Intragenetische IKZF1-Deletionen bei Erwachsenen mit BCR-ABL-negativer akuter lymphatischer Leukämie (ALL)

Hintergrund: Mutationen des Transkriptionsfaktors IKZF1 wurden in den letzten Jahren bei Patienten mit akuten Leukämien der B-Zell-Reihe (B-ALL) nachgewiesen. Neben komplettem Genverlust und Punktmutationen unterscheidet man zwei Typen von intragenetischen Deletionen: mono-allelischer Funktionsverlust (loss-of-function) und komplette Unterdrückung der Proteinfunktion (dominant-negativ). Für die große Patientengruppe von Erwachsenen mit BCR-ABL-negativer B-ALL gibt es nur begrenzte Daten zur Häufigkeit und der prognostischen Bedeutung von IKZF1-Alterationen. Methodik: Wir untersuchten DNA-Proben von 482 Patienten mit BCR-ABL-negativer B-ALL, die im Rahmen der GMALL-Studienprotokolle 06/99 und 07/03 behandelt wurden, mittels PCR auf intragenetische Deletionen (∆2-7, ∆2-8, ∆4-7, ∆4-8). Reverse-Transkriptase-PCRs (RT-PCR) wurden durchgeführt um ∆2-3 und andere seltene Deletionen zu erkennen. Mittels quantitativer PCRs (∆2-7, ∆4-7, ∆4-8) und Geldensitometrie wurde die relative Konzentration der Zellen mit IKZF1-Deletionen bestimmt. Es wurde zwischen Deletionen in einem Großteil der Zellen (“highdel”) und Deletionen in nur einem kleinen Teil der Zellen (“lowdel”) unterschieden. Der prognostische Effekt dieser beiden Gruppen wurde separat untersucht. Alle Deletionen wurden sequenziert und die DNA-Bruchpunkte analysiert. Ergebnisse: 128 Patienten (27%) zeigten eine intragenetische IKZF1-Deletion, 37 davon wiesen mehr als eine Deletion auf (175 Deletionen insgesamt). 56 Patienten (12%) hatten nur loss-of-function Deletionen, 50 (10%) hatten nur dominant-negative Deletionen, während 22 Patienten beide Deletionstypen aufwiesen (5%). Mindestens eine highdel IKZF1-Deletion konnte bei 98 Patienten (20%) nachgewiesen werden. Patienten mit einer loss-of-function IKZF1-Deletion zeigten ein signifikant reduziertes Gesamtüberleben (overall survival (OS) nach 5 Jahren 0.37 vs. 0.59, p=0.0012), während dominant-negative Deletionen keinen Effekt auf das Gesamtüberleben hatten (0.54 vs. 0.56, p=0.95). In der Patientengruppe mit loss-of-function Deletionen waren nur highdel-Deletionen mit einem reduzierten Gesamtüberleben assoziiert (OS 0.28 vs. 0.59, p<0.0001), während Patienten mit einer lowdel-Deletion einen klinischen Verlauf ähnlich Patienten ohne loss-of-function Deletion aufwiesen. Der Effekt der highdel loss-of-function Deletionen war auch in der Standardrisiko-Subgruppe nach GMALL-Kriterien signifikant (0.37 vs. 0.68, p=0.0002). In der Patientengruppe mit dominant-negativen Deletionen gab es keine Assoziation zwischen dem relativen Anteil an Zellen mit Deletionen und dem Gesamtüberleben (p=0.62). Die Sequenzierung von 193 Deletionen ergab eine Häufung der Bruchpunkte innerhalb vier großer Bruchpunkt-Cluster. Bei 183 der 193 Sequenzen waren sowohl am proximalen als auch am distalen Bruchpunkt kryptische Rekombinations-Signal-Sequenzen (cRSS) nachweisbar. Diskussion: In der Patientengruppe der Erwachsenen mit BCR-ABL-negativer B-ALL sind loss-of-function IKZF1-Deletionen mit einem schlechteren klinischen Verlauf assoziiert, wenn sie in einem großen Anteil der leukämischen Zellen auftreten. Diese Patienten sollten engmaschig auf Rezidive überwacht werden. Die unterschiedliche biologische Funktion der loss-of-function und dominant-negativen IKZF1-Deletionen sollte in weiteren Studien untersucht werden.Background: Mutations of transcription factor IKZF1 have recently been reported in B-cell precursor acute lymphoblastic leukemia (B-ALL). Besides deletions of the whole gene and point mutations, there are two types of intragenetic deletions (loss-of-function and dominant-negative). For the large subgroup of adult patients with BCR-ABL-negative B-ALL, there is only limited data on the frequency and the prognostic relevance of IKZF1 alterations. Methods: DNA samples from 482 patients with BCR-ABL-negative B-ALL treated within the GMALL study protocols 06/99 and 07/03 were analyzed by PCR for intragenetic deletions (∆2-7, ∆2-8, ∆4-7, ∆4-8). RT-PCR was conducted to detect ∆2-3 and other rare deletions. Quantitative PCRs (∆2-7, ∆4-7, ∆4-8) and gel densitometry were used to quantify the relative concentration of IKZF1-deleted cells. Deletions were considered either present in the majority of cells (“highdel”) or in a small fraction of cells only (“lowdel”) and their prognostic effect was evaluated separately. All deletions were sequenced and breakpoint sequences were analyzed. Results: Overall, 128 patients (27%) showed an intragenetic IKZF1 deletions, 37 of them expressing more than one deletion (175 deletions in total). Fifty-six patients (12%) carried only loss-of-function deletions, 50 (10%) had only dominant-negative deletions while 22 patients exhibited both types of deletions (5%). At least one highdel IKZF1 deletion could be found in 98 patients (20%). Patients carrying a loss-of-function IKZF1 deletion showed a significantly reduced overall survival (OS at 5 years 0.37 vs. 0.59, p=0.0012) while dominant-negative deletions had no effect on OS (0.54 vs. 0.56, p=0.95). In the group of patients with loss-of-function deletions, only highdel deletions were linked to a reduced OS (0.28 vs. 0.59, p<0.0001) while patients with lowdel deletions showed a clinical course comparable to patients without loss-of-function deletions. This effect of highdel loss-of-function deletions was also significant in a subgroup of standard-risk patients according to GMALL criteria (0.37 vs. 0.68, p=0.0002). There was no association between the relative amount of cells with dominant-negative deletions and overall survival (p=0.62). Sequencing of 193 breakpoints revealed four major breakpoint clusters. In 183 of 193 cases, both proximal and distal breakpoints were linked to putative cryptic recombination signal sequences. Discussion: In adult BCR-ABL-negative leukemia patients, loss-of-function IKZF1 deletions that are present in a large fraction of leukemic cells are linked with an inferior clinical outcome. These patients should be monitored closely for relapses. Consecutive research is needed to further investigate the different biological function of non-functional and dominant-negative IKZF1 deletions

