Identification of cyclin B1 as a shared human epithelial tumor-associated antigen recognized by T cells.

We eluted peptides from class I molecules of HLA-A2.1(+) breast adenocarcinoma and loaded reverse phase high-performance liquid chromatography (HPLC) fractions onto dendritic cells to prime naive CD8(+) T cells. Fractions that supported growth of tumor-specific cytotoxic T lymphocytes were analyzed by nano-HPLC micro-ESI tandem mass spectrometry. Six HLA-A2.1-binding peptides, four 9-mers (P1-P4) differing in the COOH-terminal residue, and two 10-mers (P5 and P6) with an additional COOH-terminal alanine, were identified in one fraction. Peptide sequences were homologous to cyclin B1. We primed CD8(+) T cells from another HLA-A2.1(+) healthy donor with synthetic peptides and generated P4-specific responses. We also detected memory T cells specific for one or more of these peptides in patients with breast cancer and squamous cell carcinomas of the head and neck (SCCHN). T cells from one patient, restimulated once in vitro, could kill the tumor cell line from which the peptides were derived. Immunohistochemical analysis of tumor lines and tissue sections showed cyclin B1 overexpression and aberrant localization in the cytoplasm instead of the nucleus. Sequencing genomic DNA and cDNA corresponding to P1-P6 region showed that differences in COOH-terminal residues were not due to either DNA mutations or errors in transcription, suggesting a high error rate in translation of cyclin B1 protein in tumors.

Expression pattern of alpha-protein kinase C in human astrocytomas indicates a role in malignant progression.

Protein kinase C (PKC) is a family of isoenzymes which play an important role in regulating cell proliferation and differentiation. Constitutive activation of PKC, either by phorbol esters or overexpression of specific isoenzymes, leads to growth abnormalities in vitro and tumor promotion in vivo. Since stimulation of PKC in cultured astrocytes results in biochemical and morphological alterations associated with the transformed phenotype, we wanted to determine whether abnormal expression of specific isoenzymes of PKC was important in development of human astrocytomas in vivo. We have detected a specific pattern of alpha-PKC expression in human astrocytomas which is noteworthy because the highest transcript levels were detected in well-differentiated (Grade 1) tumors, with intermediate expression in anaplastic (Grade 2) astrocytomas and low or nondetectable levels in glioblastomas (Grade 3 astrocytomas) and normal controls. In comparison, the beta-PKC transcript was not detected in any of the tumors, while the gamma-PKC transcript was present in only one Grade 2 tumor. Immunohistochemistry, using a monoclonal antibody to alpha-PKC, revealed diffuse, positive cytoplasmic signals in most cells of the Grade 1 tumors. Grade 2 tumors exhibited heterogeneity of alpha-PKC expression, although a significant percentage of cells showed positivity. In contrast, only a small number of differentiated cells within Grade 3 tumors were positive for alpha-PKC expression, with the more malignant, dedifferentiated cells uniformly negative. Throughout all tumor grades, the staining pattern of alpha-PKC closely paralleled that of glial fibrillary acidic protein. Taken in conjunction with the established role of PKC in tumor promotion, these results suggest that the alpha-PKC isoenzyme plays a specific role in facilitating clonal expansion of transformed astrocytes in low-grade astrocytomas. Analysis of alpha-PKC may therefore serve as a direct biological marker of malignancy which may serve to enhance the current histopathological grading system.

Age and TP53 mutation frequency in childhood malignant gliomas: results in a multi-institutional cohort.

Malignant astrocytoma is one of the most deadly primary central nervous system tumors. Although significant progress has been made in understanding the molecular pathways that lead to the development of these tumors in adults, comparatively little analysis has been done in childhood astrocytomas, which are less common and have a more favorable prognosis. Our previous studies of an institutional cohort of children with malignant gliomas suggested the existence of distinct molecular pathways of tumorigenesis in younger versus older children, based on the finding of a high frequency of TP53 mutations in tumors from children >3 years of age at diagnosis, compared with those from younger children. In the current study, the association between TP53 mutations and age was examined in greater detail using the multi-institutional group of children enrolled in Children's Cancer Group Study 945, the largest cohort of childhood high-grade gliomas analyzed to date. Seventy-seven tumors with centrally reviewed diagnoses of anaplastic astrocytoma or glioblastoma multiforme had sufficient archival histopathological material for microdissection-based genotyping. Sections were examined histologically, and topographic targets that contained malignant tissue were isolated by microdissection and subjected to PCR-based amplification and sequencing of TP53 exons 5-8. Twenty-six tumors (33.8%) had mutations in those exons. Mutations were observed in 2 of 17 tumors (11.8%) from children <3 years of age at diagnosis versus 24 of 60 tumors (40%) from older children, a difference that was statistically significant (P = 0.04), in agreement with our previous results. Whereas malignant gliomas in older children have a frequency of mutations comparable to tumors that arise in young adults, those from children <3 years old do not. The association between age and frequency of TP53 mutations among pediatric malignant gliomas indicates the probable existence of two distinct pathways of molecular tumorigenesis in younger versus older children.

