Transferable anticancer innate immunity in spontaneous regression/complete resistance mice.

Spontaneous regression/complete resistance (SR/CR) mice resist very high doses of cancer cells that are lethal to WT mice even at low doses. In this study, we show that this resistance is mediated by rapid infiltration of leukocytes, mostly of innate immunity, in both primary and repeated challenges. Formation of rosettes with infiltrating natural killer cells, neutrophils, and macrophages was required for the subsequent destruction of cancer cells through rapid cytolysis. Highly purified natural killer cells, macrophages, and neutrophils from the SR/CR mice independently killed cancer cells in vitro. The independent killing activity by each subset of effector cells is consistent with the observation that the resistance was abolished by depleting total infiltrating leukocytes but not by depleting only one or two subsets of leukocytes. The resistance was completely transferable to WT recipient mice through SR/CR splenocytes, bone marrow cells, or enriched peritoneal macrophages, either for prevention against subsequent cancer challenges or eradication of established malignancy at distant sites.

Trisomy of chromosome 18 in the baboon (Papio hamadryas anubis).

Trisomy 18 is usually a lethal chromosomal abnormality and is the second most common autosomal trisomy in humans, with an incidence of 1:8000 live births. It is commonly associated with abnormalities of the lower and upper extremities, having the frequency of 95% and 65%, respectively. A newborn female olive baboon (Papio hamadryas anubis) was diagnosed with intrauterine growth retardation and severe arthrogryposis-like congenital joint deformities. Cytogenetic analysis including G-banding and fluorescence in situ hybridization (FISH) revealed that the congenital abnormalities were associated with chromosomal mosaicism for trisomy 18. Genetic analysis with microsatellites from chromosome 18 confirmed the maternal origin of the extra chromosome 18. This is the first report of trisomy 18 in the baboon, which may be a promising animal model of human disease.

The HA-2 minor histocompatibility antigen is derived from a diallelic gene encoding a novel human class I myosin protein.

Human minor histocompatibility Ags (mHag) present significant barriers to successful bone marrow transplantation. However, the structure of human mHag and the basis for antigenic disparities are still largely unknown. Here we report the identification of the gene encoding the human mHag HA-2 as a previously unknown member of the class I myosin family, which we have designated MYO1G. The gene is located on the short arm of chromosome 7. Expression of this gene is limited to cells of hemopoietic origin, in keeping with the previously defined tissue expression of the HA-2 Ag. RT-PCR amplification of MYO1G from different individuals led to the identification of two genetic variants, designated MYO1G(V) and MYO1G(M). The former encodes the peptide sequence previously shown to be the HA-2 epitope (YIGEVLVSV), whereas the latter shows a single amino acid change in this peptide (YIGEVLVSM). This change has only a modest effect on peptide binding to the class I MHC-restricted element HLA-A*0201, and a minimal impact on recognition by T cells when added exogenously to target cells. Nonetheless, as detected using either T cells or mass spectrometry, this amino acid change results in a failure of the latter peptide to be presented at the surface of cells that express MYO1G(M) endogenously. These studies have thus identified a new mHag-encoding gene, and thereby provide additional information about both the genetic origins of human mHag as well as the underlying basis of an Ag-positive vs Ag-negative state.

Spermatid-specific expression of the novel X-linked gene product SPAN-X localized to the nucleus of human spermatozoa.

Formation of mature spermatozoa involves a series of dramatic molecular and morphological changes in the male germ cell lineage. These changes result from the temporally regulated transcription and translation of several testis-specific gene products. Here, we describe a novel, testis-specific protein designated SPAN-X for sperm protein associated with the nucleus on the X chromosome. SPAN-X sequences showed no significant similarity with known cDNA or peptide sequences. The SPAN-X peptide sequences contained three overlapping consensus nuclear localization signals, a high percentage (33%-37%) of charged amino acid residues, and a relatively acidic isoelectric point (pI; 4.88-6.05). Northern analysis of mRNA from multiple human tissues identified a SPAN-X transcript exclusively in the testis. In situ hybridization of human testes sections showed SPAN-X mRNA expression in haploid, round, and elongating spermatids. The SPANX gene was mapped to chromosome Xq27. 1 by fluorescence in situ hybridization and by Southern blot analysis of human/mouse somatic cell hybrids. On Western blots of human sperm proteins, antirecombinant SPAN-X antibodies reacted with broad bands migrating between 15-20 kDa. Immunofluorescent labeling of human spermatozoa demonstrated SPAN-X localization to nuclear craters and cytoplasmic droplets. Expression of SPAN-X, an X-linked gene product, exclusively in haploid spermatids leads to interesting questions regarding the transcription of sex-linked genes during spermiogenesis.

