Molecular cytogenetic characterization of the 11q13 amplicon in head and neck squamous cell carcinoma.

Amplification of 11q13 DNA sequences and overexpression of CCND1 are common findings in head and neck squamous cell carcinoma (HNSCC), identified in about 30% of the cases. However, little is known about initiation of the amplification and the organization of the amplicon. In order to study the structure of the amplicon in more detail and to learn more about the mechanisms involved in its initiation, prometaphase, metaphase, and anaphase fluorescence in situ hybridization (FISH) with 40 BAC clones spanning a 16-Mb region in chromosome bands 11q12.2 to 11q13.5 was performed in nine HNSCC cell lines with homogeneously staining regions. FISH analysis showed that the size of the amplicon varied among the nine cell lines, the smallest being 2.12 Mb and the largest 8.97 Mb. The smallest overlapping region of amplification was approximately 1.61 Mb, covering the region from BAC 729E14 to BAC 102B19. This region contained several genes previously shown to be amplified and overexpressed in HNSCC, including CCDN1, CTTN, SHANK2, and ORAOV1. The cell lines were also used to study the internal structure of the amplicon. Various patterns of amplified DNA sequences within the amplicon were found among the nine cell lines. Even within the same cell line, different amplicon structures could be found in different cell populations, indicating that the mechanisms involved in the development of the amplicons in HNSCC were more complex than previously assumed. The frequent finding of inverted repeats within the amplicons, however, suggests that breakage-fusion-bridge cycles are important in the initiation, but the fact that such repeats constituted only small parts of the amplicons indicate that they are further rearranged during tumor progression.

Cytogenetic and fluorescence in situ hybridization characterization of chromosome 1 rearrangements in head and neck carcinomas delineate a target region for deletions within 1p11-1p13.

Cytogenetic analyses have revealed structural rearrangements of chromosome 1 in a large fraction of head and neck carcinomas (HNCA). These aberrations frequently affect chromosomal band 1p13 and the centromeric region, the latter often in the form of isochromosome i(1q) and whole-arm translocations. To delineate the critical region involved in rearrangements of proximal 1p, we have undertaken a more precise breakpoint mapping in 13 HNCAs, using metaphase fluorescence in situ hybridization with 11 yeast artificial chromosome (YAC) clones spanning 1p. All of the tumors had chromosome 1 changes at G-banding analyses. Fluorescence in situ hybridization showed that in almost all of the cases, at least one copy of chromosome 1 was affected by centromeric rearrangement. By the use of YAC clones mapped to juxtacentromeric regions and a centromere-specific alpha-satellite probe, we detected variable breakpoints in the whole-arm translocations. At the cytogenetic level, 1p13 rearrangements were frequent. However, molecular breakpoints within this band varied among the HNCAs tested. The lack of consistently rearranged chromosome segments indicates that the pathogenetically important consequence of 1p rearrangements in HNCAs is loss and/or gain of genes outside the breakpoint regions. In an assessment of the genomic imbalances, partial or complete overrepresentation of 1q was seen in eight cases. Loss of 1p material was also identified in eight cases; and in four of them, the deleted segments were too small to be discovered by G-banding analysis. The minimal overlapping deleted region was in the interval between YAC 959C4 (band p11-p12) and the centromere (p10). Our findings indicate that a target region potentially harboring tumor suppressor gene(s) crucial for HNCA is located within chromosomal bands 1p11-p13.

Chromosome abnormalities in eighty-three head and neck squamous cell carcinomas: influence of culture conditions on karyotypic pattern.

