Detection of isolated tumor cells in bone marrow is an independent prognostic factor in breast cancer.

PURPOSE: This study was performed to disclose the clinical impact of isolated tumor cell (ITC) detection in bone marrow (BM) in breast cancer. PATIENTS AND METHODS: BM aspirates were collected from 817 patients at primary surgery. Tumor cells in BM were detected by immunocytochemistry using anticytokeratin antibodies (AE1/AE3). Analyses of the primary tumor included histologic grading, vascular invasion, and immunohistochemical detection of c-erbB-2, cathepsin D, p53, and estrogen receptor (ER)/progesterone receptor (PgR) expression. These analyses were compared with clinical outcome. The median follow-up was 49 months. RESULTS: ITC were detected in 13.2% of the patients. The detection rate rose with increasing tumor size (P =.011) and lymph node involvement (P <.001). Systemic relapse and death from breast cancer occurred in 31.7% and 26.9% of the BM-positive patients versus 13.7% and 10.9% of BM-negative patients, respectively (P <.001). Analyzing node-positive and node-negative patients separately, ITC positivity was associated with poor prognosis in the node-positive group and in node-negative patients not receiving adjuvant therapy (T1N0). In multivariate analysis, ITC in BM was an independent prognostic factor together with node, tumor, and ER/PgR status, histologic grade, and vascular invasion. In separate analysis of the T1N0 patients, histologic grade was independently associated with both distant disease-free survival (DDFS) and breast cancer-specific survival (BCSS), ITC detection was associated with BCSS, and vascular invasion was associated with DDFS. CONCLUSION: ITC in BM is an independent predictor of DDFS and BCSS. An unfavorable prognosis was observed for node-positive patients and for node-negative patients not receiving systemic therapy. A combination of several independent prognostic factors can classify subgroups of patients into excellent and high-risk prognosis groups.

Detection of isolated tumor cells in bone marrow in early-stage breast carcinoma patients: comparison with preoperative clinical parameters and primary tumor characteristics.

UNLABELLED: PURPOSE/EXPERIMENTAL DESIGN: The importance of detection of disseminated isolated tumor cells (ITCs) in bone marrow (BM) is still not settled. BM aspirates from 920 patients with primary breast cancer were analyzed for tumor cells by standardized direct immunocytochemical analysis (ICC) of 2 x 10(6) mononuclear cells (MNCs) using anticytokeratin monoclonal antibody (AE1/AE3). Samples (637) were analyzed by negative immunomagnetic enrichment (IMS) followed by ICC (10 x 10(6) MNCs). Analyses of the primary tumor specimens have been performed, including histomorphology, tumor-node-metastasis (TNM) staging, grading, and immunohistochemical analyses. RESULTS: Of the patients with infiltrating carcinoma, 63% were node negative (N0) and 33%, node positive (N+). The results show the presence of tumor cells in 13.4% of the evaluable patients after direct ICC analysis. The presence of tumor cells correlated to the nodal- and tumor stage, showing BM positivity in 9.9% of the N0 cases and 20.6% in the N+ group (P < 0.0005), 11.2% of the stage T(1) were positive, and 15.0% and 22.6% were positive in the T(2) and T(3/4) groups, respectively (P = 0.013). No correlation between detection of ITC and detection of p53 and cathepsin D expression was found. Vascular invasion and c-erbB2 expression were associated with ITCs in BM (P = 0.045 and P = 0.024, respectively). Node-negative patients with estrogen receptor (ER)+ and/or progesterone receptor (PgR)+ tumors had lower frequency of ITCs than ER-/PgR- (P = 0.004). The use of negative IMS increased the frequency of positive BM by 63% (P < 0.0005). CONCLUSIONS: The direct ICC detection of ITCs in BM correlated with primary tumor stage, nodal stage, vascular invasion, c-erbB2 expression, and ER/PgR status. Analysis of larger BM samples by negative IMS resulted in increased number of ITC-positive patients.

Identification of two potential suppressor gene regions on chromosome arm 14q that are commonly lost in advanced colorectal carcinomas.

