Turid Knutsen

Chromosome Translocation in Peripheral Neuroepithelioma

PERIPHERAL neuroepithelioma (peripheral neuroblastoma) is an uncommon malignant tumor of the peripheral nervous system with a histologic appearance similar to that of classical childhood neuroblast...

Cytogenetic characterization of selected small round cell tumors of childhood

Small, round, blue-cell tumors (SRCT), including rhabdomyosarcoma, Ewings sarcoma of bone and soft tissue, mesenchymal chondrosarcoma, small cell osteosarcoma, hemangiopericytoma, neuroblastoma, peripheral neurectodermal tumor (peripheral neuroepithelioma of bone and soft tissue), and the malignant small cell tumor of the thoracopulmonary region described by Askin (Askins tumor), are often difficult to distinguish by light microscopy. We have evaluated the cytogenetics of these tumors by studying 24 tumor explants in short-term culture and 22 tumor cell lines. In Ewings sarcoma (a tumor of unknown histogenesis), and in peripheral neuroepithelioma and Askins tumor (tumors with evidence of neural origin), we have observed an indistinguishable t(11;22) translocation.

Cytogenetic studies in ovarian cancer

Cytogenetic studies of ovarian cancer have been conducted in the Medicine Branch, NCI, National Institutes of Health for 5 years. A total of 72 patients were studied by direct preparation and/or 1- to 3-day short-term culture of ascites (86 samples), pleural fluid (4 samples), and tumor (2 samples). Repeat examinations (1-24 months later) were performed in 7 of the 72 patients. Forty-four patients (62%) were successfully analyzed with banding techniques: 6 patients had adenocarcinoma, 7 had serous adenocarcinoma, 13 had serous papillary adenocarcinoma, 7 had serous papillary cystadenocarcinoma, 2 had mucinous adenocarcinoma, 6 had undifferentiated or poorly differentiated adenocarcinoma, 1 had clear cell adenocarcinoma, and 2 were not classified. Of these 44 patients, 29 had received prior chemotherapy, 14 were untreated, and in 1 patient the treatment status was unknown. Aneuploidy was observed in all patients and there was considerable variation in the chromosome numbers (even within single samples), often ranging from diploidy to triploidy to tetraploidy. All 44 patients had numerical abnormalities and 39 had structural abnormalities. The chromosomes most frequently involved in structural abnormalities (in decreasing order according to the number of patients involved) were #1, #3, #2, #4, #9, #10, #15, #19, #6, and #11; the least involved chromosomes were #21 and #5. Clone formation and the number of chromosomes involved in structural abnormalities increased with duration of disease and were more extensive in patients treated with chemotherapy than in patients treated with surgery alone. Our data did not show a deletion of chromosome #6 (6q-) to be specific for ovarian cancer.

Proteomic Analysis of Apoptotic Pathways Reveals Prognostic Factors in Follicular Lymphoma

Follicular lymphoma (FL) is the second most common non-Hodgkins lymphoma and generally is incurable. Reliable prognostic markers to differentiate patients who progress rapidly from those who survive for years with indolent disease have not been established. Most cases overexpress Bcl-2, but the pathogenesis of FL remains incompletely understood. To determine whether a proteomic approach could help overcome these obstacles, we procured lymphoid follicles from 20 cases of FL and 15 cases of benign follicular hyperplasia (FH) using laser capture microdissection. Lysates were spotted on reverse-phase protein microarrays and probed with 21 antibodies to proteins in the intrinsic apoptotic pathway, including those specific for posttranslational modifications such as phosphorylation. A panel of three antibodies [phospho-Akt(Ser473), Bcl-2, and cleaved poly(ADP-ribose) polymerase] segregated most cases of FL from FH. Phospho-Akt(Ser473) and Bcl-2 were significantly increased in FL (P = 0.001 and P < 0.0001, respectively). Additionally, the Bcl-2/Bak ratio completely segregated FL from FH. High ratios of Bcl-2/Bak and Bcl-2/Bax were associated with early death from disease with differences in median survival times of 7.3 years (P = 0.0085) and 3.8 years (P = 0.018), respectively. Using protein microarrays, we identified candidate proteins that may signify clinically relevant molecular events in FL. This approach showed significant changes at the posttranslational level, including Akt phosphorylation, and suggested new prognostic markers, including the Bcl-2/Bak and Bcl-2/Bax ratios. Proteomic end points should be incorporated in larger, multicenter trials to validate the clinical utility of these protein microarray findings.