Short-term cultures from 115 squamous cell carcinomas (SCC) of the head and neck were cytogenetically investigated. Thirty-six of the tumors have been reported previously, whereas 79 are new cases. The material was divided into two series based on the medium used. The 80 tumors of series I were cultured in RPMI 1640 supplemented with fetal calf serum, glutamine, antibiotics, insulin, cholera toxin, and epidermal growth factor. The 35 tumors of series II were cultured in a chemically defined, serum-free medium with a low calcium concentration, MCDB 153, which stimulates epithelial growth while inhibiting fibroblasts. A total of 83 tumors with clonal karyotypic abnormalities were detected in the two series. Series II had a higher proportion of tumors with complex karyotypic changes than series I (43% versus 15%), a lower proportion of tumors with pseudo- or neardiploid clones characterized by simple rearrangements (3% versus 34%), and a lower frequency of unrelated clones (3% versus 24%), indicating that the different culture conditions favored growth of different cell populations. Except for rearrangements of 1p22, which were mainly found in series I, the distribution of breakpoints in structural aberrations was similar in the two series and clustered to several chromosomal bands or regions, in particular 11q13, 1p22, 1p11-12, 3p11-q11, 5q13, 1q25, 15q10, and 8q10. Unbalanced structural aberrations were more common in series II, frequently leading to loss of segments from chromosome arms 3p, 7q, 8p, 11q, 13p, 14p, and 15p, whereas gain of genetic material often involved chromosome arms 1q, 3q, 8q, and 15q.

Cytogenetic analysis of 33 basal cell carcinomas.

Cytogenetic analysis of short-term cultures from 33 basal cell carcinomas (BCC), a type of neoplasm for which no previous karyological data exist, revealed clonal chromosome aberrations, all of them different, in 8 tumors. In 2 cases, 2 cytogenetically unrelated clones were detected, suggesting a multicellular origin in at least a subset of BCC. A remarkably high level of nonclonal structural rearrangements, mostly in the form of seemingly balanced translocations, was found in 23 tumors; namely, in 6 of 8 BCC with clonal karyotypic abnormalities and in 17 of 25 without. It is possible that some of these aberrations represent additional neoplastic clones, thus indicating an even higher level of cytogenetic heterogeneity in BCC. We think that the most likely interpretation of the results is that BCC may have a multicellular origin, reflecting field cancerization of the skin. During subsequent tumor development, the selection pressure narrows down the number of clones that infiltrate the surrounding tissue. The finding by karyotypic analysis of some apparently monoclonal, some polyclonal BCC, may reflect that different tumors have been examined at different points in the clonal evolution of the neoplastic cells.

Nonrandom chromosome abnormalities in short-term cultured primary squamous cell carcinomas of the head and neck.

We report the finding of clonal chromosome abnormalities in short-term cultures from 44 squamous cell carcinomas of the head and neck region. Eleven tumors had gain or loss of the Y chromosome, sometimes one clone with +Y and another with -Y, as the sole anomaly, whereas the remaining 33 all carried structural rearrangements and usually were cytogenetically complex with multiple aberrations. The chromosomal bands most frequently involved were, in decreasing order of frequency, 8p11-q11, 1p11-q11, 3p11-q11, 11q13, 13p11-q11, 1p13, 5p11-q11, 7p11-q11, 15p11-q11, and 14p11-q11. Almost one-half of the breakpoints were located in centromeric or juxtacentromeric bands. Recurrent aberrations included i(8q), i(5p), i(1q), del(3)(p11-12), del(5)(p11), t(1;1)(p13;q25), and der(14;15)(q10;q10). To see whether the karyotypic features of head and neck squamous cell carcinoma differ depending on exact tumor site, we added to the present series our previously published 23 karyotypically abnormal head and neck squamous cell carcinomas that had been cultured in the same way as the tumors of the present series. In the ensuing correlation analysis, tumors of the oral cavity and oropharynx and hypopharynx were found to share many features: highly complex karyotypes were frequent, often containing isochromosomes such as i(8q) and i(5p), and also rearrangements of 11q13 (often as homogeneously staining regions) and loss of genetic material from the short arms of chromosomes 3, 13, 14, and 15 were repeatedly seen. Laryngeal carcinomas, on the other hand, often had simple karyotypic changes.

A relationship between retinol and cellular retinol-binding protein concentrations in human squamous cell carcinomas.