BACKGROUND: Remarkably little is known about the molecular alterations contributing to the establishment of a distant metastasis from a primary colorectal carcinoma. Previous studies on primary colorectal carcinomas have suggested an association between loss of chromosome 14 sequences and cancer progression. METHODS: In the present study, we analyzed 20 distant metastases and peripheral blood samples from 18 patients using 24 microsatellite markers spanning chromosome arm 14q. In addition, DNA from microdissected corresponding primary tumors (formalin-fixed and paraffin-embedded) was analyzed at selected 14q loci. RESULTS: Sixty-five percent (13/20) of the metastases, from 11/18 patients, showed loss of one or more markers at 14q, and the majority (94%) of the primary carcinomas showed identical 14q genotypes to those found in the metastasis. Two minimal common deleted regions were delineated in the metastases, one between markers D14S288-D14S52 at 14q13-21 and the other between D14S284-D14S81 at 14q24-31, pinpointing two previously unrecognized map positions for potential target genes. The genotype pattern of five tumors was consistent with monosomy or large chromosomal deletions spanning both potential suppressor regions. The reasons for monosomy in cancer remain unknown, but our data support the hypothesis that deletions of several tumor suppressor genes are more readily obtained by one chromosome loss than by several molecular events, and through this unison loss a growth advantage may be provided. CONCLUSION: Our data suggest that 14q loss is not a rate-limiting event in colorectal metastasis formation, but the high frequency of this alteration in primary tumors with metastatic ability, as well as in the metastases themselves, suggests it is part of the tumor clone with selective growth capacity.

Evaluation of 1p losses in primary carcinomas, local recurrences and peripheral metastases from colorectal cancer patients.

Cytogenetic and molecular genetic analyses of colorectal adenomas and carcinomas have shown that loss of the distal part of chromosome arm 1p is common, particularly in tumors of the left colon. Because the importance of 1p loss in colorectal cancer metastases is unknown, we compared the frequency, exact site and extent of 1p deletions in primary carcinomas (n=28), local recurrences (n=19) and metastases (n=33) from 67 colorectal cancer patients using 14 markers in an allelic imbalance study. Loss of 1p was found in 50% of the primary carcinomas, 33% of the local recurrences, and 64% of the metastases, revealing a significant difference between the local recurrences and the metastases (P=.04). The smallest region of 1p deletion overlap (SRO) defined separately for each group of lesions had the region between markers D1S2647 and D1S2644, at 1p35-36, in common. The genes PLA2G2A (1p35.1-36) and TP73 (1p36.3) were shown to lie outside this consistently lost region, suggesting that neither of them are targets for the 1p loss. In the second part of the study, microdissected primary carcinomas and distant metastases from the same colorectal cancer patients (n=18) were analyzed, and the same 1p genotype was found in the majority of patients (12/18, 67%). The finding that primary carcinoma cells with metastatic ability usually contain 1p deletions, and that some cases lacking 1p alterations in the primary tumor acquire such changes during growth of a metastatic lesion, supports the notion that 1p loss may be important both early and late in colorectal carcinogenesis, with the apparent exception of local recurrences.

Detection of low-frequency mutations in exon 8 of the TP53 gene by constant denaturant capillary electrophoresis (CDCE).

We extended our development of the means to measure point mutations at the DNA level to the problem of detecting TP53 gene variants in the area around tumors where mutant fractions are expected to be as low as one mutant per 1000 wild-type (WT) sequences. We met this criterion by using the following methods. The TP53 exon 8 sequence was amplified from genomic DNA samples under conditions of high polymerase fidelity using a fluorescein-labeled primer. This mixture of WT and mutant sequences was converted to heteroduplexes of mutant and WT sequences by melting and re-annealing. The mixture was resolved by capillary gel electrophoresis under appropriate constant denaturing conditions. Using laser-induced fluorescence, we found that fractions as low as 1/1000 could be detected without any prior enrichment for mutant sequences, and that the melting properties of the amplified DNA will leave "fingerprints" in the electropherogram that can be used to identify the specific mutation. This method is fast and robust and should be applicable to clinical analyses in which rapid scanning for any mutation in an exonic sequence is important.

