Paula M. DeAngelis

Identification of human papillomavirus in keratoacanthomas.

Background:  Keratoacanthomas are benign, clinically distinct skin tumors that may infiltrate and show cellular atypia. A viral etiology has been suggested, and the aim was to search for human papillomavirus (HPV) in keratoacanthomas.

Loss of chromosome 11q21-23.1 and 17p and gain of chromosome 6p are independent prognostic indicators in B-cell non-Hodgkin's lymphoma

Comparative genomic hybridization (CGH) was employed to study chromosomal aberrations in relation to cell proliferation, apoptosis, and patient survival in 94 cases of B-cell non-Hodgkin’s lymphoma diagnosed between 1983 and 1993. Eighty cases had aberrations by CGH. Chromosomal regions 1p21–31.1 (10%), 6cen-q24 (12%), 8p (11%), 9p21-ter (14%), 11q21–23.1 (11%), 13q13–21.1 (12%), and 17p (15%) were frequently lost. Gains were found at 3q21-ter (22%), 6p (11%), 7p (12%), 8q23-ter (13%), 12cen-q15 (17%), 17q24-ter (13%), and 18q13.3–21 (20%). A high number of aberrations (≥ 4, 33 cases) was associated (P ≤ 0.001) with the mantle cell and diffuse large B-cell lymphoma subtypes, a high fraction of tumour cells in S phase, and short survival (RR (relative risk) = 3.7). Loss of 1p21–31.1, 8p, 9p21-ter, 11q21–23.1, and 13q13–21.1 were associated with mantle cell lymphoma (P ≤ 0.03), while gain of 6p and 12cen-q15 were more frequent in diffuse large B-cell and small lymphocytic lymphoma, respectively (P = 0.04). Loss of 8p and 17p, and gain of 3q21-ter, 6p, 7p, and 8q23-ter were associated with a high S phase fraction (P ≤ 0.03), but none of the aberrations were associated with tumour apoptotic fraction (P ≥ 0.13). The most important prognostic CGH parameters (P < 0.001) were losses of 11q21–23.1 (RR = 3.8) and 17p (RR = 4.4), and gain of 6p (RR = 4.2). The latter parameters and IPI were the only ones with independent prognostic value (RR = 10, 5.0, 6.7, and 3.7, respectively; P < 0.001) when assessed together with lymphoma sub-type, primary versus relapse cases, treatment, B symptoms, S phase fraction, and presence of BCL1 and BCL2 translocations. A combined CGH/IPI binary parameter had high prognostic value for patients receiving different treatments, with various lymphoma sub-types, and for primary as well as relapse cases.

Spindle proteins Aurora A and BUB1B, but not Mad2, are aberrantly expressed in dysplastic mucosa of patients with longstanding ulcerative colitis

Background: Long term ulcerative colitis (UC) increases the risk of colorectal cancer (CRC). DNA aneuploidy is a common feature of both dysplastic and non-dysplastic colonic epithelia from patients with longstanding UC, and is regarded as an early sign of possible malignant transformation. The spindle proteins Aurora A, BUB1B and Mad2 have been implicated as contributors to aneuploidy and carcinogenesis. Aims: To investigate the role of these spindle proteins in relation to DNA aneuploidy and during the progressive morphological changes in ulcerative colitis associated colorectal cancer (UCCRC). Methods: Tissue microarrays were made from 31 colectomy specimens from patients with longstanding UC. Expression of Aurora A, BUB1B and Mad2 was investigated by immunohistochemistry and their relation to ploidy status, mucosal morphology and Ki67 levels was explored. Results: Expression of Aurora A and BUB1B was significantly associated with the progressive morphological changes of UCCRC. In the progression from non-dysplastic to dysplastic mucosa, Aurora A expression decreased while BUB1B expression increased. There was an increasing incidence of aneuploidy with progression towards cancer; expression of all spindle proteins was associated with the level of Ki67 but not with aneuploidy. Conclusion: Due to the significant differences in Aurora A and BUB1B expression in dysplastic compared non-dysplastic mucosa, these proteins may serve as putative biological markers for the progressive morphological changes in UC associated carcinogenesis. The close relationship to Ki67 levels reflect that spindle proteins are expressed in tissues with a high proliferative rate; a role for these proteins in the development of aneuploidy was not found.