The retinol and retinyl ester concentrations in human xenografted squamous cell carcinomas, with various concentrations of cellular retinol-binding protein (CRBP), were studied, as well as the in vivo uptake and esterification in these tumours of labelled retinol, presented as a complex with plasma RBP. The mean retinol concentration in the different tumours was in the range 3.7-6.2 nmol/g protein, and the mean CRBP concentration was between 16 and 69 nmol/g protein. There was a statistically significant correlation between the retinol and the CRBP concentrations in the same tumour (P less than 0.001; r = 0.622). Calculation of the maximal extent of retinol-saturation of CRBP showed low values (range: 9-26%). Retinyl palmitate, the predominant retinyl ester, comprised approx. 70% of the retinyl esters in the tumours. There was no correlation between the concentration of CRBP and that of retinyl palmitate. The uptake of [3H]retinol from intravenously injected retinol-RBP complex was similar in the four human squamous cell carcinomas studied, and not related to their CRBP concentration. 20% of the radioactivity in tumour specimens was lipid soluble, as compared to 96% in liver specimens, showing that in the former a higher fraction metabolised to polar compounds. Taken together, our results suggest that in these squamous carcinoma cells, factors other than cellular CRBP content are the major determinants of net cellular uptake and esterification of retinol. The cellular retinol concentration, on the other hand, appears proportional to CRBP content.

Fluorescent in situ hybridisation (FISH) characterisation of pericentromeric breakpoints on chromosome 5 in head and neck squamous cell carcinomas.

Pericentromeric rearrangements, such as isochromosomes and whole-arm translocations, are frequently encountered in short-term cultures from head and neck squamous cell carcinomas (HNSCC). To characterise further the localisation of the breakpoints in such rearrangements, metaphase cells from seven HNSCC known to carry structural rearrangements of the pericentromeric region of chromosome 5 were investigated using fluorescent in situ hybridisation (FISH) techniques. With a whole chromosome painting probe it could be confirmed that all chromosome 5 rearrangements identified at cytogenetic analysis contained chromosome 5 material. By using a centromere-specific alpha satellite probe it could be shown, however, that cytogenetically identical derivative chromosomes had different breakpoints. Thus, we conclude that the results of the present investigation add further support to the hypothesis that the essential outcome of near-centromeric chromosome rearrangements is the creation of genomic imbalances, i.e. gain and/or loss of neoplasia-associated genes.

In vitro toxicity of (191)Pt-labeled cisplatin to a human cervical carcinoma cell line (ME-180).

PURPOSE: The aim of the present work was to examine the effect of (191)Pt-cisplatin, and to study the manner in which radiation and cisplatin interact, in a human cervical carcinoma cell line (ME-180). METHODS AND MATERIALS: The cells were incubated for 1 hour with nonradioactive cisplatin or (191)Pt-cisplatin with specific activities in the range 48-167 MBq/mg. The surviving fraction of the cells after 7 days' growth was determined with a nonclonogenic tetrazolium-based (MTT) assay. The uptake of platinum into the cell and the amount of platinum bound to DNA was measured. RESULTS: The 50% inhibition concentration (IC(50)) decreased with increasing specific activity of the (191)Pt-cisplatin. For the specific activities 0 (nonradioactive), 48, 89, 143, 157, and 167 MBq/mg, IC(50) was found to be 3.24 +/- 0.08, 2.77 +/- 0.55, 2.17 +/- 0.34, 1.15 +/- 0.04, 1.02 +/- 0.03, and 0.76 +/- 0.13 respectively. Isobologram analysis showed a supra-additive (synergistic) interaction between the radiotoxicity and chemotoxicity for specific activities over 100 MBq/mg. CONCLUSION: The cytotoxic effect of cisplatin may be enhanced by labeling the drug with the radionuclide (191)Pt.

Metoclopramide enhances the effect of ionizing radiation on xenografted squamous cell carcinoma of the head and neck.