Increased sensitivity for detection of micrometastases in bone-marrow/peripheral-blood stem-cell products from breast-cancer patients by negative immunomagnetic separation.

Immunocytochemical detection (ICC) of isolated tumor cells in bone marrow (BM) is currently the most established method for monitoring early dissemination in epithelial cancer. However, the low sample size that can practically be analyzed restricts the sensitivity and reliability of the ICC method. To be able to analyze larger samples, a negative immunomagnetic separation (IMS) technique, utilising anti-CD45-conjugated Dynabeads, has been developed. Tumor-cell enrichment by depletion of CD45-expressing mononuclear cells (MNC) is followed by ICC for detection of the cytokeratin (CK)-positive (+) epithelial cells. In this study, bone-marrow samples (n = 165) and peripheral-blood-progenitor-cell (PBPC) apheresis products (n = 22) from breast-cancer patients were analyzed. The negative IMS analysis of 1 to 2 x 10(7) MNC was compared with ICC analysis of 2 x 10(6) unseparated MNC. Negative IMS resulted in 85% mean depletion of MNC. The results showed that 11.7% of the samples were positive by ICC analysis of unseparated MNC, as compared with 23.5% after negative IMS. In samples presenting > 10 CK+ cells, a 4-fold higher number of positive cells was detected by the negative IMS technique. Moreover, there was no evidence for general enrichment of false-positive cells. Altogether our results show that negative IMS is an efficient enrichment method for sensitive detection of CK+ cells in BM/PBPC products from breast-cancer patients. This opens the possibility for further characterization of micrometastatic tumor cells.

Immunocytochemical detection of isolated epithelial cells in bone marrow: non-specific staining and contribution by plasma cells directly reactive to alkaline phosphatase.

Detection of isolated tumour cells (TCs) in bone marrow (BM) from epithelial cancer patients by immunocytochemical (ICC) analysis seems to predict future relapse, but the reported percentages of positive BMs among patients with localized cancer show large variations and the number of detected TCs is low. This emphasizes the importance of thoroughly testing the methods in use. This study was performed to clarify to what extent positive staining of haematopoietic cells (HCs) interferes with the ICC detection of epithelial cells in BM. BM mononuclear cells (MNCs) from normal donors and stage I-II breast cancer patients were stained with anti-cytokeratin (CK) and isotype control monoclonal antibodies (MAbs) followed by alkaline phosphatase (AP)-based visualization of immunolabelled cells. In the ICC staining of normal donors by the anti-CK MAbs AE1/AE3 or A45-B/B3, rare immunoreactive cells were detected in 7/20 and 8/19 BMs, respectively. Morphological examination recognized all these cells as typical HCs. In the breast cancer patients (n = 257), anti-CK-positive cells were detected in 26.6 per cent, excluding cells with HC morphology. Using the same morphological criteria, isotype control-positive cells were detected in 5.4 per cent of patients. Some of the false-positive events were further analysed and cells with strong reactivity against the AP enzyme alone were detected. Double ICC staining recognized the majority of these AP directly-reactive cells as CD45-negative and human Ig kappa/lambda-positive, in accordance with the phenotype of mature plasma cells. Morphological evaluation and adequate controls are important to ensure the diagnostic specificity of micrometastases in BM. It is recommended that the number of BM MNCs included in negative controls should equal the number of cells in the diagnostic specimens.

Chromosome banding analysis of gynecomastias and breast carcinomas in men.