Loss-of-function but not dominant-negative intragenic IKZF1 deletions are associated with an adverse prognosis in adult BCR-ABL-negative acute lymphoblastic leukemia

Genetic alterations of the transcription factor IKZF1 (“IKAROS”) are detected in around 15–30% of cases of BCR-ABL-negative B-cell precursor acute lymphoblastic leukemia. Different types of intragenic deletions have been observed, resulting in a functionally inactivated allele (“loss-of-function”) or in “dominant-negative” isoforms. The prognostic impact of these alterations especially in adult acute lymphoblastic leukemia is not well defined. We analyzed 482 well-characterized cases of adult BCR-ABL-negative B-precursor acute lymphoblastic leukemia uniformly treated in the framework of the GMALL studies and detected IKZF1 alterations in 128 cases (27%). In 20%, the IKZF1 alteration was present in a large fraction of leukemic cells (“high deletion load”) while in 7% it was detected only in small subclones (“low deletion load”). Some patients showed more than one IKZF1 alteration (8%). Patients exhibiting a loss-of-function isoform with high deletion load had a shorter overall survival (OS at 5 years 28% vs. 59%; P<0.0001), also significant in a subgroup analysis of standard risk patients according to GMALL classification (OS at 5 years 37% vs. 68%; P=0.0002). Low deletion load or dominant-negative IKZF1 alterations had no prognostic impact. The results thus suggest that there is a clear distinction between loss-of-function and dominant-negative IKZF1 deletions. Affected patients should thus be monitored for minimal residual disease carefully to detect incipient relapses at an early stage and they are potential candidates for alternative or intensified treatment regimes. (clinicaltrials.gov identifiers: 00199056 and 00198991)

Plasma EBV DNA as a prognostic factor in EBV associated gastric cancer: a multicenter, prospective study (EBV PRESAGE study)

Purpose: The Cancer Genome Atlas Research Network identified Epstein-Barr-Virus (EBV)-positive gastric cancer as a distinct molecular subtype. The prevalence is 8-9% and the histological examination shows pronounced lymphocytic infiltration, elevated levels of IFN-γ and consequently overexpression of PD-L1. The role of plasma EBV DNA load as a prognostic factor in patients with this cancer subtype is still to be defined. Methods and analysis: The present multicenter prospective observational study "EBV PRESAGE", involving German and Italian cancer centers, aims to evaluate the prognostic role of plasma EBV DNA in EBV-related gastric cancer (GC). The objective is to study the association between plasma EBV DNA load at different consecutive time points and the patient's prognosis. Every patient with a new diagnosis of gastric cancer (including gastroesophageal junction adenocarcinoma) will be screened for Epstein-Barr encoded small Region (EBER) on tissue biopsies using in situ hybridization (ISH). If EBER ISH is positive, blood analysis for plasma EBV DNA will be conducted. The plasma EBV quantitative analysis will be centralized, and extraction, detection, and quantification of EBV DNA in plasma samples will be performed using real-time PCR. Discussion: We hypothesized that plasma EBV DNA represents a non-invasive tool for monitoring EBV-related GC and might be valuable as a prognostic marker