K-ras gene mutations in normal colorectal tissues from K-ras mutation-positive colorectal cancer patients.

K-ras gene mutations have been reported as early events in colorectal tumorigenesis, but their role in tumor initiation and development is still unclear. To analyze and compare K-ras mutational patterns between colorectal tissues at different stages of tumor progression in individual patients, 65 colorectal tissue samples, including carcinoma, adenoma, histologically normal mucosa, submucosal muscularis propria, and histologically normal mucosa distant from tumor, were obtained from 13 patients with colorectal cancer. In addition, normal mucosal tissues obtained from four normal individuals were analyzed. Each of the 13 tumors was shown previously to harbor a mutation in either codon 12 or 13 of the K-ras gene by direct sequencing. These tissues were reanalyzed, using the recently established mutant allele enrichment + denaturing gradient gel electrophoresis method, which can detect one mutant allele in 10(4)-10(5) normal alleles, thus allowing for the analysis of infrequent cells bearing mutations against the background of wild-type cells. No K-ras codon 12 mutation was detected by this method in the histologically normal mucosal tissues sampled at the margin of resection distant from the tumor or in those obtained from four normal individuals. On the other hand, these mutations were detected in 9 of 10 adenoma and 6 of 10 mucosa samples from 10 patients with known K-ras codon 12 mutations, and also in 2 of 3 carcinoma, 2 of 3 adenoma, and 1 of 3 mucosa samples obtained from 3 patients with known K-ras codon 13 mutations. Thus, K-ras codon 12 mutations were found to occur with a high frequency (53.8%) in histologically normal mucosa adjacent to tumors of patients with K-ras mutation-positive colorectal cancer, suggesting that they may be useful biomarkers for early detection of colorectal cancer. Furthermore, multiple K-ras mutations were found in tissues of nearly half of the 13 patients, indicating that distinct evolutionary subclones may be involved in the development of tumor in some patients with colorectal cancer.

The relationship between TP53 mutations and overexpression of p53 and prognosis in malignant gliomas of childhood.

The prognosis for children with high-grade gliomas remains somewhat unpredictable. Although prolonged disease control is sometimes achieved after surgery, radiotherapy, and chemotherapy, most patients exhibit rapid disease progression. Because p53-dependent apoptosis mechanisms are involved in the cytotoxic effects of irradiation and chemotherapy, we questioned whether p53 status might be associated with outcome in childhood malignant gliomas. Therefore, we examined p53 status, both immunohistochemically and by direct sequencing of exons 5-8, in a series of 29 archival pediatric malignant non-brainstem gliomas treated consecutively at our institution between 1975 and 1992. Eighteen tumors had dense p53 staining in the majority of cells, although only 11 had mutations of the p53 gene (TP53). On univariate analysis, there was a significant association between p53 overexpression and a shorter progression-free survival (PFS) and overall survival (OS; P = 0.019 and 0.013, respectively; rank sum test). In addition, there was a significant association between TP53 mutations and a poorer PFS (P = 0.04), and a strong trend toward a shorter OS among patients with TP53 mutations (P = 0.06). Median PFS and OS for patients with TP53-mutated tumors were 6 months and 16 months, respectively, and for those with p53 overexpression 5.5 months and 14 months, respectively, versus 16 months and 25 months, respectively, for those without TP53 mutations and 25 months and >4 years, respectively, for those without p53 overexpression. The percentage of patients in this series with TP53 mutations (37.9%) was substantially higher than in previous studies of childhood gliomas and comparable to the frequency of mutations noted in adult gliomas. However, both TP53 mutation and p53 overexpression were significantly less frequent in tumors from children younger than 4 than from older children (P = 0.02 and 0.01, respectively). These results indicate that p53 mutation and expression status may be associated with prognosis in childhood malignant gliomas, and thus may provide a basis for stratifying patients biologically in future malignant glioma studies.