Clinical implications of cytogenetic studies in cutaneous t-cell lymphoma (CTCL)

Detailed cytogenetic studies were performed in 41 patients with cutaneous T‐cell lymphoma (CTCL): four patients had limited plaques, 13 patients had generalized plaques, eight patients had cutaneous tumors, 16 patients had generalized erythroderma, and four additional patients, who had relatively benign chronic dermatosis, served as controls. Correlating the histologic and cytogenetic results in the various tissues, it was observed that 62% of the peripheral blood specimens were cytogenetically positive but only 49% were morphologically positive; in the lymph node the ratio was 80 versus 45%, and in the bone marrow, 6 versus 3%. These studies demonstrate that chromosome abnormalities are frequently detectable before morphologic changes become apparent. Chromosome banding preparations showed extensive and wide‐ranging heteroploidy; the #1 chromosome was most frequently involved in structural abnormalities while chromosomes #11, 21, and 22 were most frequently involved in numerical abnormalities. These cytogenetic findings support the impression that CTCL is a disease whose various clinical manifestations represent a chronologic sequence, with the cytogenetic findings paralleling the clinical symptoms: patients with minimal chromosomal changes had the best survival and the more extensive the chromosome abnormalities, the more advanced the clinical disease. Clone formation was seen in eight patients and this phenomenon, along with hyperdiploidy and near‐tetraploidy, was associated with a poor prognosis and short survival. We conclude that CTCL progresses from an early phase with extensive chromosomal abnormalities and lack of clone formation to a terminal phase with clone selection. Cytogenetic studies can, therefore, be of significant diagnostic and prognostic value in CTCL.

Amplification of 4q21–q22 and the MXR gene in independently derived mitoxantrone‐resistant cell lines

Molecular cytogenetic studies were conducted on three multidrug‐resistant cancer sublines which are highly resistant to the chemotherapeutic agent mitoxantrone, an anthracenedione. The three independently selected sublines were derived by exposure to mitoxantrone or Adriamycin and do not overexpress MDR1 or MRP. Two sublines, MCF‐7 AdVp3000 and MCF‐7 MX, showed an amplification peak at 4q21–q22, as demonstrated by comparative genomic hybridization (CGH), while the third, S1‐M1–80, did not. FISH using a whole chromosome 4 paint demonstrated multiple rearrangements involving chromosome 4 in MCF‐7 AdVp3000 and MCF‐7 MX, while S1‐M1–80 contained only a simple reciprocal translocation. The parental cell lines had no chromosome 4 rearrangements and no copy number gain or amplification of chromosome 4. Spectral karyotyping (SKY) analysis revealed a balanced translocation, t(4;17)(q21–q22;p13) in S1‐M1–80 and multiple clonal translocations involving chromosome 4 in MCF‐7 AdVp3000 and MCF‐7 MX. A novel cDNA, designated MXR, which encodes an ABC half‐transporter and is highly overexpressed in the three sublines, was localized to chromosome 4 by somatic cell hybrid analysis. Southern blot analysis demonstrated amplification of the MXR gene in MCF‐7 AdVp3000 and MCF‐7 MX, but not in S1‐M1–80. FISH studies with a BAC probe for MXR localized the gene to 4q21–22 in the normal chromosome 4 and revealed in both MCF‐7 AdVp3000 and MCF‐7 MX amplification of MXR at one translocation juncture, shown by SKY to be t(4;5)(4qter→4cen→4q21–22::5q13→5qter) in MCF‐7 AdVp3000 and t(6;4;6;3)(6pter→6q15::4q21–q22::hsr::6q?::3q?27→3qter) in MCF MX; neither of the breakpoints in the partner chromosomes showed amplification by CGH. The data are consistent with the hypothesis of a transporter, presumably that encoded by the MXR gene, mediating mitoxantrone resistance. The MXR gene encodes a half‐transporter and the absence of cytogenetic evidence of coamplification of other regions suggests that a partner may not be overexpressed, and instead the MXR half‐transporter homodimerizes to mediate drug transport. Genes Chromosomes Cancer 27:110–116, 2000. Published 2000 Wiley‐Liss, Inc.