Male breast cancer is 100 times less frequent than its female counterpart and accounts for less than 1% of all cancers in men. Although men with breast cancer also often have gynecomastia, it is still unknown whether gynecomastia per se predisposes the male breast to malignant disease. We describe the cytogenetic analysis of three gynecomastias and four breast cancers in men. No chromosome abnormalities were detected in two cases of gynecomastia, with no other concomitant breast disease. The third gynecomastia sample, taken from a site where a breast carcinoma had previously been removed, had a t(2;11)(p24;p13) as the sole chromosome change; this is the first time that an abnormal karyotype has been described in gynecomastia. All four cancers had clonal chromosome abnormalities. Several cytogenetically unrelated clones were found in the breast tumor and in a metastasis from case 1. In the carcinoma of case 2, a single abnormal clone was found, characterized by loss of the Y chromosome, monosomy 17, and a deletion of the long arm of chromosome 18. In the carcinoma of case 3, a clone with loss of the Y chromosome as the sole change dominated, accompanied by the gain of an X chromosome in a subclone. In the lymph node metastasis examined from case 4, a single clone carrying trisomies for chromosomes 5 and 16 was detected. Our findings, especially when collated with data on the six karyotypically abnormal breast carcinomas in men described previously, indicate that gain of the X chromosome, gain of chromosome 5, loss of the Y chromosome, loss of chromosome 17, and del(18)(q21) are nonrandom abnormalities in male breast carcinomas.

Cytogenetic comparison of primary tumors and lymph node metastases in breast cancer patients.

Chromosome banding analysis of primary tumors and axillary lymph node metastases from 10 breast cancer patients revealed abnormal karyotypes in all samples with cytogenetic similarities between the primary tumor and the metastasis in all informative pairs. Although karyotypically unrelated clones were also found in the lymph node samples, they were less numerous than in the primary tumors, indicating that there was more genetic heterogeneity among the neoplastic cells in the primary than in the secondary tumors. On the other hand, some of the clones had become more complex in the metastases as a result of clonal evolution, and by and large these metastatic breast cancer cases had more karyotypic anomalies than do unselected primary breast carcinomas. Among the aberrations occurring more frequently, and that consequently may predispose to disease spread, were losses of chromosomes 17 and 22 and homogeneously staining regions, a cytogenetic sign of gene amplification.

Cytogenetic analysis shows that carcinosarcomas of the breast are of monoclonal origin.

Carcinosarcoma of the breast is a rare biphasic neoplasm composed of a carcinomatous component contiguous or admixed with a pleomorphic spindle cell component. The issues of the histogenesis and clonal composition of carcinosarcomas have long been debated. We present the first cytogenetic characterization of mammary carcinosarcomas by analysis of eight tumor samples from two patients with this disease. In the first case, the same karyotypically complex clone, as well as evidence of clonal evolution, was found in samples from three separate areas of the primary tumor. The analysis of one intramammary and one axillary lymph node metastasis from the same patient, both showing only the sarcomatous tumor component, also revealed the common complex stemline and one of the two sidelines found in the primary tumor. The carcinosarcoma of the second patient contained six complex but karyotypically related clones unevenly distributed among the three samples examined. From this case, cells belonging to the carcinomatous and sarcomatous tumor components were separated by differential sedimentation and culturing in specific growth media. Analysis of both fractions showed largely the same karyotype, although one of the subclones was restricted to the epithelial component. Our findings indicate that the epithelial and mesenchymal components of mammary carcinosarcomas are both part of the neoplastic parenchyma and that they have evolved from a single common stem cell, in agreement with the hypothesis that the tumors are of monoclonal origin.

The TATA-less, GC-rich porcine dipeptidylpeptidase IV (DPPIV) promoter shows bidirectional activity.