Prognostic value of quantitative reverse transcription-polymerase chain reaction in lymph node-negative esophageal cancer patients.

PURPOSE: In esophageal cancer, lymph node metastases are the strongest predictor of recurrence and poor outcome. However, many node-negative patients still recur despite a potentially curative resection. This is probably the result of microscopically occult metastases missed by histological examination. In this study, we used both standard, gel-based reverse transcription-PCR (RT-PCR) and Taqman quantitative RT-PCR (QRT-PCR) for carcinoembryonic antigen (CEA) mRNA to detect occult micrometastases in 387 lymph nodes from 30 histologically node-negative esophageal cancer patients. EXPERIMENTAL DESIGN: CEA expression was compared with clinical outcomes to determine correlation with disease recurrence. For quantitative data, an optimum CEA expression level cutoff value was defined as the value that most accurately classified patients on the basis of disease recurrence. Kaplan-Meier survival curves were generated, and multivariate analyses were performed to evaluate the prognostic value of QRT-PCR. RESULTS: CEA expression levels were above the optimum cutoff level in 12 tissue blocks, resulting in the identification of 11 CEA-positive patients. Of these patients, 9 suffered disease recurrence and 2 remain disease free. Of the 19 CEA-negative patients, there was 1 disease recurrence. The sensitivity and specificity for predicting disease recurrence were 90 and 90%, respectively. Kaplan-Meier analysis showed that CEA positivity resulted in significantly lower disease-free and overall survival (P <0.0001 and 0.0006 respectively). In multivariate analyses, CEA positivity measured by QRT-PCR was the strongest independent predictor of disease recurrence among other clinical and pathological factors examined. CONCLUSIONS: QRT-PCR offers significant benefits over standard RT-PCR and identifies node-negative patients at high risk for recurrence.

Cold-temperature plastic resin embedding of liver for DNA- and RNA-based genotyping.

The standard practice of tissue fixation in 10% formalin followed by embedding in paraffin wax preserves cellular morphology at the expense of availability and quality of DNA and RNA. The negative effect on cellular constituents results from a combination of extensive cross-linking and strand scission of DNA, RNA, and proteins induced by formaldehyde as well as RNA loss secondary to ubiquitous RNase activity and negative effects of high temperature exposure during paraffin melting, microscopic section collection, and tissue adherence to glass slides. An effective strategy to correlate cellular phenotype with molecular genotype involves microdissection of tissue sections based on specific histopathological features followed by genotyping of minute representative samples for specific underlying molecular alterations. Currently, this approach is limited to short-length polymerase chain reaction amplification (<250 bp) of DNA, due to the negative effects of standard tissue fixation and processing. To overcome this obstacle and permit both cellular morphology and nucleic acid content to be preserved to the fullest extent, we instituted a system of cold-temperature plastic resin embedding based on the use of the water-miscible methyl methacrylate polymer known as Immunobed (Polysciences, Warminster, PA). The system is simple, easy to adapt to clinical practice, and cost-effective. Immunobed tissue sections demonstrate a cellular appearance equivalent or even superior to that of standard tissue sections. Moreover, thin sectioning (0.5-1.0 microm thickness) renders ultrastructural evaluation feasible on plastic-embedded blocks. Tissue microdissection is readily performed, yielding high levels of long DNA and RNA for genomic and transcription-based correlative molecular analysis. We recommend the use of Immunobed or similar products for use in molecular anatomical pathology.

Relationship of p53 gene alterations with tumor progression and recurrence in olfactory neuroblastoma.

