Cyclin D1 overexpression versus response to induction chemotherapy in squamous cell carcinoma of the head and neck--preliminary report.

The aim of this study was to investigate whether there is an association between overexpression of cyclin D1 and response to therapy. Immunohistochemical overexpression of cyclin D1 was determined in paraffin-embedded specimens from diagnostic biopsies of 89 primary cases of squamous cell carcinoma of the head and neck (SCCHN), using a polyclonal antiserum. The tumor response rates were estimated after curative treatment (i.e. surgery and/or radiotherapy and/or chemotherapy). Patients whose tumors were overexpressing cyclin D1 showed complete or partial response to neoadjuvant chemotherapy with cisplatin/5-FU. In addition, a majority of cyclin D1 negative tumors did not respond at all to this treatment (p = 0.02, Fisher's exact test). This study indicates that immunohistochemical assessment of cyclin D1 expression in SCCHN could be a new predictive marker to select a subgroup of patients that will benefit from neoadjuvant chemotherapy.

Pain treatment after tonsillectomy: advantages of analgesics regularly given compared with analgesics on demand.

The aim of the present prospective study was to evaluate pain treatment during the first postoperative 24 h for 40 patients (age over 18) undergoing tonsillectomy. Patients were divided into two groups: group A (n = 20) received analgesics on demand and group B (n = 20) on a regular basis. Basic pain treatment consisted of paracetamol 750 mg x 6 and diclofenac 50 mg x 3. Pain measurement was performed using a visual analogue scale (VAS): a 10 cm line with 0 cm equalling no pain and 10 cm equalling the worst pain ever felt. The following parameters were studied: VAS values, the need for rescue analgesics, intra- and postoperative bleeding, nausea and vomiting, postoperative food intake and hospital time. Only 4 of 20 (20%) patients in group B needed rescue analgesics in the postoperative ward compared with 15 of 20 (75%) in group A (p < 0.01, chi2 test). In group B, 13 of 20 (65%) patients could eat solid food before they were discharged from the ward, compared with 7 of 19 (37%) monitored patients in group A (p < 0.01, chi2 test). The observed VAS values were generally rather low in both groups. The mean value for all observed VAS values was less than 4 in both study groups. However, no significant difference in VAS values was observed between the two study groups. Our results suggest that regularly given postoperative pain treatment after tonsillectomy, starting intraoperatively with paracetamol and diclofenac, has significant advantages compared with a regimen in which patients receive analgesics only on demand.

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.

p53 mutation, but not p53 overexpression, correlates with survival in head and neck squamous cell carcinoma.

Survival in squamous cell carcinoma of the head and neck (HNSCC) was compared with overexpression and mutation of the p53 gene. Archival tissue from 77 tumours was analysed for protein expression using immunohistochemistry (IHC) with the monoclonal antibody Do-7, and for the presence of mutation in exons 5-8 using single-stranded conformation polymorphism (SSCP), followed by DNA sequencing in SSCP-positive cases. p53 expression was scored as high (>70% nuclei stained) in 25 (32%) tumours, as intermediate (10-70% nuclei stained) in 19 (25%) tumours and as low (<10% nuclei stained) in 33 (43%) tumours. Twelve (18%) tumours exhibited gene mutation (ten missense and two nonsense mutations) and an additional five tumours contained changes that could not result in amino acid substitution or protein truncation. There was no correlation between gene expression and mutation, mutations being equally frequent in tumours with either high (4/25), intermediate (4/19) or low protein expression (4/33). Fifty-eight patients were eligible for survival analysis. There was a strong correlation between p53 mutation and cause-specific survival; median survival among mutated cases was 12.5 months compared with >160 months among non-mutated patients (P < 0.005). There was no correlation between p53 overexpression and survival. The results suggest that p53 mutation status is an important prognostic factor in HNSCC, and that IHC analysis of protein overexpression is an inadequate measure of gene mutation in these tumours.

Cytogenetic and molecular genetic demonstration of polyclonality in an acinic cell carcinoma.

The paradigm that human malignancies are monoclonal has been questioned during recent years by the finding of unrelated, cytogenetically aberrant clones in short-term cultures from certain tumour types, notably carcinomas of the breast, skin and upper aerodigestive tract. In order to analyse whether cytogenetically unrelated clones are also unrelated at the molecular level, we analysed the X-chromosome inactivation status in cell cultures from a cytogenetically highly polyclonal acinic cell carcinoma of the parotid gland. By using cell cultures dominated by a single abnormal clone, obtained through in vitro culturing for 3-5 passages, we showed that the different clones must indeed have originated from different cells.

FISH characterization of head and neck carcinomas reveals that amplification of band 11q13 is associated with deletion of distal 11q.

