Andreas Willer

Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR

We sought to establish a rapid and reliable RT-PCR approach for detection and quantification of BCR-ABL fusion transcripts using the LightCycler technology. This device combines rapid thermocycling with online detection of PCR product formation and is based on the fluorescence resonance energy transfer (FRET) between two adjacent hybridization probes carrying donor and acceptor fluorophores. A pair of probes was designed that was complementary to ABL exon 3, thus enabling detection of all known BCR-ABL variants and also normal ABL as an internal control. Conditions were established to amplify less than 10 target molecules/reaction and to detect one CML cell in 105cells from healthy donors. To determine the utility of the assay, we quantified BCR-ABL and ABL transcripts in 254 samples (222 peripheral blood, 32 bone marrow) from 120 patients with CML after therapy with IFN-α (n = 219), allogeneic BMT (n = 17), chemotherapy (n = 11), or at diagnosis (n = 7). The level of residual disease in the 245 BCR-ABL positive specimens was expressed as the ratio of BCR-ABL/ABL. This ratio was compared to results obtained by three established methods from contemporaneous specimens. A highly significant correlation was seen between the BCR-ABL/ABL ratios determined by the LightCycler and (1) the BCR-ABL/ABL ratios obtained by nested competitive RT-PCR (n = 201, r = 0.90, P < 0.0001); (2) the proportion of philadelphia chromosome positive metaphases determined by cytogenetics (n = 81, P < 0.0001); and (3) the bcr ratio determined by southern blot analysis (n = 122, P < 0.0001). we conclude that real-time pcr with hybridization probes is a reliable and sensitive method to monitor cml patients after therapy. the major advantages of the methodology are (1) amplification and product analysis are performed in the same reaction vessel, avoiding the risk of contamination; (2) the results are standardized by the quantification of housekeeping genes; and (3) the complete pcr analysis takes less than 60 min.

Cetuximab-Based Treatment of Metastatic Anal Cancer: Correlation of Response with KRAS Mutational Status

Background: No standard chemotherapy regimen can be defined for patients with metastatic squamous cell carcinoma of the anus due to the low incidence of this disease and the high cure rate of localized tumors. Anal cancers universally express the epidermal growth factor receptor (EGFR) and KRAS mutations have not been reported in anal cancer thus far. Methods: We report on 7 patients with metastatic anal cancer treated with cetuximab – a chimeric antibody against EGFR – on a compassionate use basis along with the results of KRAS mutational analysis. Results: Marked tumor shrinkage was noted in several patients using cetuximab monotherapy or cetuximab/irinotecan combination as first or subsequent treatment line (usually after failure of cisplatin-based regimens). Two out of seven patients harbored KRAS mutations. Both patients had progressive disease receiving cetuximab, while the remaining 5 patients had either a partial remission (n = 3), a minor remission (n = 1) or no change lasting ≥6 months after previous rapid tumor progression. Conclusion: Cetuximab-based treatment appears to be a valuable treatment option for patients with metastatic KRAS wild-type anal cancer after failure of or as an alternative to cisplatin/5-fluorouracil-based therapy.

Genomic anatomy of the specific reciprocal translocation t(15;17) in acute promyelocytic leukemia

The genomic breakpoints in the t(15;17)(q22;q21), associated with acute promyelocytic leukemia (APL), are known to occur within three different PML breakpoint cluster regions (bcr) on chromosome 15 and within RARA intron 2 on chromosome 17; however, the precise mechanism by which this translocation arises is unclear. To clarify this mechanism, we (i) assembled the sequence of RARA intron 2, (ii) amplified and sequenced the genomic PML‐RARA junction sequences from 37 APL patients, and (iii) amplified and sequenced the reverse RARA‐PML genomic fusion in 29 of these cases. Three significant breakpoint microclusters within RARA intron 2 were identified, suggesting that sequence‐associated or structural factors play a role in the formation of the t(15;17). There was no evidence that the location of a breakpoint in PML had any relationship to the location of the corresponding breakpoint in RARA. Although some sequence motifs previously implicated in illegitimate recombinations were found in the microcluster regions, these associations were not significant. Comparison of forward and reverse genomic junctions revealed microhomologies, deletions, and/or duplications of either gene in all but one case, in which a complex rearrangement with inversion of the PML‐derived sequence was found. These findings are consistent with the hypothesis that the t(15;17) occurs by nonhomologous recombination of DNA after processing of the double‐strand breaks by a dysfunctional DNA damage‐repair mechanism.