Olfactory neuroblastoma (ONB) is a rare neuroectodermal tumor whose clinical course is not effectively predicted by initial stage or grade; p53 tumor suppressor gene alterations have not been determined concerning the ONB pathobiology and recurrence. We analyzed 18 formalin-fixed, paraffin-embedded ONB specimens (12 primary tumors and six recurrences or metastases) from 14 patients for p53 alterations using immunohistochemistry for p53 and WAF1 together with topographic genotyping (selection of minute tissue targets from unstained sections, PCR [polymerase chain reaction] amplification of exons 5-8 followed by direct DNA sequencing). Sequential material representing tumor recurrence or metastasis was available in four cases to compare genetic alterations over time in the same patient. None of the cases showed strong, diffuse p53 immunostaining. Focal weak to moderate intensity staining was evident in nine of 14 cases. Mutations in p53 were not detected in any of the cases, suggesting hyperexpression of p53 wild-type protein. Hyperexpression was further confirmed by correlation of WAF-1 and p53 immunopositivity. Importantly, in four cases with recurrence or metastasis, tumors manifested p53 wild-type hyperexpression. It appears that p53 point mutation does not play an important role in the initial development of ONB; however, p53 wild-type hyperexpression may occur in subsets of ONB likely to show local aggressive behavior and a tendency for recurrence. Wild-type p53 hyperexpression may be an important event in later stages of ONB growth and progression.

Molecular genotyping of BK and JC viruses in human polyomavirus-associated interstitial nephritis after renal transplantation.

The human polyomaviruses BK virus (BKV) and JC virus (JCV) have been linked to ureteric stenosis and allograft interstitial nephritis, but molecular characterization of the species involved has not been performed. We studied paraffin-embedded renal tissue from 19 cases of allograft viral interstitial nephritis. Histological sections were subjected to polymerase chain reaction amplification using consensus, BKV-, and JCV-specific primers, with subsequent DNA sequencing for strain determination. BKV was present in all (100%) interstitial nephritis kidneys and placed in genotypes corresponding to serological groups I (n = 11), II (n = 1), and IV (n = 5). Fourteen of 17 isolates (82%) showed sequence variations in the viral capsid protein-1 (VP1) capsid region, with predicted changes in the encoded amino acids and sometimes with potential implications for the secondary and tertiary structure of the corresponding protein molecules. An additional case showed a previously reported glutamine-->leucine T-antigen region mutation. JCV was seen in seven interstitial nephritis kidneys (37%), with types 4 (n = 3), 3A (n = 2), and 2A (n = 1) identified. Most white individuals with asymptomatic infection are reported to shed type 1 JCV in the urine. Simian 40 polyomavirus was not identified in any case. These observations may have pathogenic relevance to the development of an extremely refractory form of polyomavirus interstitial nephritis seen after kidney transplantation.

Intrahepatic cholestasis with predominant pericentral deposition in systemic amyloidosis.

Liver involvement in amyloidosis is rarely associated with intrahepatic cholestasis. The cases recorded in the literature indicate that there is a tendency toward periportal deposition, leading to centrilobular cholestasis. Our case was most interesting in that the amyloid deposition was most severe in the centrilobular area. Presumably there was still sufficient compression of canaliculi at the periportal level to produce obstruction of the bile flow. This would appear to be a less common but distinct variant of systemic amyloidosis with associated obstructive jaundice.

Fat embolism to the cardiac conduction system associated with sudden death.

During a brief hospital stay, a patient under treatment with corticosteroids for lymphoma died suddenly and unexpectedly. At autopsy, fat emboli were identified in the lungs, myocardium, liver, and brain. Examination of the cardiac conduction system revealed fat emboli in the vessels of the bundle of His as well as those accompanying the bundle branches, a finding believed to have resulted in the sudden death. This report is the first of fat embolization to the cardiac conduction system and emphasizes the importance of examination of this system in cases of unexplained or sudden death.

Lymphoepithelial carcinoma of the larynx and hypopharynx: study of eight cases with relationship to Epstein-Barr virus and p53 gene alterations, and review of the literature.

Eight cases of lymphoepithelial carcinoma (LEC) of the larynx and hypopharynx were evaluated for clinicopathologic features, and the presence of the Epstein-Barr virus (EBV) and p53 alterations. The seven men and one woman, all of non-Asian descent, averaged 64 years of age. Eighty-eight percent had histologically confirmed cervical lymph node metastasis at diagnosis. None had systemic disease. Seven of eight patients available for follow-up (mean, 17.7 months) were alive and free of disease, although one did develop recurrent tumor in the neck. Four tumors were composed, histologically, of pure LEC. Four others had foci of both LEC and conventional squamous cell carcinoma. All eight tumors exhibited alterations in p53 expression, but none was positive for EBV. Combining these 8 cases with the 15 previously published cases in the English literature indicate that LEC in this site is a rare, rather aggressive tumor, primarily of older adults (mean, 62 years) with a propensity for early cervical lymph node metastasis and eventual distant dissemination and death from disease in about one third of patients. Although p53 alterations are common and of no apparent prognostic significance, LEC at this site seems to have little, if any, relationship to the EBV in patients of non-Asian origin.