Analysis of the human LHX3 neuroendocrine transcription factor gene and mapping to the subtelomeric region of chromosome 9.

The Lhx3 LIM homeodomain transcription factor is critical to pituitary organogenesis and motor neuron development. We determined the genomic structure and chromosomal localization of human LHX3. The gene contains seven coding exons and six introns that span 8.7 kilobases in length. The LHX3 gene codes for two functionally distinct isoforms that differ in their amino termini but share common LIM domains and a homeodomain. The functional domains of the LHX3 proteins are encoded by distinct exons. The alternate amino termini and LIM domains lie within individual exons, and the homeodomain is coded by two exons interrupted by a small intron. Human LHX3 maps to the subtelomeric region of chromosome 9 at band 9q34.3, within a region noted for chromosomal translocation and insertion events. Characterization of the genomic organization and chromosomal localization of LHX3 will enable molecular evaluation and genetic diagnoses of pituitary diseases and central nervous system developmental disorders in humans.

Genetic heterogeneity of gingival fibromatosis on chromosome 2p.

Gingival fibromatosis (GF) occurs in several genetic forms as a simple Mendelian trait, in malformation syndromes, and in some chromosomal disorders. Specific genes responsible for GF have not been identified. An autosomal dominant form of hereditary gingival fibromatosis (HGF, MIM 135300) was recently mapped to chromosome 2p21 in a large Brazilian family and there was an earlier report of GF in a boy with a cytogenetic duplication involving 2p13-->p21. We thus hypothesised that a common gene locus may be responsible for GF in both the Brazilian family and the boy with the chromosome 2p duplication. We performed additional genetic linkage studies on the Brazilian family and molecular cytogenetic studies on the patient with the cytogenetic duplication to correlate more precisely the genetic interval of the HGF phenotype with the duplicated 2p interval. Additional linkage analysis of new family members resulted in refinement of the candidate region for HGF to an 8 Mb region. Molecular cytogenetic analysis of the 2p13-->p21 duplication associated with GF showed that the duplicated region was proximal to the candidate interval for HGF. Thus, our results support the presence of two different gene loci on chromosome 2p that are involved in GF.

Gonadoblastomas in 45,X/46,XY mosaicism: analysis of Y chromosome distribution by fluorescence in situ hybridization.

Gonadoblastomas are composed of nests of neoplastic germ cells and sex cord derivatives surrounded by ovarian-type stroma. These tumors are found almost exclusively in persons with gonadal dysgenesis associated with a Y chromosome or Y chromosome fragment, and accordingly, the Y chromosome has been implicated in gonadoblastoma oncogenesis. To evaluate this association, we used two-color fluorescence in situ hybridization with chromosome-specific probes to determine the distribution of the X and Y chromosomes in the tumor nests and surrounding stromal cells in paraffin tissue sections of three gonadoblastomas in two patients with gonadal dysgenesis and 45,X/46,XY mosaicism. Statistical analysis of the data from the fluorescence in situ hybridization demonstrated that in all three gonadoblastomas, the proportion of nuclei with a Y chromosome signal was significantly higher in the tumor cells than in the nontumoral cells of the surrounding stroma (P<.001). These results suggest that Y chromosome material participates in gonadoblastoma tumorigenesis.

Molecular analysis of recombination in a family with Duchenne muscular dystrophy and a large pericentric X chromosome inversion.

It has been demonstrated in animal studies that, in animals heterozygous for pericentric chromosomal inversions, loop formation is greatly reduced during meiosis. This results in absence of recombination within the inverted segment, with recombination seen only outside the inversion. A recent study in yeast has shown that telomeres, rather than centromeres, lead in chromosome movement just prior to meiosis and may be involved in promoting recombination. We studied by cytogenetic analysis and DNA polymorphisms the nature of meiotic recombination in a three-generation family with a large pericentric X chromosome inversion, inv(X)(p21.1q26), in which Duchenne muscular dystrophy (DMD) was cosegregating with the inversion. On DNA analysis there was no evidence of meiotic recombination between the inverted and normal X chromosomes in the inverted segment. Recombination was seen at the telomeric regions, Xp22 and Xq27-28. No deletion or point mutation was found on analysis of the DMD gene. On the basis of the FISH results, we believe that the X inversion is the mutation responsible for DMD in this family. Our results indicate that (1) pericentric X chromosome inversions result in reduction of recombination between the normal and inverted X chromosomes; (2) meiotic X chromosome pairing in these individuals is likely initiated at the telomeres; and (3) in this family DMD is caused by the pericentric inversion.