The commonly used drug metoclopramide, a benzamide derivative, has been shown previously in our laboratory to enhance the effect of cisplatin on xenografted squamous cell carcinomas of the head and neck. In the present study, we show that metoclopramide also enhances the effect of ionizing radiation. Two human squamous cell carcinoma lines of the head and neck xenografted to nude mice have been used. Doses of radiation were chosen (5 and 8 Gy single doses) which caused only a slight retardation of tumor growth when administered alone. Tumor response to ionizing radiation was assessed with and without metoclopramide (2.0 mg kg-1), and administered at the time of radiation and 24 and 48 hr after treatment. The effects of these schedules on the tumors were compared using the reduction of the area under the growth curves and specific growth delay. The dose schedule with metoclopramide alone did not induce any significant reduction in the area under the growth curves. The addition of metoclopramide to the radiated groups caused a significant enhancement of the radiation-induced reduction of the area under the growth curves in both of the tumor lines studied.

Antitumor effect of radioactive cisplatin (191Pt) on nude mice.

PURPOSE: To investigate the effect of (191)Pt-cisplatin in vivo in terms of the antitumor effect and general toxicity on tumor-bearing nude mice. METHODS AND MATERIALS: Tumor-bearing (human squamous cell carcinoma, AB) nude mice were divided into four groups and given, i.p., physiological saline (controls), cisplatin, (191)Pt-cisplatin (80 MBq/mg), or (191)Pt-cisplatin (160 MBq/mg), respectively. Mortality and weight were used as parameters for monitoring general toxic effect, while specific growth delay (SGD) and the area under the logarithm of the relative tumor size curve (AUC-log[RTS]) were used to evaluate the antitumor effect of the treatments. RESULTS: Both SGD and AUC-log(RTS) values showed that (191)Pt-cisplatin was significantly (P < 0.05) more effective in retarding tumor growth than nonradioactive cisplatin. No differences in mortality between the different groups could be observed and no significant differences in weight change between the mice treated with cisplatin or (191)Pt-cisplatin could be seen. CONCLUSION: (191)Pt-cisplatin is a more effective drug than nonradioactive cisplatin in retarding tumor growth on nude mice without adding systemic toxic effects. We believe that radioactive cisplatin may prove to be an alternative to conventional cisplatin; however, the possible toxic effects on organs at risk have to be thoroughly investigated.

Growth of xenografted squamous cell carcinoma of the head and neck--possible correlation with patient survival.

Tumor biopsies from 100 cases of squamous cell carcinoma of the head and neck (HNSCC) were xenografted to athymic nude mice. To ascertain whether xenograft take might be a factor of clinical significance, it was compared with patient survival, the patients being divided into two groups (take and non-take) according to the results of transplantation. The tumor take rate was 29%. Median survival time with respect to cancer death was 18 months in the take group, as compared with over 74 months in the non-take group (p = 0.06). No significant differences were observed between the two groups with respect to age, tumor size, nodal status, clinical stage or histological differentiation. The findings suggest that take of HNSCC xenografts in nude mice may reflect the malignant potential of the original tumor. Moreover, the possibility cannot be excluded of a selection bias favoring the use of such malignant xenografts in therapeutic studies in nude mice.

Abnormal cell cycle regulation in malignancy.

The cell cycle consists of an initial growth phase (G1), DNA replication (S), a gap phase (G2), and mitosis (M), after which the cell may differentiate or enter the resting state (G0). The cycle is driven by a number of positive and negative regulatory phosphorylation and dephosphorylation events, involving protein kinases, protein phosphatases, cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors, that ultimately impinge on the activity of transcription factors. Unreplicated or damaged DNA blocks the progression of the cell cycle at checkpoints, including a late G1 checkpoint regulated by the dephosphorylated retinoblastoma protein and a late G2 checkpoint regulated by the phosphorylation of cyclin-dependent kinase 1 complexed with cyclin B. Many cell cycle regulator genes may be considered proto-oncogenes or tumor suppressor genes, and point mutations, amplifications, deletions, or rearrangements involving their loci, particularly those in the "RB pathway," are associated with various tumors. A number of molecular techniques may be used to detect genomic alterations or posttranscriptional modifications, but immunohistochemistry remains the most common method to determine expression levels of a regulatory protein. Multivariate analysis of the usefulness in prognosis has been applied most often for the general proliferation antigen Ki-67.

