Prognostic and predictive value of CA 19-9 in locally advanced pancreatic cancer treated with multiagent induction chemotherapy: results from a prospective, multicenter phase II trial (NEOLAP-AIO-PAK-0113).

BACKGROUND: The prognostic and predictive value of carbohydrate antigen 19-9 (CA 19-9) in locally advanced pancreatic cancer (LAPC) has not yet been defined from prospective randomized controlled trials (RCTs). PATIENTS AND METHODS: A total of 165 LAPC patients were treated within the NEOLAP RCT for 16 weeks with multiagent induction chemotherapy [ICT; either nab-paclitaxel/gemcitabine alone or nab-paclitaxel/gemcitabine followed by FOLFIRINOX (combination of fluorouracil, leucovorin, irinotecan, and oxaliplatin)] followed by surgical exploration of all patients without evidence of disease progression. CA 19-9 was determined at baseline and after ICT and correlated with overall survival (OS) and secondary R0 resection rate. RESULTS: From the NEOLAP study population (N = 165) 133 patients (81%) were evaluable for CA 19-9 at baseline and 81/88 patients (92%) for post-ICT CA 19-9 response. Median OS (mOS) in the CA 19-9 cohort (n = 133) was 16.2 months [95% confidence interval (CI) 13.0-19.4] and R0 resection (n = 31; 23%) was associated with a significant survival benefit [40.8 months (95% CI 21.7-59.8)], while R1 resected patients (n = 14; 11%) had no survival benefit [14.0 (95% CI 11.7-16.3) months, hazard ratio (HR) 0.27; P = 0.001]. After ICT most patients showed a CA 19-9 response (median change from baseline: -82%; relative decrease ≥55%: 83%; absolute decrease to ≤50 U/ml: 43%). Robust CA 19-9 response (decrease to ≤50U/ml) was significantly associated with mOS [27.8 (95% CI 18.4-37.2) versus 16.5 (95% CI 11.7-21.2) months, HR 0.49; P = 0.013], whereas CA 19-9 baseline levels were not prognostic for OS. Multivariate analysis demonstrated that a robust CA 19-9 response was an independent predictive factor for R0 resection. Using a CA 19-9 decrease to ≤61 U/ml as optimal cut-off (by receiver operating characteristic analysis) yielded 72% sensitivity and 62% specificity for successful R0 resection, whereas CA 19-9 nonresponders (<20% decrease or increase) had no chance for successful R0 resection. CONCLUSIONS: CA 19-9 response after multiagent ICT provides relevant prognostic and predictive information and is useful in selecting LAPC patients for explorative surgery. CLINICAL TRIAL NUMBER: ClinicalTrials.govNCT02125136; https://clinicaltrials.gov/ct2/show/NCT02125136; EudraCT 2013-004796-12; https://www.clinicaltrialsregister.eu/ctr-search/trial/2013-004796-12/results.

Epstein-Barr virus encoded latent membrane protein 1 (LMP1) and TNF receptor associated factors (TRAF): colocalisation of LMP1 and TRAF1 in primary EBV infection and in EBV associated Hodgkin lymphoma.

AIMS: Epstein-Barr virus (EBV) immortalises B cells in vitro and is associated with several malignancies. Most phenotypic effects of EBV are mediated by latent membrane protein 1 (LMP1), which interacts with tumour necrosis factor receptor associated factors (TRAFs) to activate NF-kappaB. This study examines TRAF1 and LMP1 expression in EBV associated lymphoproliferations. METHODS: TRAF1 expression was investigated in 26 Hodgkin lymphomas (HL; 18 EBV+, eight EBV-), seven EBV+ Burkitt lymphomas (BL), two infectious mononucleosis (IM) tonsils, and lymphoreticular tissue from eight chronic virus carriers. Seven anaplastic large cell lymphomas and 10 follicular B cell lymphomas were also studied. Colocalisation of TRAF1 and LMP1 was studied by immunofluorescent double labelling and confocal laser microscopy. RESULTS: TRAF1 colocalises with LMP1 in EBV infected cells in IM. EBV positive lymphocytes from chronic virus carriers were negative for TRAF1 and LMP1. In HL biopsies, TRAF1 was strongly expressed independently of EBV status, whereas all BL cases were TRAF1-. In EBV+ HL cases, TRAF1 colocalised with LMP1. Eight of 10 follicular lymphomas expressed TRAF1 in centroblast-like cells. Four of seven anaplastic large cell lymphomas weakly expressed TRAF1. CONCLUSIONS: These results suggest that in non-neoplastic lymphocytes, TRAF1 expression is dependent on the presence of LMP1, and that in IM B cells in vivo, LMP1 associated signalling pathways are active. In HL, TRAF1 is expressed independently of EBV status, probably because of constitutive NF-kappaB activation. The function of TRAF1 in HL remains to be determined.