Alterations of the cyclin D1/pRb/p16INK4A pathway in multiple myeloma

The retinoblastoma protein (pRb), p16INK4A, D-type cyclins, and their partners cyclin-dependent kinase (CDK) 4 and 6 constitute a G1 regulatory pathway commonly targeted in tumorigenesis. Several malignancies show a reciprocal correlation between genetic alterations of single members of the pRb pathway. Therefore, we determined the frequency of Rb deletions and cyclin D1 alterations by fluorescence in situ hybridization as well as 5′ CpG island hypermethylation of the p16INK4Agene using methylation-specific polymerase chain reaction in bone marrow mononuclear cells from 82 individuals with plasma cell disorders. Alterations in at least one of the components of the pathway were found in 75%. Cyclin D1 translocations or amplifications were detected in 14/82 (17.1%), Rb deletions at 13q14 in 23/82 (28%) of the cases, including three (3.6%) homozygous deletions. p16INK4A was hypermethylated in 33/57 (57.9%) of the samples. Further analysis revealed a highly significant correlation between cyclin D1 alterations and extramedullar or leukemic myeloma manifestations (P = 0.014; Fishers test). Whereas Rb deletions seemed to occur alternatively to cyclin D1 alterations, no reciprocal correlation was found between p16INK4A hypermethylations and cyclin D1 or Rb locus aberrations. Cyclin D1 locus alterations and Rb deletions were associated with a significantly worse prognosis whereas p16INK4A hypermethylation had no impact on survival. We conclude that cyclin D1 and Rb aberrations seem to occur as alternative events in plasma cell malignancies and contribute to clinical course and prognosis. In contrast, although p16INK4A hypermethylation is frequent, inactivation of p16INK4A seems not to be involved in the pathogenesis of plasma cell disorders.

Specific aneusomies in Chinese hamster cells at different stages of neoplastic transformation, initiated by nitrosomethylurea

Aneuploidy is ubiquitous in cancer, and its phenotypes are inevitably dominant and abnormal. In view of these facts we recently proposed that aneuploidy is sufficient for carcinogenesis generating cancer-specific aneusomies via a chain reaction of autocatalytic aneuploidizations. According to this hypothesis a carcinogen initiates carcinogenesis via a random aneuploidy. Aneuploidy then generates transformation stage-specific aneusomies and further random aneusomies autocatalytically, because it renders chromosome segregation and repair mechanisms error-prone. The hypothesis predicts that several specific aneusomies can cause the same cancers, because several chromosomes also cooperate in normal differentiation. Here we describe experiments on the Chinese hamster (CH) that confirm this hypothesis. (i) Random aneuploidy was detected before transformation in up to 90% of CH embryo cells treated with the carcinogen nitrosomethylurea (NMU). (ii) Several specific aneusomies were found in 70–100% of the aneuploid cells from colonies transformed with NMU in vitro and from tumors generated by NMU-transformed cells in syngeneic animals. Among the aneuploid in vitro transformed cells, 79% were trisomic for chromosome 3, and 59% were monosomic for chromosome 10, compared with 8% expected for random distribution of any aneusomy among the 12 CH chromosomes. Moreover, 52% shared both trisomy 3 and monosomy 10 compared with 0.6% expected for random distribution of any two aneusomies. Among the tumor cells, 65% were trisomic for chromosome 3, 51% were trisomic for chromosome 5, and 30% shared both trisomies. Aneuploid cells without these specific aneusomies may contain minor transformation-specific aneusomies or may be untransformed. (iii) Random aneusomies and structurally altered chromosomes increased with the generations of transformed cells to the point where their origins became unidentifiable in tumors. We conclude that specific aneusomies are necessary for carcinogenesis.

Cyclin A1 is predominantly expressed in hematological malignancies with myeloid differentiation.

