Adi F. Gazdar

Genomic alterations in cultured human embryonic stem cells

Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10−9 per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.

Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers

Somatic mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene in lung cancers have generated enormous interest, because they predict for sensitivity to TK inhibitors (TKIs). While mutational status is of great importance in determining response to TKIs, it is not the sole factor, and evidence is accumulating that EGFR gene amplification, other members of the EGFR family (HER2, HER3) and genes downstream of EGFR signaling (KRAS, BRAF), may be involved in cancer pathogenesis and the response of TKIs. EGFR mutations occur in highly selected subpopulations of lung cancer patients: adenocarcinoma histology, never‐smoker status, East Asian ethnicity and female gender. The recent finding of “a resistance associated” mutation for TKIs also provides new insights into this complicated mechanism. Thus, molecular‐based studies to analyze the biological functions and to assess TKI sensitivity depending on the type of mutations are required. Epidemiological studies to identify possible carcinogenic factor(s) affecting different subpopulations are also of interest. In addition, for optimal therapeutic approach a comprehensive understanding of the genes related to EGFR signaling pathway, including RAS/RAF/MAPK and PI3K‐AKT pathways, are required.

Aberrant promoter methylation and silencing of the RASSF1A gene in pediatric tumors and cell lines

Aberrant promoter methylation of tumor suppressor genes has not been fully investigated in pediatric tumors. Therefore, we examined the methylation status of nine genes (p16INK4A, MGMT, GSTP1, RASSF1A, APC, DAPK, RARβ, CDH1 and CDH13) in 175 primary pediatric tumors and 23 tumor cell lines using methylation-specific PCR. We studied the major forms of pediatric tumors – Wilms tumor, neuroblastoma, hepatoblastoma, medulloblastoma, rhabdomyosarcoma, osteosarcoma, Ewings sarcoma, retinoblastoma and acute leukemia. The most frequently methylated gene in both primary tumors and cell lines was RASSF1A (40, 86%, respectively). However, the rates of RASSF1A methylation in individual tumor types varied from 0 to 88%. RASSF1A methylation was tumor specific and was absent in adjacent non-malignant tissues. Methylation of the other genes was relatively rare in tumors and non-malignant tissues (less than 5%). Neuroblastoma patients with methylation of RASSF1A were significantly older than patients without methylation (P=0.008). There was no relationship between methylation status and other clinico-pathologic parameters. We treated six cell lines lacking RASSF1A mRNA with 5-aza-2′deoxycytidine to examine the relationship between methylation and transcriptional silencing. In five of six cell lines, restoration of RASSF1A mRNA was confirmed by RT–PCR. Our findings indicate that aberrant promoter methylation of RASSF1A may contribute to the pathogenesis of many different forms of pediatric tumors.

High-resolution chromosome 3p allelotyping of breast carcinomas and precursor lesions demonstrates frequent loss of heterozygosity and a discontinuous pattern of allele loss

We performed high-resolution allelotyping for loss of heterozygosity (LOH) analysis on microdissected samples from 45 primary breast cancers, 47 mammary preneoplastic epithelial foci, and 18 breast cancer cell lines, using a panel of 27 polymorphic chromosome 3p markers. Allele loss in some regions of chromosome 3p was detected in 39 of 45 (87%) primary breast tumors. The 3p21.3 region had the highest frequency of LOH (69%), followed by 3p22-24 (61%), 3p21.2-21.3 (58%), 3p25 (48%), 3p14.2 (45%), 3p14.3 (41%), and 3p12 (35%). Analysis of all of the data revealed at least nine discrete intervals showing frequent allele loss: D3S1511-D3S1284 (U2020/DUTT1 region centered on D3S1274 with a homozygous deletion), D3S1300-D3S1234 [fragile histidine triad (FHIT)/FRA3B region centered on D3S1300 with a homozygous deletion], D3S1076-D3S1573, D3S4624/Luca2.1-D3S4597/P1.5, D3S1478-D3S1029, D3S1029 (with a homozygous deletion), D3S1612-D3S1537, D3S1293-D3S1597, and D3S1597-telomere; it is more than likely that additional localized regions of LOH not examined in this study also exist on chromosome 3p. In multiple cases, there was discontinuous allele loss at several 3p sites in the same tumor. Twenty-one of 47 (45%) preneoplastic lesions demonstrated 3p LOH, including 12 of 13 (92%) ductal carcinoma in situ, 2 of 7 (29%) apocrine metaplasia, and 7 of 25 (28%) usual epithelial hyperplasia. The 3p21.3 region had the highest frequency of LOH in preneoplastic breast epithelium (36%), followed by 3p21.2-21.3 (20%), 3p14.2/FHIT region (11%), 3p25 (10%), and 3p22-24 (5%). In 39 3p loci showing LOH in both the tumor and accompanying preneoplasia, 34 (87%) showed loss of the same parental allele (P = 1.2 x 10(-6), cumulative binomial test). In addition, when 21 preneoplastic samples showing LOH were compared to their accompanying cancers, 67% were clonally related, 20% were potentially clonally related but were divergent, and 13% were clonally unrelated. Overall this demonstrated the high likelihood of clonal relatedness of the preneoplastic foci to the tumors. We conclude that: chromosome 3p allele loss is a common event in breast carcinoma pathogenesis; involves multiple, localized sites that often show discontinuous LOH with intervening markers retaining heterozygosity; and is seen in early preneoplastic stages, which demonstrate clonal relatedness to the invasive cancer.