Constellation of congenital abnormalities in an infant: a new syndrome or tissue-specific mosaicism for trisomy 18?

A newborn infant born to consanguineous (first cousin) parents was noted to have complex congenital heart defect and minor anomalies suggestive of trisomy 18. Blood lymphocyte and skin fibroblast karyotypes were normal. He died in the neonatal period of postoperative complications. On interphase fluorescence in-situ hybridization (FISH) using autopsy specimens, a significant number of cells in the liver (17%) were trisomic for chromosome 18, compared to normal control liver tissue. However, interphase FISH analyses of blood lymphocytes, skin fibroblasts, and kidney tissue were normal. It is our opinion that this apparent mosaicism for trisomy 18 in the patient's liver may be spurious, though it brings into focus the issue of possible tissue/organ-specific mosaicism. The anomalies in this infant do not resemble a previously described malformation syndrome. Parental consanguinity raises the possibility that this represents a new autosomal recessive malformation syndrome.

Ovarian granulosa cell tumors with bizarre nuclei: an immunohistochemical analysis with fluorescence in situ hybridization documenting trisomy 12 in the bizarre component [corrected].

Granulosa cell tumors with bizarre nuclei (GCT-BN) are rare lesions with a prognosis apparently similar to that of conventional granulosa cell tumors (GCT-NOS). The immunohistochemical features of GCT-BN have not been described, and the exact nature of the bizarre nuclei (BN) is unclear. Thirteen GCT-BN were studied with antibodies to cytokeratin, vimentin, epithelial membrane antigen, muscle-specific actin, alpha smooth muscle actin, desmin, and S-100 protein. Six cases were also examined by fluorescence in situ hybridization for trisomy 12, a nonrandom chromosomal aberration found in a proportion of ovarian sex-cord stromal tumors. Histologically, 12 tumors (86%) contained BN areas interspersed with large areas of GCT-NOS. The remaining tumor contained only microscopic foci of GCT-NOS. Immunohistochemically, the tumors stained for vimentin (13 tumors), S-100 protein (11 tumors), muscle-specific actin (10 tumors), cytokeratin (eight tumors), alpha smooth muscle actin (eight tumors), and desmin (one tumor), but none stained for epithelial membrane antigen. Immunostaining results for the BN and GCT-NOS areas were concordant in eight (73%) of the 11 tumors in which both areas could be independently assessed. The remaining three tumors (27%) showed discordant results for only one of the eight markers used. In five patients, trisomy 12 was detected by fluorescence in situ hybridization in areas of BN but not in areas of GCT-NOS present in the same tumor. Trisomy 12 was also present in another BN tumor in which the foci of GCT-NOS were too small to be evaluated. We conclude that within GCT-BN, areas with BN are immunohistochemically similar to areas of GCT-NOS present in the same tumor. The finding of trisomy 12 in areas with BN but not GCT-NOS in the same tumor, however, suggests that cells with BN represent a genetically distinct clone of tumor cells arising within GCT-NOS.

Loss of chromosomes 22 and 14 in the malignant progression of meningiomas. A comparative study of fluorescence in situ hybridization (FISH) and standard cytogenetic analysis.

The majority of meningiomas are classified as typical and have a relatively benign course. However, approximately 10% are diagnosed as atypical, anaplastic, or malignant and have a worse prognosis. The genetic differences between the typical and higher grade meningiomas are not well characterized, although there appear to be increasingly complex karyotypic changes associated with the higher grade tumors. Because higher grade meningiomas are not common tumors, and because of the inherent problems associated with the culturing of tumors, the use of interphase cytogenetic techniques with paraffin-embedded archival material is desirable for studying these neoplasms. To determine its accuracy in detecting aneuploidy, we performed fluorescence in situ hybridization (FISH) on 2-micron paraffin sections of nine previously karyotyped meningiomas using an alpha-satellite probe for chromosomes 14 and 22. Sections of normal tissue from six patients without malignancy were used as controls. FISH analysis detected all of the chromosome losses in the meningioma cases that had been characterized cytogenetically. In five cases, cell lines not detected by standard cytogenetics were identified by FISH. These results indicate that FISH is a reliable method for detecting chromosomal loss and may be more sensitive than standard cytogenetics alone. Furthermore, the results of this study support the concept that loss of chromosome 14 is associated with malignant progression in meningiomas.