The role of p53 mutation and protein expression in primary and recurrent adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a malignant tumor of salivary gland origin having a propensity for spread by direct extension or perineural invasion with frequent recurrences. Previous reports have shown that tumor behavior is not always predicted by histological pattern or stage. Little is known of the role of p53 tumor suppressor gene mutation and altered protein expression with respect to ACC pathobiology and recurrence. The authors analyzed a group of 14 ACC specimens (seven primary; seven recurrent) from 13 patients treated between 1987 to 1993. Formalin-fixed, paraffin-embedded specimens were reviewed and subjected to, immunohistochemistry (p53, DO-7, DAKO, Nutley, NJ; and WAF-I, Ab-1, Oncogene Sciences, Uniondale, NY) on 4-microm-thick histological sections as a prelude to p53 genotyping. In one case, sequential material representing primary and recurrent tumor was analyzed. Each tumor specimen was topographically genotyped for p53 point mutational change. Minute tissue samples were removed from unstained sections, polymerase chain reaction (PCR) amplified for p53 exons 5 to 8, and then underwent direct DNA sequencing. Six of seven primary ACCs were p53 immunostain negative. Four of seven recurrent (57%) ACCs were p53 immunopositive. These tumors showed varying degrees of p53 immunopositivity ranging from diffuse, intense staining of most tumor cells (n = 1) to interspersed, strongly positive cells mixed with predominantly p53 immunonegative cells (n = 4). All tumors were WAF-I immunostain negative. Two of the most immunopositive recurrent tumors each manifested a single type of p53 point mutation detected by p53 DNA genotyping (p53 exon 5:codon 175 and p53 exon 6:codon 199). In the case in which both primary and recurrent tumor was available, only the recurrent tumor contained point mutational damage. Negative immunostaining for p53 in primary ACC suggests that p53 mutation is not important in early events involving development of this tumor. In contrast, the frequent presence of p53-positive cells and the detection of point mutations in recurrent ACC suggests that p53 alterations are involved in later stages of tumor progression, important in the phenomenon of ACC recurrence.

Absence of jaagsiekte sheep retrovirus DNA and RNA in bronchioloalveolar and conventional human pulmonary adenocarcinoma by PCR and RT-PCR analysis.

Bronchioloalveolar adenocarcinoma (BAC) morphologically resembles sheep pulmonary adenomatosis (SPA), a contagious ovine pulmonary adenocarcinoma caused by the jaagsiekte sheep retrovirus (JSRV). Previously, positivity for JSRV by immunostaining, reverse-transcription polymerase chain reaction (RT-PCR), and Western blot was reported in most nonmucinous BACs. Our objective in this study was to analyze additional BAC subtypes and conventional adenocarcinomas (CA) to further substantiate this association. Tumor tissue was microdissected from unstained paraffin sections of 26 cases of formalin-fixed, paraffin-embedded BAC (7 mucinous, 17 nonmucinous, 2 sclerosing) and 29 cases of CA. Positive controls consisted of 2 separate paraffin blocks of known SPA. Primer sequences were derived that were capable of hybridizing to all reported strain variants of both the DNA (endogenous) and RNA (exogenous) forms of JSRV. Each sample was tested using both PCR (DNA) and RT-PCR (RNA). All BAC and CA cases were negative for JSRV. Positive controls yielded PCR products that were sequenced and precisely matched the published prototype stain of JSRV. To control for negative effects of tissue fixation, dilutions of positive control tissue were added to BAC and CA samples. Detection of JSRV was evident at 1:50 dilution. Although the possibility of a viral association with BAC cannot be excluded, this study shows that the association with JSRV is probably very weak, if present at all.

Genotypic analysis of pulmonary Langerhans cell histiocytosis.