In order to characterize homogeneously staining regions (HSR) and other 11q13 rearrangements identified cytogenetically, we performed fluorescence in situ hybridization (FISH) using a CCND1 cosmid and five YAC clones spanning chromosomal bands 11q13-14 on metaphase cells from 14 primary and one metastatic head and neck carcinomas. At the cytogenetic level, a total of 17 HSR were detected in ten cases: five were in derivative chromosomes 11 in band 11q13, and 12 were located in other derivative chromosomes. Other forms of 11q13 rearrangements were observed in five cases, whereas two cases had normal chromosomes 11. FISH analysis demonstrated that all HSR but two were derived from the 11q13 band. The size of the amplicon varied from case to case, but the amplification always included the region covered by YAC 55G7, which contains the CCND1 locus. The amplification of CCND1 was confirmed by use of a CCND1 cosmid. We also showed that most of the cases (9 of 11) with 11q13 amplification had lost material from distal 11q. The breakpoints were mapped by FISH and were shown to cluster to the region between YACs 55G7 and 749G2. We conclude that loss of gene(s) in distal 11q may be as important as amplification of genes in 11q13 for the biological aggressiveness of head and neck carcinomas.

Complex karyotypes in flow cytometrically DNA-diploid squamous cell carcinomas of the head and neck.

In squamous cell carcinoma of the head and neck (SCCHN), DNA ploidy as determined by flow cytometry (FCM) has been found to yield prognostic information but only for tumours at oral sites. Cytogenetic findings have indicated complex karyotype to be a correlate of poor clinical outcome. In the present study, 73 SCCHN were investigated with the two techniques. Aneuploid cell populations were identified in 49 (67%) cases by FCM but in only 21 (29%) cases by cytogenetic analysis. The chromosome index (CI), calculated as the mean chromosome number divided by 46, was compared with the respective DNA index (DI) obtained by FCM in 15 tumours, non-diploid according to both techniques, DI being systematically 12% higher than CI in this subgroup. Eight (33%) of the 24 tumours diploid according to FCM had complex karyotypes, three of the tumours being cytogenetically hypodiploid, three diploid and two non-diploid. The findings in the present study may partly explain the low prognostic value of ploidy status as assessed by FCM that has been observed in SCCHN. In addition, we conclude that FCM yields information of the genetic changes that is too unspecific, and that cytogenetic analysis shows a high rate of unsuccessful investigations, thus diminishing the value of the two methods as prognostic factors in SCCHN.

Clonal chromosome aberrations accumulate with age in upper aerodigestive tract mucosa.

Short-term cultured non-neoplastic upper aerodigestive tract (UAT) mucosa samples from 36 patients with squamous cell carcinoma of the head and neck (SCC) and 53 patients with benign UAT disorders were cytogenetically analyzed. The cell cultures were divided into two series: in series A, cells were cultured in a medium stimulating outgrowth of mesenchymal cells; whereas the cultured cells in series B were of epithelial morphology. Series A was further subdivided into three different age groups (< or = 15 years, 16-59 years, and > or = 60 years) of non-SCC patients and one SCC group. Series B was composed of two groups; one with and one without SCC. Among the non-SCC patients in series A, there was an increase with age in the frequency of cells/sample with numerical and structural chromosomal changes as well as in the incidence of clonal chromosomal aberrations. No differences could, however, be detected between cancer patients and age-matched controls. In series B, the frequency of cells/sample with numerical changes and the incidence of clonal numerical aberrations were significantly higher among SCC patients. Three main conclusions could be drawn. First, the frequencies of clonal and non-clonal chromosome aberrations in UAT mucosa were age dependent. Second, the cytogenetic support for the validity of the field cancerization hypothesis was restricted to increased levels of numerical chromosome changes in epithelial cell cultures from cancer patients. Third, clonal chromosome aberrations, including autosomal and sex chromosome aneuploidies as well as structural rearrangements, are not restricted to neoplastic mucosal cells.

Cytogenetic analysis of inverted nasal papillomas and demonstration of genetic convergence during in vitro passaging.

