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Important biological and pathologic properties are often conserved across species. Although several mouse leukemia models have been well established, the genes deregulated in both human and murine leukemia cells have not been studied systematically. We performed a serial analysis of gene expression in both human and murine MLL-ELL or MLL-ENL leukemia cells and identified 88 genes that seemed to be significantly deregulated in both types of leukemia cells, including 57 genes not reported previously as being deregulated in MLL-associated leukemias. These changes were validated by quantitative PCR. The most up-regulated genes include several HOX genes (e.g., HOX A5, HOXA9, and HOXA10) and MEIS1, which are the typical hallmark of MLL rearrangement leukemia. The most down-regulated genes include LTF, LCN2, MMP9, S100A8, S100A9, PADI4, TGFBI, and CYBB. Notably, the up-regulated genes are enriched in gene ontology terms, such as gene expression and transcription, whereas the down-regulated genes are enriched in signal transduction and apoptosis. We showed that the CpG islands of the down-regulated genes are hypermethylated. We also showed that seven individual microRNAs (miRNA) from the mir-17-92 cluster, which are overexpressed in human MLL rearrangement leukemias, are also consistently overexpressed in mouse MLL rearrangement leukemia cells. Nineteen possible targets of these miRNAs were identified, and two of them (i.e., APP and RASSF2) were confirmed further by luciferase reporter and mutagenesis assays. The identification and validation of consistent changes of gene expression in human and murine MLL rearrangement leukemias provide important insights into the genetic base for MLL-associated leukemogenesis. [Cancer Res 2009;69(3):OF1109–16]

The tumor suppressor gene INK4b (p15) is silenced by CpG island hypermethylation in most acute myelogenous leukemias (AML), and this epigenetic phenomenon can be reversed by treatment with hypomethylating agents. Thus far, it was not investigated whether INK4b is hypermethylated in all cytogenetic subtypes of AML. A comparison of levels of INK4b methylation in AML with the three most common cytogenetic alterations, inv(16), t(8;21), and t(15;17), revealed a strikingly low level of methylation in all leukemias with inv(16) compared with the other types. Surprisingly, the expression level of INK4b in inv(16)+ AML samples was low and comparable with that of the other subtypes. An investigation into an alternative mechanism of INK4b silencing determined that the loss of INK4b expression was caused by inv(16)-encoded core binding factor β-smooth muscle myosin heavy chain (CBFβ-SMMHC). The silencing was manifested in an inability to activate the normal expression of INK4b RNA as shown in vitamin D3–treated U937 cells expressing CBFβ-SMMHC. CBFβ-SMMHC was shown to displace RUNX1 from a newly determined CBF site in the promoter of INK4b. Importantly, this study (a) establishes that the gene encoding the tumor suppressor p15INK4b is a target of CBFβ-SMMHC, a finding relevant to the leukemogenesis process, and (b) indicates that, in patients with inv(16)-containing AML, reexpression from the INK4b locus in the leukemia would not be predicted to occur using hypomethylating drugs. [Cancer Res 2007;67(3):992–1000]

Background: Prognostically relevant biomarkers in breast cancers can be assayed using nucleic acid (e.g. OncoType DX) or protein based (e.g. immunohistochemistry (IHC)) detection stratagies. Nucleic acid tests measure many more markers compared to immunohistochemistry, but are weighted heavily for ER, PR, and HER2 in their prognostic scoring algorithms. Recent data suggest that a panel of 4 IHC stains (IHC4) may have equivalent prognostic ability to multigene assays. The aim of this study was to retrospectively compare IHC4 scores to OncoType DX recurrence scores in a large cohort of patients in our statewide health system. Methods: Formalin-fixed paraffin-embedded breast cancer samples previously submitted for OncoType DX testing between 2008 and 2011 were obtained and sections stained for ER, PR, HER2, and Ki67 (the IHC4 staining panel). Stained slides were scanned digitally and scored using customized pathologist assisted image analysis algorithms (Leica Biosystems Aperio, Vista CA). The resulting data were used in the modified “Magee Equation 3” (Klein et al. Mod Pathol. 2013. PMID: 23503643) to calculate an IHC4 recurrence score on a numerically comparable scale to that used in the OncoType score. The IHC4 and Oncotype DX scores were directly compared for concordance and correlation. Results: 280 breast cancer samples comprised the study cohort. The mean (median) IHC4 score was 20.13 (19), range: 11-42. The mean (median) OncoType score was 18.48 (16), range: 0-75. Using cut-offs of 0-17 for low risk, 18-30 for intermediate risk, and >30 for high risk, the concordance data is shown in Table 1. Comparison of scores from IHC4 and OncoType by Risk Category OncoType RS LowIntermedHigh Low80201IHC4 RSIntermed816819 High029RS = Recurrence Score Pearson's correlation coefficient for the entire set was 0.6669. The overall concordance was 56.1%; one-step discordance 43.6%; and two-step discordance: 0.3%. Eliminating the intermediate scores, the concordance was 98.8%. Conclusions: Reasonable agreement was seen between risk scores derived by IHC4 and OncoType DX. Only one discrepancy among 280 scores would have resulted in a different treatment recommendation (low risk by IHC4 and high risk by OncoType). If the IHC4 score is clearly high or low, one would not expect a dramatically different result from Oncotype DX, and the Oncotype DX test may not be needed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-34.