Cyclin A is a cell cycle regulatory protein that functions in mitotic and S phase control in mammalian cells. However, in contrast to other G1 phase regulatory proteins, such as cyclin D, retinoblastoma protein and p16INK4A, cyclin A seems not to be commonly involved in tumorigenesis. Recently, a second human cyclin A – cyclin A1 – has been identified. In contrast to cyclin A which is expressed throughout embryonic development and in adult tissue, the expression of cyclin A1 has been reported to be restricted to embryonic and germ line cells. We have confirmed the absence of cyclin A1 mRNA from normal peripheral blood leukocytes of seven healthy donors by single step reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, we have examined the expression of cyclin A1 mRNA in 173 peripheral blood samples of 162 patients with various hematological malignancies. Cyclin A1 mRNA was detectable in 11 of 11 patients with acute myeloid leukemia, three of three patients with acute biphenotypic leukemia, eight of eight patients with myelodysplastic syndrome, 59 of 69 patients with chronic myelogenous leukemia (CML) at diagnosis, 13 of 15 patients with CML in blastic transformation, 10 of 18 patients with chronic lymphocytic leukemia, two of nine patients with essential thrombocythemia, and only two of 10 patients with acute lymphoblastic leukemia (ALL) with both cyclin A1 RT-PCR positive ALL leukemias being undifferentiated relapses. In addition, cyclin A1 mRNA was found in one of six leukapheresis products, harvested from individuals without hematological disorders. Taken together, cyclin A1 is expressed in the majority of myeloid and undifferentiated hematological malignancies as well as in normal hematopoietic progenitor cells. We conclude that cyclin A1, a protein potentially involved in G1/S phase progression of immature cells, might be necessary for proliferation of early hematopoietic progenitor cells and their leukemic counterparts being blocked at that stage of differentiation.

Expression of interferon regulatory factor (IRF) genes and response to interferon-α in chronic myeloid leukaemia

Interferon regulatory factors (IRF) 1 and 2 are DNA-binding proteins which control interferon (IFN) gene expression. IRF1 functions as an activator for IFN and IFN-inducible genes, whereas IRF2 represses the action of IRF1. Expression of the two regulatory genes is itself IFN-inducible. Because therapeutic responses of chronic myeloid leukaemia (CML) patients to IFN-α may be determined by intracellular levels of these two mutually antagonistic transcription factors, we have devised a competitive reverse-transcription polymerase chain reaction (RT-PCR) assay which provides an estimate of the ratio of IRF1 to IRF2 expression in a given cell population. Analysis of peripheral blood leucocytes from 25 normal individuals showed that the IRF1:IRF2 ratio varied between 1.13 and 2.30 (mean ± s.d. 1.49 ± 0.33). Similar values were obtained for normal bone marrow specimens, with no significant difference between CD34+ and CD34− cells. In contrast, the IRF1:IRF2 ratio in leucocytes from CML patients showed a much wider variation (0.53–5.11). Eleven out of 130 patients in chronic phase had ratios above the normal range, whereas none of the 33 blast crisis samples had a ratio >2.5. Analysis of diagnostic specimens in 59 CML patients treated subsequently with IFN-α showed a high IRF1:IRF2 ratio of 5.11 in one of two patients who became complete responders; all the 53 patients with minimal or no cytogenetic response had ratios below 2.5. In a separate series of 97 CML patients studied after IFN-α therapy a highly significant correlation was found between the IRF1:IRF2 ratio and both the cytogenetic and the molecular response (ie low concentration of BCR-ABL transcripts) to treatment: 53 out of 115 prospectively analysed samples of good cytogenetic responders had ratios above 2.0, as opposed to only 13 out of 91 samples from poor responders (P < 0.0001; χ2 test). We conclude that a high ratio of IRF1/IRF2 expression may be associated with good cytogenetic and molecular response to IFN- α in CML.