Three inverted nasal papillomas were cytogenetically investigated after short-term culture. Two of the cases were characterized by a single abnormal clone with t(1;8)(p36;q11) and trisomy 7, respectively, whereas the third papilloma showed extreme cytogenetic heterogeneity: of 852 analyzed cells, 329 belonged to 36 unrelated clones, 344 had non-clonal changes, and 179 had a normal chromosome constitution. The polyclonal papilloma was further analyzed during in vitro passage of 3 lines (L1-L3) cultured independently since initiation of the primary cultures and found to have 6, 16 and 6 unrelated clones at analysis of primary cultures. At passage 1, each line was further subdivided into 2 sub-lines (L1A and B, L2A and B, and L3A and B), which were cultured separately until the cells spontaneously stopped dividing. After 4 to 7 passages, each sub-line was dominated (83-98% of the cells) by a single clone. The cell populations that took over the cultures were the same within each set of sub-lines (A and B lines), demonstrating that clonal overgrowth in vitro is not random. The difference in clonal selection among the L1-L3 lines further shows that genetic convergence during in vitro growth in stable conditions is dependent not only on the clones' ability to adapt to the culture conditions, but also on the nature of the neighboring cells with which they collaborate and compete.

Amplification of cyclin D1 in squamous cell carcinoma of the head and neck and the prognostic value of chromosomal abnormalities and cyclin D1 overexpression.

BACKGROUND: Abnormalities of chromosome band 11q13 are frequent in squamous cell carcinoma of the head and neck (SCCHN). The oncogene CCND1 is located at 11q13 and encodes cyclin D1, a cell cycle-regulating protein. The authors investigated the clinical relevance and associations between amplification and overexpression of cyclin D1 and 11q13 rearrangements. METHODS: The study involved two series of patients. In Series 1, overexpression of cyclin D1 and 11q13 rearrangements, assessed by immunohistochemistry and cytogenetics, respectively, were compared with clinical data in 75 patients with SCCHN. Patients were monitored for at least 18 months or until death. In another 23 patients (Series 2), the authors investigated the association between DNA amplification (by slot blot hybridization), overexpression of cyclin D1, and cytogenetics. RESULTS: In Series 1, 9 of 75 tumors (12%) had 11q13 aberrations, 6 of which manifested elevated expression of cyclin D1. Patients with tumors strongly positive for cyclin D1 (n = 9) and those with tumors showing 11q13 rearrangements had poorer survival (P = 0.047 and 0.005, respectively). However, the correlation between these two variables was weak (P = 0.12). In Series 2, 17 of 23 tumors (74%) showed elevated cyclin D1 protein expression, and 6 of these showed gene amplification as well. Of these six, only one revealed 11q13 rearrangements. CONCLUSIONS: Overexpression of cyclin D1 and 11q13 rearrangements are independent prognostic factors for SCCHN. In general, DNA amplification results in overexpression of cyclin D1, but additional genetic mechanisms are involved in the deregulation. Furthermore, oncogenes at 11q13 besides CCND1 may be involved in the tumorigenesis.

Chromosomal abnormalities involving 11q13 are associated with poor prognosis in patients with squamous cell carcinoma of the head and neck.

BACKGROUND: In individual patients with squamous cell carcinoma of the head and neck (SCCHN), established prognostic factors do not satisfactorily predict clinical outcome. Although the karyotype is an independent prognostic factor in certain hematologic malignancies and solid tumors, no data have been reported concerning the possible relationship between chromosomal abnormalities and clinical outcome in patients with SCCHN: METHODS: In 116 cases of primary SCCHN, short term cultures were analyzed cytogenetically during 1987 through 1991, the karyotypes were divided into four groups: k1, normal (n = 35); k2, numeric changes only (n = 31); k3, simple structural abnormalities (n = 27); and k4, complex karyotypes (n = 23). The patients were followed for at least 18 months after diagnosis or until death. RESULTS: The 2-year survival rate was lower in the k4 subgroup (35%) than in the k1, k2, and k3 subgroups taken together (61%), both in the series as a whole (P = 0.02), and in the largest tumor site subgroup, laryngeal squamous cell carcinoma (n = 32), (P = 0.04). The most prevalent breakpoint was in chromosome band 11q13, occurring in 11 tumors, 10 of which belonged to the k4-subgroup. The 2-year survival rate was lower for patients with 11q13 rearrangements (20%) than for those without (60%), both in the series as a whole (P = 0.001), and in the k4-subgroup (P = 0.02). CONCLUSIONS: The results suggest that in SCCHN the presence of a complex karyotype is associated with poor prognosis, particularly when 11q13 rearrangements are present.

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.

Are tumor necrosis factor (TNF) or cortisol of value for the diagnosis of acute septicemia?

Early diagnosis is of great importance to improve the prognosis of septicemia. Traditional laboratory tests are either delayed like blood cultures, or unspecific like WBC count or ESR. In this retrospective pilot study we have assayed plasma cortisol, blood sugar and serum tumor necrosis factor (TNF) from patients with verified septicemia. With the approach used in this study none of the tests were able to differentiate between septicemia and other infectious febrile illnesses, or to predict if the causing organism was gram-positive or gram-negative.