The p27Kip1 (p27) gene encodes an inducible inhibitor of cyclin-dependent kinase activity. Using a murine p27 cDNA as probe, we obtained a human cDNA clone and subsequently used it to isolate a genomic clone of this gene. The coding region of the human p27 gene was contained in two exons. Both the amino acid sequence and intron-exon organization of p27 were similar to those previously found for the related cyclin-dependent kinase inhibitor p21Waf1 (p21). The p27 gene was localized to chromosome band 12p13 by a combination of somatic cell hybrid and fluorescence in situ hybridization analyses. The p27 gene product is thought to control the leukocyte cell cycle and the 12p13 chromosomal band is known to be deleted in leukemias, suggesting that the p27 gene may act as a tumor suppressor gene in leukemias. Although p27 was found to reside in the minimal region of chromosomal loss in hematological malignancies, no mutations of p27 were observed in leukemia samples. Haploinsufficiency of p27 may confer a growth advantage to leukemia cells.

Previous studies have suggested that structural abnormalities involving the short arm of chromosome 9 are frequently associated with gliomas. The alpha-, beta-, and omega-interferon (IFNA, IFNB1, and IFNW, respectively) and the methylthioadenosine phosphorylase (MTAP) genes have been mapped to the short arm of chromosome 9, band p22. Homozygous deletions of these genes have been reported in many leukemia- and glioma-derived cell lines. In this report, we present a detailed analysis of partial and complete homozygous or hemizygous deletions of DNA sequences on 9p in human cell lines and primary tumor samples of glioma patients. Ten of 15 (67%) glioma-derived cell lines had hemizygous or homozygous deletion of IFN genes or rearrangement of sequences around these genes, while 13 of 35 (37%) primary glioma tumor samples had hemizygous (8 tumors) or homozygous (5 tumors) deletion of the IFN genes. The shortest region of overlap of these deletions maps in the interval between the centromeric end of the IFN gene cluster and the MTAP gene. In the cell lines and primary tumors examined, these gross genomic alterations were seen only in association with high grade or recurrent gliomas. Our observations confirm that loss of DNA sequences on 9p, particularly the IFN genes, occurs at a significant frequency in gliomas, and may represent an important step in the progression of these tumors. These results are consistent with a model of tumorigenesis in which the development or progression of cancer involves the loss or inactivation of a gene or several genes that normally act to suppress tumorigenesis. One such gene may be located on 9p; this gene may be closely linked to the IFN genes. Nevertheless, loss of the IFN genes, when it occurs, may play an additional role in the progression of these tumors.

Background:Oncotype DX has been validated to quantify the risk of distant recurrence and predict the benefit of chemotherapy (CT) in ER positive, node negative breast cancer treated with tamoxifen. A retrospective study was undertaken to assess the use of the Oncotype DX test at Intermountain Healthcare. Intermountain is a not-for-profit healthcare system with 21 inpatient facilities, 9 comprehensive cancer centers and 42 affiliated medical oncologists ranging from single practitioners to multiphysician groups.Methods: This study contains a group of T1-3 N0 ER+ breast cancer patients who received an Oncotype DX test paired with a control group of non-tested T1-T3 N0 ER+ patients receiving hormone therapy (HT) from the same period. Data comes from a supplemental database containing treatment and follow-up data from individual physician offices combined with data from Intermountain's cancer registry. To ensure data completeness, Genomic Health provided a list of relevant Oncotype DX results for study patients. The analysis was done using multivariate logistic regression and controlled for age, tumor size, grade and T stage.Results: From 2005 to 2008, Oncotype DX testing was performed on tumor specimens from 285 patients. 8 patients had positive nodes and 4 patients were ER negative. In addition, 9 patients did not and will not receive HT (5 refused, 4 contraindicated) and 9 patients have yet to begin HT. 11 patients were lost to follow-up. Of the remaining 244 patients who form the study group, six patients were Her-2 neu positive (1 high recurrence score (RS), 3 intermediate and 2 low). Tumor size ranged from 2 cm. Compared to our control group of 688 patients, Oncotype DX-tested patients are younger (p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6058.