Cytogenetic and fluorescence in situ hybridization characterization of chromosome 8 rearrangements in head and neck squamous cell carcinomas.

Structural rearrangements of chromosome 8 are frequently encountered in squamous cell carcinomas of the head and neck (HNSCC). These aberrations often affect the centromeric region, resulting in the formation of isochromosome i(8q) and whole arm translocations. Some tumors may display structural rearrangements of 8p23. To characterize further the localization of the breakpoints in such rearrangements, 12 HNSCC known to carry pericentromeric rearrangements of chromosome 8 and 8p23 abnormalities were investigated with fluorescence in situ hybridization (FISH) by the use of 15 YAC clones spanning 8p23 and 8p11 to 8q11. FISH confirmed that all, except one, aberrations cytogenetically interpreted to be i(8q) were true, monocentric i(8q). Similarly, all whole-arm translocations appeared as centric fusions. It could thus be concluded that the essential outcome of these rearrangements is genomic imbalances and not rearrangement of genes in the pericentromeric region. By the use of five YAC clones mapping to 8p23, different breakpoints at the molecular level were disclosed in cases with cytogenetically identical 8p23 rearrangements. An evaluation of the genomic imbalances detected in the present series revealed that overrepresentation of 8q material was present in 11 of the 12 tumors. The most commonly gained segment was 8q22 approximately qter, found in all cases with 8q overrepresentation. Loss of parts of or the entire 8p was seen in 10 tumors. The smallest overlapping deleted region was localized to the subtelomeric region of 8p.

Tetraploidization and progressive loss of 6q in a squamous cell carcinoma of the parotid gland.

Cytogenetic analysis of short-term cultures from a squamous cell carcinoma (SCC) of the parotid gland revealed one clone with loss of the Y chromosome only, as well as three related subclones: 91,XXYY,add(6)(q21), -11,t(11;22)(q13;q11),ins(15;?)(q22;?)[cp5+ ++]/91,XXYY,add (6)(q21), -11,add(11)(p11), ins(15;?)(q22;?),der(22)t(11;22)(p11;q11)[2]/91,XXYY,add(6)(q11), -11,add(11)(p11),ins(15;?)(q22;?),der(22) t(11;22)(p11;q11) [cp4]. The finding of only one copy of all structurally rearranged chromosomes in a near-tetraploid karyotype indicates that tetraploidization was an early event in tumorigenesis. Rearrangements, in particular deletions, of 6q have previously been associated with adenoid salivary gland malignancies. Our finding of progressive 6q loss with clonal evolution, combined with the fact that 6q deletions were also seen in the two previously reported SCCs of the salivary glands, indicate that loss of genetic information from this chromosome arm is characteristic for most types of salivary gland carcinomas, irrespective of their histologic differentiation.

Multiple apparently unrelated clonal chromosome abnormalities in a squamous cell carcinoma of the tongue.

We have cytogenetically examined short-term cultures from a squamous cell carcinoma of the tongue, a tumor type in which chromosome aberrations hitherto have not been reported. No less than 12 pseudodiploid clones were detected, giving the tumor karyotype 46,X,der(X)t(X;1)(q26;p32),der(1)(Xqter----Xq26::1p32 ----cen----1q42:), del(13)(q11q21),t(15;?) (q26;?)/46,XX,t(1;?)(p34;?),inv(2)(p21q11)/46,XX,t(1;10)(p32;q24)/ 46,XX, + der(1)(12pter----12p11::1p11----cen----1q32:: 11q13----11q22::1q32----1q42:), del(11)(q13q22), -12, der(17)t(1;17) (q42;p13)/46,XX,inv(1)(p22q44)/47,XX,del(1)(q32),der(17)t(1; 17)(p22;q25), der(1)inv(1) (q25q44)t(1;17)(p22;q25),ins(14;7)(q11;q22q36), + 14/46,XX,t(1;4)(q23;q35)/46, XX,t(1;21) (q25;q22),t(2;10)(q31;q26),t(22;?)(q12;?)/46,XX,del(1)(q32)/46,XX, t(1;8)(q44;q21)/46,XX, t(2;21)(q11;p11)/46,XX,t(9;11)(q34;q13). The large number of apparently unrelated abnormalities leads us to suggest that the carcinoma may have been of multiclonal origin.