ONCOGENIC ABERRATIONS IN THE p53 PATHWAY ARE ASSOCIATED WITH A HIGH S PHASE FRACTION AND POOR PATIENT SURVIVAL IN B-CELL NON-HODGKIN'S LYMPHOMA

The implications of aberrations in the p53 pathway for induction of apoptosis and regulation of S phase entry, and for patient survival, were investigated in 83 B‐cell Non‐Hodgkins lymphomas. Eight cases had missense mutations in exons 5, 7, 8 and 9 as revealed by constant denaturant gel electrophoresis and sequencing. Fifteen cases had lost 1 TP53 allele as revealed by fluorescent in situ hybridization and comparative genomic hybridization. Ten cases expressed high levels of p53 as assessed by immunoblotting and immunohistochemistry. S phase fractions were higher, apoptotic fractions were the same and survival times were shorter in all aberration groups compared with the cases with no TP53/p53 aberrations. Since many tumors had more than one TP53/p53 aberration, the tumors were divided into groups with the following characteristics: no TP53/p53 aberrations; loss of one TP53 allele only (9 cases), TP53 point mutation (8 cases), high‐level p53 expression and no TP53 mutation (3 cases). Tumors from the 3 latter groups had higher median S phase fractions (5%, 7.6%, and 5%, respectively, p<0.02) than the cases without any aberrations (1.1%), and survival time for these patients was much shorter (relative risks of 5.9, 8.9, and 6.6, respectively, p<0.003). Apoptotic fractions were similar in all these groups (p=0.09). Multivariate analysis showed that the presence of TP53/p53 aberrations is a strong and independent prognostic parameter in B‐cell Non‐Hodgkins lymphoma. Int. J. Cancer 89:313–324, 2000.

Array comparative genomic hybridization of keratoacanthomas and squamous cell carcinomas: different patterns of genetic aberrations suggest two distinct entities.

Keratoacanthoma (KA) is a benign keratinocytic neoplasm that spontaneously regresses after 3-6 months and shares features with squamous cell carcinomas (SCCs). Furthermore, there are reports of KAs that have metastasized, invoking the question of whether KA is a variant of SCC (Hodak et al., 1993). To date, no reported criteria are sensitive enough to discriminate reliably between KA and SCC, and consequently there is a clinical need for discriminating markers. Our previous study analyzed 132 KAs and 29 SCCs and revealed significantly different regions of genomic aberrations using chromosomal comparative genomic hybridization (CGH). In the present study, we applied array CGH to investigate 98 KAs and 22 SCCs from the above samples. The result shows that all KAs and SCCs have some degree of genetic aberrations. The distribution of numbers of aberrant clones per sample differed significantly between KAs and SCCs (P<0.02), which also demonstrated recurrent aberrations that differed significantly (P<0.001), as illustrated by unsupervised cluster analysis. Classifiers for clinicopathological parameters of KAs were established based on t-test statistics and permutation tests. Tumor size, fibrosis, and inflammation, which are related to the developmental stages of KAs, showed significant (t-test, permutation test) associations with aberrations of selected genomic regions. This suggests chromosomal instability during the whole life cycle of KAs.

Translocation t(14;18) and gain of chromosome 18/ BCL2 : effects on BCL2 expression and apoptosis in B-cell non-Hodgkin's lymphomas