Fine structure of translocation breakpoints in leukemic blasts with chromosomal translocation t(4;11): the DNA damage-repair model of translocation.

Chromosomal translocations t(4;11) are regularly associated with a specific type of acute leukemias and probably initiate the development of this disease. It has been proposed by others, that these translocations are mediated by recombinases of the immune system. The breakpoints on both derivative chromosomes for three t(4;11) leukemia-derived cell lines and primary blasts from two patients have been analysed here in detail. The results revealed that: (a) multiple double- or single-stranded DNA breaks must have occured near the translocation breakpoints on both participating chromosomes; and (b) DNA fragments flanked by these breaks must have either been deleted, inverted or duplicated during the translocation process. We found no evidence for the involvement of specific target sequences and recombinases of the immune system. Similar characteristic features were observed by re-interpretation of published t(6;11) and t(9;22) translocation data. Therefore we present a new model for the generation of these translocations which poses, that these translocations are reciprocal but not balanced at the fine structure level and that the DNA damage-repair machinery is likely involved in producing the final structure of the translocation breakpoint.

Exon/intron structure of the human AF-4 gene, a member of the AF-4/LAF-4/FMR-2 gene family coding for a nuclear protein with structural alterations in acute leukaemia.

The AF-4 gene on human chromosome 4q21 is involved in reciprocal translocations to the ALL-1 gene on chromosome 11q23, which are associated with acute lymphoblastic leukaemias. A set of recombinant phage carrying genomic fragments for the coding region and flanking sequences of the AF-4 gene were isolated. Phage inserts were assembled into four contigs with 21 exons, and an intron phase map was produced enabling the interpretation of translocation-generated fusion proteins. The gene contains two alternative first exons, 1a and 1b, both including a translation initiation codon. The translocation breakpoint cluster region is flanked by exons 3 and 6 and two different polyadenylation signals were identified. Polyclonal antisera directed against three different portions of the AF-4 protein were produced and used to detect a 116 kD protein in cellular extracts of human B-lymphoblastoid and proB cell lines. In mitogen-stimulated human peripheral blood mononuclear cells the AF-4 antigen was predominantly located in the nucleus. The AF-4 gene is a member of the AF-4, LAF-4 and FMR-2 gene family. The members of this family encode serine-proline-rich proteins with properties of nuclear transcription factors. Comparison of AF-4 protein coding sequences with the LAF-4 and FMR-2 sequences revealed five highly conserved domains of potential functional relevance.

The structure of the human ALL-1/MLL/HRX gene.

The human ALL-1/MLL/HRX gene on chromosome 11q23 is the site of many locally clustered chromosomal alterations associated with several types of acute leukemias, including deletions. partial duplications and reciprocal translocations. Structurally variant proteins derived from an altered ALL-1 gene presumably make essential contributions to the malignant transformation of hematopoietic progenitor cells. The ALL-1 gene is spread over approximately 92 kb and consists of at least 37 exons. An exon/intron map including the position of the 3'-end of the gene and a detailed restriction map were produced and an updated map is presented. Data from other laboratories were incorporated where compatible. Exon/intron boundaries were sequenced and an intron-phase analysis was performed. The results are expected to contribute to a better understanding of those structural alterations of the gene that conserve the open reading frame and produce presumably oncogenic variants of the ALL-1 protein. They will also facilitate the rapid molecular diagnosis of structural alterations of this gene and the choice of therapeutic options. Mechanisms that may potentially account for the striking clustering of the translocation breakpoints in the breakpoint cluster region of the gene are discussed.