Reported studies show that the systemic form of Langerhans cell histiocytosis (LCH) is a clonal expansion of Langerhans cells (LC) associated with aberrant expression of several oncogenes or tumor-suppressor genes. LCH of the lung is a heterogenous group of lesions thought to be a reactive rather than neoplastic process. The histogenesis of the LCH of the lung is uncertain, and to date there are no studies investigating its underlying molecular abnormalities. We performed comparative genotypic analysis by using allelic loss (LOH) of polymorphic microsatellite markers associated with tumor suppressor genes. Fourteen cases of formalin-fixed, paraffin-embedded LCH of the lung were studied. Microdissection of a total of 26 nodules from 14 patients and paired reference lung tissue was performed under stereomicroscopic visualization. To evaluate allelic loss, we used a panel of 11 polymorphic microsatellite markers that were situated at or near tumor suppressor genes on chromosomes 1p, 1q, 3p, 5p, 9p, 17p, and 22q. The PCR products were analyzed by using capillary electrophoresis to identify germline heterozygous alleles and LOH. Allelic loss at 1 or more tumor suppressor gene loci was identified in 19 of 24 nodules. The total fractional allelic loss (FAL) ranged from 6% (1q) to 41% (22q), with a mean of 22%. The FAL in individual cases ranged from 0 (7 nodules) to 57% (1 nodule). Fifteen discordant allelic losses at 1 to 3 chromosomal loci were identified in 8 patients with multiple synchronous nodules. Our results show that LOH of tumor suppressor genes is present in the LCH of the lung, and they indicate that the putative tumor suppressor genes situated on chromosomes 9p and 22q may play a role in the development of a subset of the LCH of the lung.

Carcinosarcomas (malignant mixed mullerian tumors) of the female genital tract: comparative molecular analysis of epithelial and mesenchymal components.

Female genital tract carcinosarcomas (FGTCS) are biphasic neoplasms composed of an admixture of malignant epithelial and mesenchymal elements. Histogenesis of FGTCS centers on two theories: (1) simultaneous formation of independent tumors (biclonal theory), (2) multidirectional differentiation of a single neoplasm (monoclonal theory). In an attempt to resolve this histogenetic controversy, we determined the presence, specific genotype, and timing of p53 mutational change in each component of FGTCS using a topographic genotyping (TG) approach. We selected 43 FGTCS from the files of Magee-Womens Hospital, Pittsburgh, and initially immunostained them for p53 protein. Strong p53 immunopositivity was detected in 35 (82%) of 43 tumors. Subsequently, topographic genotyping (TG) was performed on a subset of nine immunopositive tumors with sufficiently distinct malignant components to enable effective sampling. All nine tumors showed point mutations in p53 exons 5 through 8. In each case, the identical point mutational genotype was present in both components. Furthermore, in all nine cases mutations were present with loss of the wild-type allele. P53 gene mutation is a frequent event in progression of FGTCS. Of importance, both p53 mutation and allelic loss occur before the differentiation into separate epithelial and mesenchymal malignant components. These molecular findings strongly support monoclonal, multidirectional histogenesis of FGTCS.

Primary mediastinal and testicular seminomas: a comparison of K-ras-2 gene sequence and p53 immunoperoxidase analysis of 26 cases.

Primary mediastinal seminomas (MS) are rare tumors. Histologically, they are similar to their counterpart in the gonads. The survival rate has varied from 50% to 85% in different series. However, large series of these tumors primarily in the mediastinum are lacking. At the molecular level, a few reports document K-ras mutations in up to 40% of testicular seminomas (TS), localized predominantly to codon 12. Reports on TS p53 immunohistochemistry (IHC) range from negative to overexpression approaching 90% of cases, and by sequence analysis one small series showed a 23% mutation rate. To date, no analyses have been performed for either K-ras mutations or p53 immunohistochemical expression in primary MS. The authors studied 13 cases each of primary MS and TS from archival formalin-fixed, paraffin-embedded sections in which adequate tumor sampling and clinical history, including serological studies, and histological, histochemical, and IHC staining, were performed to confirm the diagnosis. p53 immunoperoxidase staining using citrate buffer/microwave antigen retrieval was performed. Topographic genotyping was performed on 5-microns-thick tissue sections up to 17 years old, in which the neoplastic cell population was sampled. Additionally, multiple sites within a given cases were sampled to determine clonality of the tumor cell population. Polymerase chain reaction and subsequent sequence analysis of the K-ras-2 exon-1 gene was used for mutation analysis. Focal weak staining with p53 IHC was observed in 4 of 13 (31%) MS and 10 of 13 (77%) TS cases, with all remaining cases being negative (P < .05). Only one MS case (8%) showed K-ras mutation (codon 13 GGC > GAC; glycine > aspartate), which is in contrast to 2 of the TS cases (15%), showing codon 12 mutations. All the remaining cases were wild type. Therefore, primary mediastinal seminomas appear to be different in their K-ras sequence and p53 immunostain profile from TS. Codon mutation type may be useful in determining primary versus metastatic origin of a mediastinal seminoma.