Gain of chromosome 18q and translocation t(14;18) are] frequently found in B-cell non-Hodgkins lymphomas (B-NHL). Increased BCL2 transcription and BCL2 protein expression have been suggested to be the result of the gain. We utilized FISH, PCR and array CGH to study BCL2 and chromosome 18 copy number changes and rearrangements in 93 cases of B-NHL. BCL2 protein was expressed in >75% of the tumor cells in 92% of the cases by immunohistochemistry. Gain of BCL2 was associated with a 25% increase in BCL2 expression levels (immunoblotting), whereas t(14;18) resulted in a 55% increase in BCL2 levels compared to cases without BCL2 alterations. The tumor cell (spontaneous) apoptotic fractions were similar for the cases with different BCL2 genotypes. However, the normal cell apoptotic fractions were higher for the tumors with t(14;18) compared to the tumors without BCL2 alterations, while the tumors with gain of BCL2 only showed intermediate levels. Low-level gains of parts of chromosome 18 were found in 14 of the 38 B-NHL cases with t(14;18), with a consensus region 18pter-q21.33 that did not include the BCL2 gene. The 11 cases with 18q gain only showed a consensus region encompassing 18q21.2–18q21.32 and 18q21.33, which contain PMAIP1/MALT1 and BCL2, respectively.

Response of malignant B lymphocytes to ionizing radiation: Gene expression and genotype

The human malignant B‐lymphocyte cell lines Reh and U698 show arrest in G2 phase after ionizing radiation (IR), but only Reh cells arrest in G1 phase and die by apoptosis. We have used cDNA microarrays to measure changes in gene expression at 2, 4 and 6hr after irradiation of Reh and U698 cells with 0.5 and 4 Gy in order to begin exploring the molecular mechanisms underlying the phenotypic changes. We also investigated whether gene expression changes could be caused by possible aberrations of genes, as measured by comparative genomic hybridization. Reh cells showed upregulation of CDKN1A that likely mediated the G1 arrest. In contrast, U698 cells have impaired function of TP53 protein and no activation of CDKN1A, suppressing the arrest in G1. The G2 arrest in both cell lines was likely due to repression of PLK1 and/or CCNF. IR‐induced apoptosis in Reh cells was probably mediated by TP53 and CDKN1A, whereas a high expression level of MCL1, caused by gene amplification, and activation of the NFKB pathway may have suppressed the apoptotic response in U698 cells. Genes suggested to be involved in apoptosis were activated long before this phenotype was detectable and showed the same temporal expression profiles as genes involved in cell cycle arrest. Our results suggest that differences in functionality and/or copy number of several genes involved in IR‐regulated pathways contributed to the phenotypic differences between Reh and U698 cells after IR, and that multiple molecular factors control the radiation response of malignant B lymphocytes.

Propidium iodide quenches the fluorescence of TdT-incorporated FITC-labeled dUTP in apoptotic cells

Apoptotic cells with frequent DNA strand breaks may be detected by tagging with directly or indirectly labeled nucleotides incorporated by the use of terminal deoxynucleotidyl transferase (TdT). Propidium iodide (PI) is typically added for the simultaneous assessment of DNA content. PI was found to quench the specific in situ FITC-fluorescence of apoptotic cells which were labeled by TdT with FITC-conjugated dUTP, biotin-dUTP followed by streptavidin-FITC, or digoxigenin-dUTP followed by FITC-labeled anti-digoxigenin antibodies as measured by flow cytometry. The effect was concentration-dependent, with 50% quenching occurring at 0.8 microg/ml, 1.5 microg/ml, and 5 microg/ml PI, respectively, at approximately 1 x 10(6) cells/ml. Spectrofluorimetry in solution revealed that 15 microg/ml PI was required to quench 50% of the fluorescence of ss FITC-labeled poly(dU)35. In contrast, the fluorescence of ds FITC-labeled poly(dU)35-poly(dA) was quenched to 50% at 3 microg/ml PI. The maximum of the fluorescence excitation spectrum of PI shifted from 490 nm to 535 nm upon binding to ds DNA as well as ss poly(dU)35, and the fluorescence yield of PI at 610 nm increased, but the binding required 10-fold higher concentrations of poly(dU)35 as compared to ds DNA. The spectroscopic properties of PI are therefore similar whether bound to poly(dU) or to double-stranded DNA, but the binding to poly(dU) is much weaker. The observed quenching in situ therefore cannot be explained by direct binding of PI to the poly(dU) tails synthesized by TdT in situ in apoptotic cells, but may rather be due to radiationless energy transfer from FITC to PI bound to double-stranded DNA close to the nicks where TdT is known to start polymerization.