Dipeptidylpeptidase IV (DPPIV) is an exopeptidase highly expressed in the brush-border membrane of the small intestine and in the proximal renal tubules. In this paper the 5'-flanking region of the DPPIV gene containing the promoter was sequenced and functionally characterized. The porcine DPPIV promoter lacks a consensus TATA-box, but contains two TATA-like sequences. Evidence for multiple transcription initiation sites was found. Different deletion constructs of the DPPIV 5'-flanking region in front of the CAT gene were analyzed for transient CAT-expression after transfection of the intestinal Caco-2 cell line. These experiments showed that a 89 base pair construct (-91 to -3 relative to the translation initiation site) is sufficient for promoter activity. In the reverse orientation this construct also stimulates transcription with a similar effectivity indicating that the DPPIV promoter has a bidirectional function. The bidirectional function was further demonstrated by the introduction of the -91 to -3 construct into the bidirectional vector system pLUC/CAT-3. In the hepatoma cell line HepG2 two selected deletion constructs (-857 to -3; -282 to -3) were analyzed in the normal orientation using the CAT gene as a reporter gene. The transfection experiments showed that deletion of the region -857 to -282 raised the promoter activity 3-fold. The GC-rich 5'-flanking region was further analyzed and we demonstrate that the DPPIV promoter contains a region with the characteristics of an unmethylated CpG island.

Discrimination between multicentric and multifocal breast carcinoma by cytogenetic investigation of macroscopically distinct ipsilateral lesions.

Whether macroscopically distinct carcinomas in the same breast are clonally related (multifocal breast carcinoma) or unrelated (multicentric breast carcinoma) is no longer only a scientific-pathological issue but, because different therapeutic strategies may be preferable for cases with intramammary metastatic disease compared with cases of multiple primary breast carcinomas, one that may have profound clinical implications. We studied the evolutionary relationship among macroscopically distinct, ipsilateral breast carcinomas by cytogenetic analysis of 26 tumorous lesions from 12 patients. Sixteen of the 26 foci (62%) were found to contain clonal chromosome abnormalities. Two carcinoma foci were karyotypically abnormal in each of seven patients. Four of these cases had an evolutionarily related, cytogenetically abnormal clone in the two lesions from the same breast, whereas the remaining three cases had completely different clonal karyotypic aberrations in the separate foci. These results, together with our previous findings in five other informative cases, show that multiple, synchronous breast tumors sometimes arise through intramammary spreading of a single primary carcinoma, whereas on other occasions they are the result of the simultaneous emergence of pathogenetically independent carcinomas within the breast. In the total material, an association was seen between the proximity of the foci and the likelihood of them being karyotypically related.

Cytogenetic analysis of several pseudomyxoma peritonei lesions originating from a mucinous cystadenoma of the appendix.

Epithelial proliferative lesions of the appendix are rare and have never been studied cytogenetically. We present the chromosomal banding analysis of four successfully short-term cultured samples from pseudomyxoma peritonei lesions originating from a cystadenoma of the appendix. All four sample contained clonal chromosome abnormalities. In three of them, the clone 46,XX,der(6)?del(6)(q16q21)?del(6)(q27) was found, whereas a clone with the karyotype 46,XX,t(2;17)(p21;p13),t(6;12)(p21;q13),t(12;15)(q24;q15) was detected in the fourth sample. Our findings support the view that pseudomyxoma peritonei originates by spreading from a primary mucinous neoplasm of an intraperitoneal organ rather than through mucinous metaplasia or multifocal primary neoplastic transformation of the peritoneum.

Cytogenetic findings in phyllodes tumors of the breast: karyotypic complexity differentiates between malignant and benign tumors.

Clonal karyotypic abnormalities were detected in short-term cell cultures from six phyllodes tumors of the breast. Whereas all five benign tumors had simple chromosomal changes, the highly malignant one had a near-triploid stemline, indicating that karyotypic complexity is a marker of malignancy in phyllodes tumors. Interstitial deletions of the short arm of chromosome 3, del(3)(p12p14) and del(3)(p21p23),were the only aberrations in two benign tumors. Cytogenetic polyclonality was detected in three benign tumors: two had cytogenetically unrelated clones, whereas the third had three different, karyotypically related cell populations as evidence of clonal evolution. The finding of clonal chromosome abnormalities in both the epithelial and connective tissue components of the phyllodes tumors indicates that they are genuinely biphasic, that is, that both components are part of the neoplastic parenchyma.

Comparison of genetic changes in frozen biopsies and microdissected archival material from the same colorectal liver metastases.

