidence of any significant increase in post-implantation lethality in the postmeiotic phase of spermatogenesis attributable to ethanol treatment. A moderate but significant reduction in mean total implants indicating pre-implantation losses was seen in Swiss but not in CBA mice. Prolonged feeding of ethanol up to 40% in drinking water failed to provide any evidence of dominant lethal mutations in C57BL6 males at the pre-implantation level and the post-implantation lethals were also not significantly higher than in controls. In Swiss mice, however, the mutagenic index based on both pre- and post-implantation lethality was consistently positive.

Malignant pleural mesothelioma: a clinicopathological study.

In this paper the results of a retrospective review of 58 patients with malignant pleural mesothelioma treated at our Institute are reported. There were 50 males and 8 females; the mean age was 56.3 years (range: 13-77). History of asbestos exposure was ascertained in 25 patients (43%). The most common finding in chest X-ray was pleural effusion which was seen in 47/58 patients on presentation. The cytological examination of pleural effusion was most of the time nondiagnostic. Pleural biopsy was needed for the correct diagnosis. Pathologically, 26 patients (44.8%) had epithelial type, 24 patients (41.4%) had mixed type, and 8 patients (13.8%) had fibrous or sarcomatous type of pleural mesothelioma. Most of the patients on presentation had Stage I disease by Butchart's classification. The overall survival time ranged from 1 month to as long as 17 years with a median of 12.5 months. The mean survival of patients who received nonsurgical therapies was 7-13.4 months. Thirteen patients were treated surgically: three patients survived over 5 years, but the median survival was 15 months. Six patients received no treatment, and the median survival was seven months.

Complex karyotypic aberrations, including i(12p), in malignant mixed mullerian tumor of uterus.

We report the cytogenetic findings in a malignant mixed mullerian tumor of the uterus in a 59-year-old woman. Karyotypic analysis of short-term cultures revealed two abnormal clones of cells characterized by extensive structural and numerical rearrangements. An i(12p) maker chromosome was present in addition to other changes in both clones. This marker, characteristically associated with testicular germ cell tumors in males, has recently been reported in ovarian germ cell tumors, a mediastinal germ cell tumor and in a mixed mullerian tumor of the ovary.

Cytogenetic findings in a malignant fibrous histiocytoma of the gallbladder.

We report the cytogenetic findings in a rare tumor, a malignant fibrous histiocytoma of the gallbladder. Four related clones, two near-diploid and two near-tetraploid, which appeared to have been formed by a doubling of the near-diploid clones, were present. Numerous structural and numerical abnormalities characterized the tumor. Structural rearrangements included reciprocal translocations, translocations of unidentified material onto chromosomes, and deletions. Chromosomes involved in the rearrangements included 1, 3, 10, 12, 14, 16, and 19. Numerical changes included trisomy of chromosomes 2, 8, 10, and 20. Double minute chromatin bodies ranging in number from 5 to several were present in over a third of the cells.

Clonal chromosomal abnormalities in hemangiopericytoma.

We report the cytogenetic findings in nine hemangiopericytomas studied after short-term culture. Clonal chromosome abnormalities were present in four cases. One case had a simple translocation (12;19)(q13;q13.3) as the sole abnormality whereas complex and multiple chromosomal abnormalities involving almost all chromosomes in the complement characterized tumors from the three other cases.

Adjuvant, specific, active immunotherapy for resectable squamous cell lung carcinoma: a 5-year survival analysis.

In 1976 Stewart et al. (Annals of the New York Academy of Sciences 277:436-466) reported the effectiveness of adjuvant specific active immunotherapy of lung carcinoma in improving the postoperative survival of stage I lung carcinoma patients in a phase II study using lung carcinoma-associated antigen (TAA) and complete Freund's adjuvant (CFA). A phase III study was then designed by the authors to see the effects of specific active immunotherapy compared to the conventional management (no treatment) and to nonspecific immunotherapy. From 1976 to 1981, 85 patients with resectable (stages I and II) squamous cell lung carcinoma were entered into a randomized study: 1) control group; 2) specific immunotherapy group--three monthly doses of 500 micrograms of TAA emulsified with CFA; 3) nonspecific immunotherapy group--three monthly doses of CFA emulsified in saline. All the patients in the study received skin tests with 100 micrograms of the same TAA used for the immunotherapy. Recently, a 5-year follow-up of all the patients became available: The life table 5-year survival of group 1 was 34%, of group 2 was 75%, and of group 3 was 53%. The median survivals for the three groups were group 1, 38 months; group 2, 106 months; and group 3, 71 months. The difference was significant at P = .007 (Cox-Mantel test).