Background: Hormone receptor testing is important in the management of women with breast cancer. We previously reported potential adverse effects of variable specimen and prolonged specimen handling conditions on ER (estrogen receptor) test results (2005 SABCS, abstract#5107).Objective: To compare prevalence of ER and PR (progesterone receptor) negative test results following standardization of pre-analytical specimen handling conditions at Intermountain facilities.Methods: Prospective, quasi-experimental study design of 6 Intermountain facilities. Facilities were separated into 2 categories: experimental (2 facilities) and control (4 facilities) groups. Pre-analytical specimen handling conditions (including recording of time to fixative and duration of fixation in neutral buffered formalin) were standardized at experimental facilities but not at control facilities. Standardization consisted of educating operating and grossing room staff about appropriate specimen handling and the value of recording time to fixation and fixative duration as a way to improve pre-analytic standardization. OR staff was called in any cases where times were not recorded. Study population includes women undergoing breast cancer surgery and who were tested for ER/PR status between January 2008 and January 2009. Specimen handling conditions and ER/PR test results were collected manually. Covariates were retrieved from cancer registry and included age, grade, positive lymph nodes, specimen type, and tumor stage. Multivariate logistic regression was used to compare prevalence of ER and PR negativity between experimental and control facilities after controlling for covariates.Results: 1054 women with breast cancer were tested for ER/PR status during the study period. The average age was 60.2 years (59.2 years for control cases and 61.1 years for the experimental group). The overall prevalence of ER and PR negative tests was respectively 18.5% and 27.3%. Average time to fixative at experimental facilities was 18.4 minutes (SE=3.1; 95% CI, 12.2-25.6) and average time in fixative was 18.0 hours (SE=0.4; 95% CI, 17.2-18.8). Compared to experimental facilities, both the prevalence of ER and PR negativity was higher (16.9% vs 19.7%) and (23.9% vs 30.0%) at control facilities. After controlling for covariates there was no difference in prevalence of ER negativity (p=0.13) between the two groups. However, the prevalence of PR negativity remains significantly higher (p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4154.

An interstitial deletion of the short arm of a chromosome 11 was found in Wilms' tumor cells of a patient without aniridia. The chromosomes in her peripheral blood lymphocytes were normal. The karyotype of the tumor cells is similar to that reported for the somatic cells of patients with aniridia-Wilms' tumor association. We showed that the same cytogenetic change as that seen in patients with this condition, namely, an interstitial deletion of 11p13, can occur in patients without aniridia as a mutation that is confined to tumor cells.

Karyotypes were analyzed by routine Giemsa and quinacrine fluorescence for 16 patients with acute lymphocytic leukemia [ten adults (18 to 51 years) and six children (3 to 15 years)]. Four patients had received previous therapy, but all 16 had active disease when they were first studied. Eight patients (five untreated) had a normal karyotype initially; however, three of these developed a chromosomal abnormality during relapse. Eight patients had a chromosomal abnormality in their initial samples. Each of the 11 patients had different abnormalities. All chromosomes except Nos. 3, 5, 15, 16, and Y were involved in the various aneuploidies. One patient had a Ph1 chromosome due to a translocation with No. 21: t(21;22)(q22;q11). A patient with B-cell acute lymphocytic leukemia had a 14q+ marker in addition to other abnormalities. The median survival of patients with initially normal karyotypes may be longer than that of patients whose karyotypes are abnormal initially.