Microdissection of tissue sections from formalin-fixed, paraffin-embedded tumor material allows separation of microscopic sites within a sample. DNA can easily be extracted, and polymerase chain reaction (PCR) technology makes it possible to perform different molecular biologic analyses on small cell populations. The presence of normal cells or tumor heterogeneity may cause false negatives in allelic imbalance (AI) studies. Microdissected well-defined cell populations from a tumor section are assumed to increase the sensitivity of AI analyses. The present study has evaluated this in colorectal liver metastases by comparing genotypes in frozen biopsies with genotypes in microdissected archival samples from the same patients. Constitutional genotypes were obtained from corresponding peripheral blood leukocytes as well as normal liver tissue. Archival samples (n = 43) from 16 patients were analyzed after microdissection with 2-5 of 10 selected microsatellite markers. Frozen biopsies from one metastasis of each patient had previously been investigated at numerous microsatellite loci. From those results we selected, for the comparable analysis of archival samples, 41 tumor genotypes at 10 loci representing 11 heterozygotes, 13 AI, 7 losses of heterozygosity (LOHs), 8 homozygotes, and 2 microsatellite unstable cases. The microdissected samples revealed AI or a complete loss of one allele (LOH) in 5 of 11 (45%) genotypes that were previously evaluated as unchanged (retained heterozygosity) in the frozen biopsies, and LOH in 8 of 13 (62%) genotypes at loci known to exhibit AI in the frozen biopsies. Microsatellite instability, LOH, and homozygosity found in the frozen samples were all confirmed by analyses of the archival material. Intertumoral genetic heterogeneity was found in samples from two patients. The same allelic intensities were seen in DNA from tumor-close liver tissue as in blood DNA from the same patient except in one sample. The present study shows a 54% increase in sensitivity of genetic alterations if pure tumor cell components are used (five "new" AIs and LOHs and eight "new" LOHs among previously scored heterozygotes [n = 11] and AI [n = 13], respectively). In total, a 93% success rate (108/115 analyses) was obtained using standard PCR conditions for the 10 selected markers. The fact that standard PCR conditions and 5-micron tumor sections are used shows how easy these analyses are to perform, and that only minor amounts of valuable archival material is used.

Allelotype profiles of local recurrences and distant metastases from colorectal-cancer patients.

Several genetic alterations have been described in benign and malignant primary tumors of the colorectum, but few such associations have been made with the progression of these tumors. This study compares genetic changes found in distant metastases (n = 22) with local recurrences (n = 15) as well as with primary carcinomas (n = 12). Complete allelotypes of the tumors were obtained by analyzing 43 microsatellite loci, representing all non-acrocentric chromosome arms and mapping to the mid-portion of the arms. Allelic imbalances in the tumor DNA were evaluated by comparison with the patient's constitutional pattern in blood DNA. The allelotype profile of the distant metastases was different from those found in the local recurrences and in the primary carcinomas. More than 20% of the distant metastases exhibited allelic imbalances at loci representing 20 chromosome arms. The majority of these regions were less frequently changed in the local recurrences and in the primary tumors. The markers that most often were altered in the metastasis (>40%) represented chromosome arms 14q, 17p, 18p and 18q. Only two regions, 10p and 19p, were unaltered in all tumors analyzed. We found that the median value of fractional allelic imbalance was twice as high in the distant metastases as in the recurrent tumors. Novel alleles at microsatellite loci were observed in all 3 tumor types, but in the advanced tumors this phenotype was characterized by only a single novel allele seen at less than 10% of the analyzed loci.

Multiple glove powder granulomas masquerading as peritoneal carcinomatosis.