Analysis of p73 and p53 gene deletions in multiple myeloma

Recently, p73, a protein with structural and functional similarities to p53, an extensively studied tumor suppressor gene, has been cloned. After being mapped to the chromosomal region 1p35–1p36, it has been postulated to act as a tumor suppressor gene, too, as this region is altered in several human malignancies. Deletions of the short arm of chromosome 1 have frequently been described in multiple myeloma (MM) whereas structural abnormalities of the 17p13 region including p53 are rare events in this disease. Since it has been proposed that especially neoplasias lacking p53 alterations might show a loss of heterozygosity at 1p35–1p36, we studied the frequency of p53 and p73 deletions in bone marrow mononuclear cells of 68 patients with MM, two patients with monoclonal gammopathy of undetermined significance and four patients with plasma cell leukemia. Dual-color fluorescence in situ hybridization (FISH) for p53 and p73 was performed using commercially available DNA probes for 17p13.3 and the microsatellite marker D1Z2, respectively. Centromeric DNA probes served to distinguish gene deletions from whole chromosome losses. In contrast to recently published FISH results, we only detected heterozygous p53 deletions in eight out of the 74 patients, three of them showing a monosomy 17. Heterozygous deletions of the D1Z2 region at 1p36 were found in six cases with one patient having a monosomy 1. Neither homozygous deletions of either chromosomal region nor nullisomies 1 or 17 could be detected. These results argue against a major role of p73 deletions in MM. As MM patients with 1p structural abnormalities have a significantly poorer survival rate than those with normal karyotypes, the role of other putative tumor suppressor genes located at the chromosomal region 1p36 in the pathogenesis of MM has to be determined.

Reduction of the Individual Cancer Risk by Physical Exercise

The great variety of different types of human malignancies and the equally variable individual history of physical activity militate against finding any simple relationship between the risk of developing cancer and physical activity. Due to an obvious lack of prospective randomized trials, any evidence for a correlation is currently based on cohort and case control studies. Furthermore, the study results are sometimes inconsistent, and only for selected cancers, the available data are sufficient to draw any conclusions, resulting in a level of evidence of 2–3 (level of recommendation ‘B’). Thus, a convincing risk reduction was found for colon cancer (40–50%) and estrogen-dependent malignancies such as breast (40–50%) and endometrial cancer (35–40%). Risk reduction is likely for some others, e. g. ovarian, lung or prostate cancer; but no definite conclusions can be drawn for hematological malignancies. Plausible explanations for reduction of the individual’s cancer risk by increased physical activity are currently available for estrogen dependent cancers (breast, ovarian, endometrial) and colon cancers. The currently available data allow the recommendation of adopting a ‘healthy life style’ including physical activity for prevention of certain cancers.

Frequent polymorphism in BCR exon b2 identified in BCR-ABL positive and negative individuals using fluorescent hybridization probes.

Recently, a polymorphic base in exon 13 of the BCR gene (exon b2 of the major breakpoint cluster region) has been identified in the eighth position before the junctional region of BCR-ABL cDNA. Cytosine replaces thymidine; the corresponding triplets are AAT (T allele) and AAC (C allele), respectively, both coding for asparagine. Therefore, this polymorphism has no implication in the primary structure of BCR and BCR-ABL proteins. However, since the alteration is located close to the fusion region it may have a significant influence on the annealing of PCR primers, probes for real time PCR, and antisense oligonucleotides. We have developed a RT-PCR-based screening method to easily identify polymorphic BCR and BCR-ABL alleles in CML patients and normal individuals in order to estimate their frequency. After amplification from cDNA, a melting curve of a specific fluorogenic probe mapping to the 3′ end of BCR exon b2 and spanning the polymorphism readily discriminates between normal and polymorphic BCR and BCR-ABL alleles. This reporter probe is 3′ labeled with fluorescein and placed next to 5′ LC Red640-labeled anchor probes mapping to the 5′ ends of BCR exon b3 or ABL exon a2 so that resonance energy transfer occurs when the probes are hybridized (LightCycler technology). T and C alleles were discriminated by a melting temperature difference of the reporter probe of 3.2 K. We have investigated cDNAs derived from leukocytes from seven cell lines and a total of 229 individuals: normal donors, n = 15; BCR-ABL negative chronic myeloproliferative disorders, n = 30; BCR-ABL negative acute leukemias, n = 11; b2a2BCR-ABL positive CML, n = 93; and b3a2BCR-ABL positive CML, n = 80. The frequency of the C allele was 33.0% in BCR-ABL negative individuals, 30.6% in b2a2BCR-ABL, and 23.8% in b3a2BCR-ABL positive CML. In CML patients, 27.7% of BCR-ABL and 27.2% of BCR alleles had the C allele (NS). In total, 132 of 458 (28.8%) exons b2 of BCR or BCR-ABL alleles demonstrated this polymorphism. We conclude that a thymidine/cytosine replacement occurs frequently in BCR exon b2. Probes for real time quantitative RT-PCR should be designed not to map to the critical region in order to avoid underestimation of the number of BCR-ABL transcripts.