Exon/intron structure of the human ALL-1 (MLL) gene involved in translocations to chromosomal region 11q23 and acute leukaemias.

The acute lymphoblastic leukaemia (ALL)-1 gene on human chromosome 11q23 is the site of many locally clustered chromosomal alterations associated with several types of acute leukaemias, including deletions, partial duplications and translocations. Structurally variant proteins derived from the altered gene presumably cause the malignant transformation of early haemopoietic progenitor cells. According to previously published reports, the gene consisted of at least 21 exons spread over approximately 100 kb. In this report a set of genomic fragments was isolated that represent a total of 35 exons (exons 3-37) encompassing > 95% of the protein-coding region (except exons 1 and 2) and the 3'-non-translated region of the gene. The distances between these exons were determined and a detailed restriction map was produced. The majority of the exon/intron boundaries were sequenced and an intron-phase analysis was performed. The results form the basis for a greater understanding of the translocations and other structural alterations of the gene that conserve the open reading frame and thus produce presumably oncogenic variants of the ALL-1 protein.

A specific deletion in the breakpoint cluster region of the ALL-1 gene is associated with acute lymphoblastic T-cell leukemias.

A variety of chromosomal translocations to the ALL-1 gene are regularly observed in acute leukemias and are thought to play a key role in the leukemogenic process. Chimeric proteins are encoded by the breakpoint regions of the derivative chromosomes have been proposed to be the relevant oncogenic agents. In addition, internal duplications of the ALL-1 gene have been observed in patients with specific acute myeloid leukemias. Thus, it has been hypothesized that oncogenic variants of the ALL-1 protein may be generated by both chimerization and self-fusion, but the critical structural features endowing the altered proteins with their oncogenic potential are still unknown. Here a novel structural alteration of the ALL-1 gene was observed in three patients presenting with acute T-cell leukemia (ALL) without chromosomal translocations or self-fusions of the ALL-1 gene. These unrelated patients carried an internal deletion in one of the two alleles of the ALL-1 gene that eliminated parts of introns 7 and 8, together with exon 8. The deletion was found in 3 of 74 ALL patients, but not in acute myeloid leukemias, follicular lymphomas, or peripheral blood leukocytes from healthy donors. One ALL patient showed the deletion at diagnosis but no longer at remission or at 9 months after remission. These findings support the hypothesis that the ALL-1 protein may be converted to an oncogenic variant, not only by chimerization or self-fusion, but also by deletion of sequences coded by exon 8. They further suggest that these three different types of structural alterations of the ALL-1 protein may each cause a distinct disease phenotype. Alternatively spliced mRNA species omitting exon 8 were observed in 14 of 24 ALL patients without detectable macroscopic alterations of the ALL-1 gene and also in peripheral blood leukocytes from healthy donors.

Molecular analysis of the chromosomal breakpoint and fusion transcripts in the acute lymphoblastic SEM cell line with chromosomal translocation t(4;11).

The chromosomal breakpoint and fusion transcripts of the pre-B-leukaemia-derived SEM cell line carrying a reciprocal t(4;11)(q21;q23) translocation were analysed. The breakpoint from derivative chromosome der4 was cloned and sequenced. The crossover site was localized in intron 7 of the ALL-1 gene on chromosome 11q23 and in a large intron of the AF-4 (FEL) gene. RNA transcripts from both wild-type genes and both hybrid genes were detected by reverse transcriptase polymerase chain reaction (RT-PCR) assays. In addition, alternatively spliced mRNA species derived from the der4 chromosome were found. They were generated by using the exon 5' of the breakpoint on der4 as a common splice donor site and the 5' boundaries of exons 8 or 9 of the ALL-1 gene as alternative splice acceptor sites. The hypothesis is proposed that selective pressure operators to maintain the presence of both derivative chromosomes as important elements in the leukaemogenic process.