Differential methylation of genes that regulate cytokine signaling in lymphoid and hematopoietic tumors

The perturbations of the cytokine signaling pathway play an important role in lymphoid/hematopoietic tumors. Aberrant promoter methylation is the major mechanism of gene silencing in tumors. We examined 150 lymphoid/hematopoietic tumors or potential premalignant specimens, 55 control specimens and 12 EBV-transformed B lymphoblastoid cultures and 10 lymphoma/leukemia (L/L) or multiple myeloma (MM) cell lines for the methylation (and, in cell lines, of the expression status) of three genes involved in the cytokine signaling pathway. The genes were: SHP1, a protein tyrosine phosphatase; SYK, a protein kinase; and SOCS1, a suppressor of cytokine signaling. Our major findings were: (1) one or more of the three genes was frequently methylated in L/L and MM cell lines and there was good concordance (90–100%) between methylation and loss of gene expression; (2) treatment of L/L cell lines with a demethylating agent resulted in re-expression of SHP1 protein and downregulation of phosphorylated STAT3 in L/L cell lines; (3) all 55 control specimens and the lymphoblastoid cultures were negative for methylation of the three genes; (4) non-Hodgkins lymphomas (100%), and leukemias (94%) had almost universal methylation of SHP1 and relatively less frequent (<30%) methylation of SOCS1 and SYK; (5) MM and monoclonal gammopathy of unknown significance (MGUS) had infrequent methylation of SHP1 (<20%), and occasional methylation of SOCS1 and SYK; and (6) comparable methylation frequencies for SOCS1 were observed in MM and MGUS, suggesting that SOCS1 methylation is an early event in MM pathogenesis. At least one gene was methylated in 119 of 130 (93%) of the malignant and 12 of 20 (60%) of the MGUS samples. Our findings demonstrate that the perturbations of cytokine signaling via silencing of these three genes are almost universal in lymphoid/hematopoietic tumors but the patterns of gene methylated for L/L and plasma cell dyscrasias are different.

Targeted therapies for killing tumor cells.

In this issue of PNAS, Su et al. (1) present evidence that transfection and expression of the mda-7 gene in human pancreatic cancer cells combined with antisense inhibition of expression of a mutant K-ras gene results in tumor cell growth suppression and induction of apoptosis. They show that both of these treatments are required for this effect and also indicate that these treatments should not affect normal cells having wild-type K-ras. These results suggest translating the laboratory findings into the clinic as a new rational approach for this deadly disease. How can this translation take place, what are the potential problems, and can this approach be applied to other types of cancer?

Tissue microdissection and processing.

The accurate analysis of molecular changes associated with tumors and their precursor lesions requires the precise isolation of the specific cell types from a heterogeneous background of non-neoplastic elements such as normal epithelium, desmoplastic stroma, inflammatory cells and blood vessels(1).In the absence of a prior cell enrichment technique, the results of molecular analysis are undoubtedly confounded by genetic material not derived from cancer cells alone(2).The need for obtaining pure samples of tumor tissue has resulted in the genesis of several methods of cell enrichment including xenograft enrichment,tumor cell lines, cell sorting and microdissection. Xenograft enrichment involves the serial passage of tissues through immunodeficient rodents such asnu/nuor SCID mice to obtain human tumor cell populations whose non-malignant cells are of rodent origin(3, 4, 5).Despite their potential for being an unlimited self-replicating source of high quality genetic material, the ability to propagate xenografts requires considerable expertise, a reasonable animal facility,and time for establishment (between 2 to 6 months)(5).Moreover,there is a possibility that additional genetic changes may be introduced in the tumor cells during serial passage, or a subset of tumor cells with a selective growth advantage may propagate, which may not necessarily be representative of the primary lesion(4,6).Further, the presence of large numbers of stromal cells of rodent origin may complicate molecular analyses. On a similar note, tumor cell lines have been used for a long time to study genetic changes in neoplasia, and are an excellent source of unlimited reagents for this purpose(7,8).Like xenografts however,the establishment of a human tumor cell culture facility requires time,considerable expertise and resources;in addition, the introduction of additional genetic alterations or subset selection are always possibilities(9). Moreover, both cell lines and xenografts are virtually limited to the study of tumor cells only,and preneoplastic lesions have rarely been cultured (10).Cell sorting techniques have also been used at times as a means of cell enrichment, using density gradients, fluoroscence-activated cell sorting or antibody-labeled immunobead selection(11, 12, 13).Cell sorting can be easily applied to tumors amenable to formation of suspensions,such as hematolymphoid malignancies.However,cell sorting techniques are rarely applicable in solid tissue where intercellular adhesion generally prevents the disaggregation of cells,which is a prerequisite for the formation of a cell suspension.

hTR repressor-related gene on human chromosome 10p15.1

Somatic cells express genes that suppress telomerase activity and these genes may be inactivated in tumour cells. We postulated that cancer cells acquire immortality by activation of telomerase by the loss of such a gene. We have reported recently that a telomerase repressor gene may be located on 10p15.1 by deletion mapping using microcell-mediated chromosome transfer (MMCT), radiated microcell fusion (RMF), fluorescent in situ hybridization (FISH) and STS analysis. To independently confirm this result, we correlated expression of RNA component of telomerase (hTR) as a marker of telomerase expression by in situ hybridization with allelic loss in pulmonary carcinoid tumours. Unlike most malignant tumours, pulmonary carcinoids (which are low-grade malignant tumours) are heterogeneous for telomerase expression. Loss of 5 closely spaced polymorphic markers on 10p15.1, especially D10S1728, were highly correlated with hTR expression. In an additional experiment, 10p15.1 showed higher and more significant correlation than any region of 3p where it has been predicted as another chromosomal location of telomerase repressor with allelic loss of the region. Our findings strongly suggest that 10p15.1 harbours a gene involved in repression of telomerase RNA component in human somatic cells and each putative repressor (on 3p and 10p) may act independently.