Escape from hsa-miR-519c enables drug-resistant cells to maintain high expression of ABCG2

Overexpression of ABCG2 has been reported in cell lines selected for drug resistance and it is widely believed to be important in the clinical pharmacology of anticancer drugs. We and others have previously identified and validated two microRNAs (miRNA; hsa-miR-519c and hsa-miR-520h) targeting ABCG2. In this study, the shortening of the ABCG2 3′ untranslated region (3′UTR) was found to be a common phenomenon in several ABCG2-overexpressing resistant cell lines, which as a result removes the hsa-miR-519c binding site and its repressive effects on mRNA stability and translation blockade, thereby contributing to drug resistance. On the other hand, reduced expression of hsa-miR-520h, previously thought to have allowed ABCG2 overexpression, was found to be caused by the sequestering of the miRNA by the highly expressed ABCG2. In drug-sensitive cells, inhibitors against hsa-miR-519c and hsa-miR-520h could augment the cytotoxic effect of mitoxantrone, suggesting a substantial role for both miRNAs in controlling ABCG2 level and thereby anticancer drug response. However, in drug-resistant cells, altering the levels of the two miRNAs did not have any effect on sensitivity to mitoxantrone. Taken together, these studies suggest that in ABCG2-overexpressing drug-resistant cells, hsa-miR-519c is unable to affect ABCG2 expression because the mRNA lacks its binding site, whereas hsa-miR-520h is sequestered and unable to limit ABCG2 expression. Given the recent observation that a truncated 3′UTR is also observed in ABCG2-overexpressing human embryonic stem cells, our results in drug-resistant cell lines suggest that 3′UTR truncation is a relatively common mechanism of ABCG2 regulation. [Mol Cancer Ther 2009;8(10):2959–68]

Acute non‐lymphocytic leukemia and acute myeloproliferative syndrome following radiation therapy for non‐hodgkin's lymphoma and chronic lymphocytic leukemia. Cytogenetic studies

Seven cases of acute nonlymphocytic leukemia (ANLL) and one of malignant myeloproliferative syndrome were identified from a pool of 189 cases of non‐Hodgkins lymphoma (NHL) and CLL treated primarily with extensive radiotherapy at the Clinical Center, the National Institutes of Health. Four patients also received chemotherapy, two for only short periods. The median time interval from the diagnosis of the primary malignancy to the development of leukemia was 61 months (range 33 to 98 months) and the median survival after the diagnosis of leukemia was two months (0 to 9 months). All eight patients were cytogenetically abnormal and serial chromosome studies revealed that hypodiploidy was the most commonly observed chromosomal abnormality. Abnormalities of chromosome #7 were seen in all five patients analyzed by the chromosome banding technique; four of them had monosomy 7. The next most frequently involved chromosome was #5. The complexity, extensive nature, and long duration of the cytogenetic abnormalities prior to the diagnosis of leukemia in these patients may be characteristic of secondary leukemia in radiation‐treated lymphoma and the presence of such anomalies may predict leukemic transformation.