Complete data regarding age, sex, karyotype, and French-American-British Cooperative Group classification were available for 239 unselected patients with acute nonlymphocytic leukemia. Of these, 128 were classified as having acute myeloblastic leukemia (M1 or M2); within the acute myeloblastic leukemia group, 83 (65%) of the patients were chromosomally abnormal. Except for 16 patients with a t(8;21), the percentage of patients with an abnormal karyotype increased with age, particularly above the age of 50 years. Besides the patients with t(8;21), there were 29 patients with loss of part or all of chromosomes 5 and/or 7, 11 patients with +8, and 27 patients with other abnormalities. Of 70 patients with acute myelomonocytic leukemia (M4), on the other hand, 28 (40%) were chromosomally abnormal, only three had loss of chromosomes 5 or 7, one was -7, +8, and our were +8, whereas 20 had other abnormalities. This difference may reflect different etiological factors in these two types of leukemia.

DNA-based cytophotometry was used to analyze metaphase chromosomes in four patients with chronic myelogenous leukemia. In three of these patients, both Philadelphia chromosome (Ph1)-positive and Ph1-negative cells were measured. On the basis of these three patients, the characteristic 9q+/22q- translocation of chronic myelogenous leukemia involves the net transfer of 0.325% of the autosomal genome; there is no evidence of net gain or loss of DNA (apart from duplication of the Ph1 chromosome in one patient), and no significant difference is found in the amount of DNA transferred in different patients. Significant differences are found among patients in the derived Chromosomes 9 and the Ph1 chromosomes and are ascribed to preexisting variations in the Ph1-negative cells of these patients. There is no evidence in these patients of any further cytogenetic lesion associated with chronic myelogenous leukemia.

The karyotype of nine of 11 Wilms' tumors was successfully analyzed using chromosome banding techniques. Peripheral lymphocytes had a normal karyotype in all six analyzed cases. Cultured cells from one tumor had a normal karyotype; however, they appeared to be fibroblasts. A chromosome 11 deletion, del(11)(p13p14), similar to that seen in patients with sporadic aniridia, was found as the sole abnormality in cells from one tumor. Abnormalities of chromosome 1 resulting in trisomy for the long arm (q21-q31) were found in five cases. Two of them had a translocation involving 1q and 16q, although the breakpoints in each chromosome appeared to differ in the two cases. Two patients had an isochromosome of the long arm, i(1q), and a fifth case had a duplication of the long arm as a result of karyotypic evolution. Chromosome 16 abnormalities were found in three cases, resulting in the partial monosomy of the long arm, sharing q22 as a common deletion. The same three cases also had trisomy 1q due to an unbalanced translocation of 1q or an i(1q). Trisomy for both chromosomes 9 and 12 were present in three cases. Two patients each had whole or partial trisomy of chromosomes 6, 7, 8, 17, and 18. Our data show that although an 11p deletion can occur as a mutation confined to tumor cells, the most common changes are trisomy for 1q, and less often a deletion of 16q.

We studied the clinical and karyotypic features of 50 patients with acute lymphoblastic leukemia, including 33 American and 17 Japanese patients, at two institutions. Clonal chromosome abnormalities were found in 39 of the 50 patients (78%) at diagnosis. Eleven patients had diploidy (N patients). Among the 39 aneuploid patients, 17 had pseudodiploidy (A1 patients), eight had hyperdiploidy with 47 to 49 chromosomes (A2 patients), nine had hyperdiploidy with 50 to 59 chromosomes (A3 patients), and five had other chromosome abnormalities. Of 14 patients whose chromosomes were also studied at relapse, eight had karyotypic progression, five had abnormalities identical or similar to those observed at diagnosis, and one had a change of karyotype from diploidy to aneuploidy. The median age and the median WBC of A1 patients were higher than those of any other group of patients, although one-third of the patients had WBC below 20 x 10(3)/microliters, and they often had leukemic cells of T-cell or B-cell lineage. The A2 patients were relatively old and tended to have higher WBC. The N patients were relatively young and tended to have low WBC, although these tendencies were not as marked as those in A3 patients. The A3 patients had longer survival times than the A1 (p = 0.003) or A2 (p = 0.002) patients. Also, N patients had longer survival times than A1 (p = 0.03) or A2 (p = 0.05) patients. The difference in survival times between A3 and N patients was not significant. Our study demonstrated that the karyotype is correlated with survival and with other recognized prognostic factors. However, in some A1 and A2 patients, the karyotype was a more reliable factor in indicating a poor prognosis than was the WBC or age.