Extracellular matrix expression in metastasizing and nonmetastasizing adenocarcinomas of the lung.

Alterations in extracellular matrix, cell-cell and cell-matrix adhesion, and oncogenes are thought to be important in tumor progression and metastasis. Adenocarcinomas of the lung from 31 patients were studied for immunohistochemical expression of basement membrane molecule type IV collagen, type IV collagenase, and integrins alpha2,3,v adhesion molecules to assess their diagnostic and prognostic importance in pathological stage T2 tumors. The results indicate that with decreasing tumor differentiation, there is a progressive loss of type IV basement membrane collagen (P = .06) and decreased integrin alpha2 expression (P = .03). Type IV collagenase expression was significantly associated with the presence of lymph node metastases, with moderate to strong expression present in 53% T2N1 tumors compared with none (0%) of the T2N0 tumors (P = .008). Integrin alpha(v) was increased in tumors with nodal metastases compared with those without (P = .08). Loss of alpha2 and alpha3 integrins was associated with increased alpha v expression (P = .03). Median survival was 48 months for T2N0 and 20 months for T2N1 (P = .07). In correlating expression of the immunohistochemical markers and survival, type IV collagenase expression was found to be a predictor of survival at a level of P = .07. Measurable alterations in integrins and extracellular matrix, and in particular, expression of matrix-degrading enzyme type IV collagenase may be of prognostic importance in resectable adenocarcinoma of the lung.

Naloxone and experimental spinal cord injury: effect of varying dose and intensity of injury.

We have reported previously that high-dose (10 mg/kg) and megadose naloxone (as high as 150 mg/kg) failed to promote recovery of motor function after spinal cord injury in rat. In view of these negative results, in comparison to some reports of benefit of naloxone in the literature, the present study was undertaken to assess lower doses, using a modified 3 x 4 factorial design, to evaluate a range of lower doses in relation to various intensities of cord injury. Sprague-Dawley rats were assigned randomly to 10 groups (n = 10) relating to two factors: intensity of injury and dosage of naloxone. A dynamic-load injury was induced with a 10-g weight dropped from a height of 2.5 cm, 5.0 cm, or 17.5 cm. Animals were treated with naloxone 1 mg/kg, 4 mg/kg, 10 mg/kg, or saline (control). Tests of motor recovery were carried out weekly for 4 weeks postinjury. Histopathological morphometric analysis of the spinal cords was carried out for measurement of residual gray and white matter at the epicenter of the cord injury. In general, the behavioral data showed no improvement in recovery of function, with the possible exception of naloxone at a dosage of 4 mg/kg (not statistically significant at 4 weeks). Independent of naloxone treatment, there was a significant difference among the three intensities of injury. Pathologically, a difference could not be demonstrated in relation to dosage of naloxone, but as in the case of the behavioral data, a graded response occurred as a function of intensity of injury.

Experimental spinal cord injury: qualitative and quantitative histopathologic evaluation.

This study involved a morphometric analysis of an experimental model of spinal cord injury. The spinal cords of rats were injured by a weight drop at T8 level. Animals were sacrificed 4 weeks after injury, and histopathologic examination of the spinal cords was carried out qualitatively and also quantitatively with the aid of computer-assisted morphometry. Total cross-sectional areas of residual gray and white matter were determined at five regularly spaced intervals through the injured cord segment. The histologic findings were correlated with height of weight-drop and motor recovery in the hind limbs at 4 weeks postinjury. The weight-drop injury was found to produce a longitudinally asymmetrical cavitary defect, which was better assessed by a series of cross-sectional profiles than by a single histologic cross-section through the epicenter (site of maximal impact) of the cord injury. There was a strong correlation between height of weight-drop and amount of residual tissue (gray and white matter) at the epicenter. A correlation was also found between height of weight-drop and a composite of residual tissue evaluated at multiple levels through the injury site. By comparison with cross-sectional morphometry at the epicenter, multiple cross-sections, reflecting volume of residual tissue in the longitudinal extent of injury, showed greater statistical correlation with functional (behavioral) outcome. This "volumetric" assessment of the total region of injury is therefore recommended as preferable to a histopathologic evaluation limited to the epicenter.