Cytogenetic and molecular characterization of random chromosomal rearrangements activating the drug resistance gene, MDR1/P-glycoprotein, in drug-selected cell lines and patients with drug refractory ALL

Drug resistance, both primary and acquired, is a major obstacle to advances in cancer chemotherapy. In vitro, multidrug resistance can be mediated by P‐glycoprotein (PGY1), a cell surface phosphoglycoprotein that acts to efflux natural products from cells. PGY1 is encoded by the MDR1 gene located at 7q21.1. Overexpression of MDR1 has been demonstrated in many cancers, both in patient tumors and in cell lines selected with a variety of chemotherapeutic agents. Recent studies in drug‐selected cell lines and patients samples have identified hybrid mRNAs comprised of an active, but apparently random, gene fused 5′ to MDR1. This observation indicates that random chromosomal rearrangements, such as translocations and inversions, leading to “capture” of MDR1 by constitutively expressed genes may be a mechanism for activation of this gene following drug exposure. In this study, fluorescence in situ hybridization (FISH) using whole chromosome paints (WCP) and bacterial artificial chromosome (BAC)‐derived probes showed structural rearrangements involving 7q in metaphase and interphase cells, and comparative genomic hybridization (CGH) revealed high levels of amplification at chromosomal breakpoints. In an adriamycin‐selected resistant colon cancer line (S48–3s/Adr), WCP4/WCP7 revealed t(4;7)(q31;q21) and BAC‐derived probes demonstrated that the breakpoint lay between MDR1 and sequences 500–1000 KB telomeric to it. Similarly, in a subline isolated following exposure to actinomycin D (S48–3s/ActD), a hybrid MDR1 gene composed of heme oxygenase‐2 sequences (at 16p13) fused to MDR1 was identified and a rearrangement confirmed with WCP7 and a subtelomeric 16p probe. Likewise, in a paclitaxel‐selected MCF‐7 subline where CASP sequences (at 7q22) were shown to be fused to MDR1, WCP7 showed an elongated chromosome 7 with a homogeneously staining regions (hsr); BAC‐derived probes demonstrated that the hsr was composed of highly amplified MDR1 and CASP sequences. In all three selected cell lines, CGH demonstrated amplification at breakpoints involving MDR1(at 7q21) and genes fused to MDR1 at 4q31, 7q22, and 16p13.3. Finally, in samples obtained from two patients with drug refractory ALL, BAC‐derived probes applied to archived marrow cells demonstrated that a breakpoint occurred between MDR1 and sequences 500–1000 KB telomeric to MDR1, consistent with a random chromosomal rearrangement. These results support the proposal that random chromosomal rearrangement leading to capture and activation of MDR1 is a mechanism of acquired drug resistance. Genes Chromosomes Cancer 23:44–54, 1998.

DNA Content Analysis by Flow Cytometry and Cytogenetic Analysis in Mycosis Fungoides and Sézary Syndrome: DIAGNOSTIC AND PROGNOSTIC IMPLICATIONS

Flow cytometric (FCM) analysis of DNA content was performed on 82 lymph node and peripheral blood specimens from 46 patients with mycosis fungoides and the Sézary syndrome. Overall, 32 of the 46 patients (70%) had aneuploidy detected by FCM. Aneuploidy was present in 63% of the patients at the time of diagnosis before systemic therapy. In these patients, aneuploidy was frequently detected in blood and lymph node specimens scored as negative by cytology and histology, suggesting that unsuspected extracutaneous dissemination is present in many patients at the time of diagnosis. Direct comparison with Giemsa-banded cytogenetic studies showed an excellent correlation of FCM results and cytogenetic chromosome number. However, FCM frequently detected a larger fraction of aneuploid cells, and mitogen-stimulation studies suggest this is the result of preferential stimulation of normal lymphocytes by phytohemagglutinin. Thus, mitogens with a preference for malignant T cells, such as staphylococcal protein A, should be used for cytogenetic analysis of malignant T-cell disorders. At diagnosis, some histologically positive specimens contained only diploid cells by FCM and cytogenetic analysis. These patients had a more indolent clinical course than patients with aneuploidy. Aneuploidy was detected by FCM as either wide G(1) or as discrete aneuploid peaks. The presence of aneuploidy at any time in the clinical course implied a poor prognosis. Discrete hyperdiploid peaks were associated with large cell histology, early relapse, and aggressive clinical course. The development of hyperdiploidy at relapse was documented in four patients and was associated with a transition to large cell histology and a poor prognosis. Similar studies may elucidate differences in natural history and mechanism for transition in histology in other lymphomas and solid tumors.