BACKGROUND: The occurrence of glove powder granulomas in peritoneal nodules presumed to represent carcinomatosis has not gained widespread attention and remains a challenging clinical problem even for the experienced surgeon. STUDY DESIGN: During the past four years, we have registered ten cases of multiple glove powder granulomas, believed by the surgeon to represent tumor dissemination, but diagnosed by the pathologist as glove powder granules with the typical Maltese cross pattern when viewed under polarized light. RESULTS: Patients were referred with the presumptive diagnosis of intra-abdominal malignant disease and all had undergone at least one previous laparotomy during the past few years at a hospital that used powdered gloves. The unsuspected perioperative finding of multiple peritoneal nodules was presumed to represent carcinomatosis and resulted in the surgeon erroneously cancelling the operation in one case. In six other cases, the results of perioperative frozen sections caused the surgeon to change strategy and perform the planned procedure. Three patients had starch powder peritonitis postoperatively and one of them had a fatal pulmonary embolus. In two patients, the starch powder reaction was associated with the occurrence of true carcinomatosis and one patient later had recurrence of hepatic metastases. After a mean follow-up period of 17 months, six patients are alive with no signs of disseminated malignancy. CONCLUSIONS: Based on this information, we recommend routine pathologic documentation of all peritoneal nodules appearing to be tumor dissemination. If the results influence the surgical procedure, frozen sections should always be performed. To minimize the risk of powder-induced complications, the use of particle-free gloves is strongly recommended.

The hypothalamocerebellar projection in the cat: branching and nuclear termination.

The hypothalamic projection to the cerebellar nuclei and cortex in the cat was studied by means of retrograde transport of wheat germ agglutinin-horseradish peroxidase complex and various fluorescent tracers. The hypothalamocerebellar nuclear projection originates from various parts of the posterior hypothalamus and reaches mainly the ipsilateral fastigial and interposed nuclei, but all nuclei receive some hypothalamocerebellar fibres. It appears from our double labelling experiments that at least one half of the hypothalamocerebellar nuclear neurones by means of axon collaterals also projects to the cerebellar cortex. Experiments with depositions of fluorescent tracers in both cerebellar hemispheres show that some hypothalamocerebellar fibres branch to reach different parts of the cerebellar cortex. Previous studies have shown that hypothalamocerebellar axons may be branches of hypothalamic efferents to other sites. However, experiments with combined fluorescent tracer depositions in the cerebellum and hippocampus gave no evidence for hypothalamic neurones with axon collaterals to both these regions.

Undifferentiated carcinoma: an immunohistochemical and ultrastructural study.

Twenty-eight undifferentiated carcinomas (UCs) were immunohistochemically investigated with antibodies against cytokeratins (CKs), vimentin, p53 protein, c-erbB-2 protein and CEA. The diagnoses were based on the findings of conventional histopathology, immunohistochemistry and electron microscopy. CKs8, 18 and 19 were the CKs most frequently present in these tumors, in 61%, 61% and 82% of the cases, respectively. Nine of the 28 (32%) UCs were CKs5/6 positive. Expression of CK20 was found in three (11%) cases. Four UCs were sub-type Cks negative, but one of them was confirmed AE1/AE3 positive. P53 protein overexpression was found in nine (32%) cases. One of the patients with p53 protein positive tumors has been alive for 174 months. Nine (32%) UCs expressed vimentin, which included all of the three thyroid UCs. Comparing with our previous study of squamous cell carcinomas, we found that vimentin and CK18 are more frequently expressed in UCs. The overexpression of p53 protein is similar in the two groups of carcinomas and thus, p53 protein is not a differentiation marker in these tumors. Finally, we recommend the use of a CK "cocktail of antibodies" in the diagnosis of UCs.

Assignment of the dipeptidylpeptidase IV (DPP4) gene to pig chromosome 15q21.

A porcine 2-kb partial dipeptidylpeptidase IV (DPP4, EC 3.4.14.5) cDNA clone and a porcine 16-kb genomic fragment containing parts of the DPP4 gene were isolated, characterized, and used as probes to map the DPP4 gene to pig Chr (Chr) 15q21 by fluorescence in situ hybridization. A two-allele RFLP was revealed for the DPP4 gene. This polymorphism was utilized in a linkage test against the erythrocyte antigen G (EAG), previously assigned to Chr 15, and the microsatellite S0088, which is linked to EAG. The linkage analyses revealed significant evidence for linkage confirming the assignment of DPP4 to Chr 15.