Interphase fluorescence in situ hybridization for trisomy 12 on archival ovarian sex cord-stromal tumors.

Trisomy 12 is a nonrandom chromosomal abnormality found in a large proportion of ovarian sex cord-stromal tumors (OSCTs), including thecoma-fibromas (TFs) and granulosa cell tumors (GCTs). The prognostic significance of trisomy 12 in these tumors, however, is unknown. A series of 16 OSCTs, obtained from patients with long-term follow-up, was analyzed for the presence of trisomy 12 by interphase fluorescence in situ hybridization on paraffin-embedded sections. Sections of the contralateral nonneoplastic ovary were available in five cases and utilized as controls. Evidence of trisomy 12 was detected in 9 of 10 TFs, and contrary to previous reports, in only one of six GCTs. One TF with trisomy 12 was a malignant variant that resulted in the death of the patient in 5 months, but the remaining TFs with trisomy 12 were cytologically and clinically benign in those with follow-up available. The single GCT with trisomy 12 was a nonaggressive, stage 1 lesion without evidence of recurrence after 264 months, whereas those GCTs without trisomy 12 included one stage 2 tumor and a cytologically atypical GCT with tumor necrosis and an elevated number of mitotic figures. The evidence suggests that the great majority of OSCTs with trisomy 12 is clinically benign, but not all benign OSCTs have trisomy 12. We conclude that the presence of trisomy 12 is of limited prognostic usefulness in OSCTs.

Malignant rhabdoid tumor of the kidney: involvement of chromosome 22.

Cytogenetic and molecular studies have demonstrated that involvement of 22q is a non-random finding in malignant rhabdoid tumors (MRTs) of the brain. We present an MRT of the kidney with the karyotype 47,XY, + i(1)(q10), der(8)t(8;22)(q12;q11.2),der(22)t(8;22)(q23 or q24.1;q11.2). This unbalanced reciprocal translocation was confirmed by fluorescence in situ hybridization (FISH) with chromosome-specific paints for chromosomes 8 and 22. Molecular analysis demonstrated a partial deletion of 22q in the BCR region at q11.2, strengthening the suspicion that this is a critical region for the initiation or progression of these highly malignant neoplasms. Establishing non-random cytogenetic changes in MRTs arising from the kidney may be of value in distinguishing these rare, but often fatal tumors from other renal neoplasms that mimic them histologically. The similarity in cytogenetic and molecular abnormalities between renal and extra-renal MRTs argues against the concept that extra-renal MRTs are only representative of a rhabdoid phenotype, rather than being true rhabdoid tumors.

Refinement of the localization of the gene for human intraacrosomal protein SP-10 (ACRV1) to the junction of bands q23-->q24 of chromosome 11 by nonisotopic in situ hybridization.

The human sperm antigen SP-10 is a testis-specific protein associated with the matrix of the acrosomal vesicle in developing spermatids and the acrosomal matrix and membranes of ejaculated sperm. A previous study, utilizing somatic cell hybrids, localized the gene for SP-10 to chromosome 11 and assigned the gene symbol ACRV1 (acrosomal vesicle protein-1). Although previous analysis of several somatic cell hybrids containing portions of chromosome 11 indicated that ACRV1 was in the p12-->q13 region, the present fluorescence in situ hybridization studies using cDNA, ribo, and genomic versions of probes for SP-10 coupled to analysis of an expanded series of somatic cell hybrids demonstrated the refined localization of ACRV1 to the junction of bands q23 and q24 of chromosome 11. A comparison of the three types of probes used for the in situ study demonstrated that while the genomic probe hybridized most efficiently, the riboprobe hybridized to the same location and was superior to the cDNA probe in mapping this single-copy gene. This report emphasizes the utility of riboprobes for chromosome localization of single-copy genes.