Multiple unrelated clonal chromosome abnormalities in an in situ squamous cell carcinoma of the skin.

We have cytogenetically analyzed short-term cultures from an in situ squamous cell carcinoma of the skin (Bowen's disease). The following mosaic tumor karyotype was found: 46,XX, -1, +der(1)(pter----p22::q11----cen----p22:), -9, +der(9)t(1;9)(q11; p24)/46,XX,t(3;6) (q21;p21)/46,XX,t(5;14)(q13;q24),t(7;18)(q32;q11)/46,XX,t(8;11)(p22;q13) /46, XX,t(8;11) (p22;q13),t(15;17) (q13;q24)/46,XX,t(12;15)(q12;p11). None of the rearrangements correspond to previously known cancer-associated abnormalities. Two of the clones are obviously related, and it is reasonable to assume that the t(15;17) developed as an evolutionary change in a cell that already contained t(8;11)(p22;q13). Since five clones without cytogenetic similarities were found in this in situ skin carcinoma, we suggest that the tumor was of polyclonal origin. It is impossible to decide whether all, or indeed any, of the visible abnormalities constitute pathogenetically essential primary changes, or merely represent chromosomal markers of secondary importance in tumorigenesis.

Unique karyotypic abnormalities in a squamous cell carcinoma of the larynx.

We have cytogenetically examined short-term cultures from a squamous cell carcinoma of the larynx, a type of carcinoma in which chromosome aberrations have hitherto not been reported. The tumor karyotype was 46,XY,inv(2)(p22q24),t(9;13)(q34;q12),t(11;18)(q23;q21). None of these abnormalities have been described in carcinomas before.

Near-haploid clones in a malignant fibrous histiocytoma.

Near-haploid solid tumors are very rare. In a storiform-pleomorphic malignant fibrous histiocytoma (MFH) of bone, we found three cell populations: one with a near-haploid, a second with a near-diploid, and a third with a near-tetraploid chromosome number. The near-haploid cells had few structural rearrangements: i(12p) and t(13q21q) in one clone, and these two and an additional t(19;?)(p11;?) in another clone. One structurally normal copy of all chromosomes was also present, except that the only chromosome 13 was involved in the t(13q21q). There were also two near-diploid clones, one without the t(19;?) and one with a single copy of this derivative chromosome. This is the first tumor with i(12p) among bone and soft tissue tumors.

Multiple clonal chromosome aberrations in squamous cell carcinomas of the larynx.

Short-term cultures from five squamous cell carcinomas of the larynx were subjected to cytogenetic analysis. In the first three cases, two, three, and 10 chromosomally abnormal clones were detected. Single clonal abnormalities were found in cases 4 and 5. In addition to the clonal aberrations, a number of nonclonal changes were also present in all five tumors. None of the aberrations, clonal or nonclonal, was found in more than one tumor, nor did the rearrangements correspond to any of the consistently cancer-associated aberrations known from other tumors. The remarkably diverse karyotypic picture of the five squamous cell larynx carcinomas, in particular the finding of cytogenetically unrelated clones in three of them, suggests that some of these neoplasms are polyclonal rather than monoclonal.

Two unrelated clonal chromosome rearrangements in a nasal papilloma.

We have cytogenetically examined short-term cultures from a nasal papilloma, a tumor type in which chromosome aberrations have hitherto not been reported. Two pseudodiploid clones were detected, giving the tumor karyotype 46,XY,t(1;3)(p31;p12)/46,XY,t(11;?)(q25;?).