Single-Centre Experience of Systemic Treatment with Vincristine, Ifosfamide, and Doxorubicin Alternating with Etoposide, Ifosfamide, and Cisplatin in Adult Patients with Ewing Sarcoma.

The treatment of Ewing sarcoma (ES) in adult patients requires a multidisciplinary approach. Systemic therapy remains an important component of clinical management of this disease. ES is extremely rare in adult patients. Due to the rarity of the disease, no standard of care in terms of chemotherapy for the adult population exists, and the level of evidence for individual agents or some multidrug combinations is limited. Most regimens that are used in both adults and children include anthracyclines, etoposide, vincristine, cyclophosphamide, and ifosfamide. In this report, we describe our experience with the alternating use of triple combination therapies based on vincristine, ifosfamide, and doxorubicin (VIA) and an etoposide, ifosfamide, and cisplatin combination (VIP). We retrospectively evaluated the response rates, outcome, and tolerance of adult patients (n = 64) treated with VIA/VIP between 1990 and 2014. The patients included were treated with perioperative chemotherapy (53.1% neoadjuvant therapy and 17.2% adjuvant therapy) or had synchronous metastases at diagnosis (29.7%). Five-year overall survival rate was 52.2% for all patients, 72.2% for patients with localized disease, and 5.3% in patients with synchronous metastases. Overall response rate (ORR) was 37% after 2 cycles of VIA and 2 cycles of VIP. There were no patients with progressive disease (PD).

Telomeric recombination in mismatch repair deficient human colon cancer cells after telomerase inhibition.

The majority of human malignancies use telomerase to maintain telomere homeostasis. Antitelomerase therapy is therefore a promising approach for a cancer-specific therapy. The alternative lengthening of telomeres pathway (ALT) is a recombination-based, telomerase-independent mechanism of telomere length control. It is widely believed that ALT could be engaged when cancer cells escape from telomerase inhibition. However, no reports exist that would support this concept of therapy resistance. We inhibited telomerase in a human cancer cell line with a mismatch repair defect and observed a telomerase-independent, ALT-like telomere elongation. This is the first report of inducing a telomerase-independent telomere elongation in human cancer cells when telomerase is inhibited, thus describing a novel mechanism of resistance to antitelomerase therapy.

Phase I safety and pharmacokinetic dose-escalation study of pilaralisib polymorph E, a phosphoinositide 3-kinase inhibitor in tablet formulation, in patients with solid tumors or lymphoma.

PURPOSE: Pilaralisib (SAR245408), a pan-class I PI3K inhibitor, has been investigated in Phase I/II trials in several solid tumors and lymphomas in capsule and tablet formulations of polymorph A (capsule-A and tablet-A). This Phase I study was conducted to determine the recommended Phase II dose (RP2D) of a more thermodynamically stable form of pilaralisib (polymorph E), in tablet formulation (tablet-E), in patients with advanced solid tumors or relapsed/refractory lymphoma. METHODS: A modified '3 + 3' dose-escalation design was employed. Patients received pilaralisib once daily (QD; starting dose 400 mg) for two 28-day cycles. Primary endpoints were safety and pharmacokinetics (PK). Exploratory endpoints were pharmacodynamics and efficacy. RESULTS: Eighteen patients were enrolled: Six patients received pilaralisib 400 mg QD and 12 patients received pilaralisib 600 mg QD. Two patients in the 600 mg QD cohort had dose-limiting toxicities (DLTs) (one patient with Grade 3 maculopapular rash and one patient with Grade 3 generalized rash and Grade 4 lipase increased). The most frequently occurring treatment-related, treatment-emergent adverse events were decreased appetite (22 %), dry skin (22 %), nausea (22 %) and vomiting (22 %). In PK analyses, individual exposures observed with 600 mg tablet-E were within the range of data at steady state from previous studies of 400 mg tablet-A and 600 mg capsule-A. Five patients (28 %) had stable disease as best response. CONCLUSIONS: With pilaralisib tablet-E, the RP2D was 600 mg QD, drug exposure was similar to the 400 mg tablet-A and 600 mg capsule-A formulations, and safety was consistent with the known safety profile of pilaralisib.

The frequency of homologous recombination in human ALT cells.

Instead of telomerase, some immortal cells use the alternative lengthening of telomeres pathway (ALT) to maintain their telomeres. There is good evidence that homologous recombination contributes to the ALT mechanism. Using an inducible GFP reporter system to measure the frequency of homologous recombination, we asked whether or not ALT cells exhibited a general change of the recombination machinery. Our results show that the frequency of homologous recombination for non-telomeric sequences in ALT cells is identical to that in telomerase positive cells, irrespective of whether the reporter was present at an intra-chromosomal location or next to a telomeric sequence. We conclude that the underlying recombination defect in ALT cells is restricted to telomeric sequences.

CpG island methylation of the hTERT promoter is associated with lower telomerase activity in B-cell lymphocytic leukemia.

OBJECTIVE: Expression of the catalytic subunit of the telomerase enzyme hTERT is essential for prolonging the replicative lifespan and is the rate-limiting step in cellular immortalization and carcinogenesis. Because hTERT expression is positively correlated with telomerase activity, its regulation is suggested as the major determinant of enzymatic activity. The hTERT promoter region contains two CpG islands, which are known to be target sites for de novo DNA methylation. To elucidate the impact of this epigenetic mechanism on telomerase activity, we analyzed the degree of hTERT promoter methylation in 30 patients with B-cell chronic lymphocytic leukemia. MATERIALS AND METHODS: hTERT promoter methylation was assessed using a methylation-specific competitive polymerase chain reaction assay. The assay is based on digestion of genomic DNA with a methylation-sensitive restriction enzyme before amplification with an internal standard. RESULTS: Patients exhibiting high telomerase activity showed significantly less methylation of the hTERT promoter core domain than patients with low enzyme activity. In addition, telomerase activity was significantly associated with telomere length and overall survival. CONCLUSIONS: Our data show that the degree of CpG island methylation of the hTERT promoter exhibits an impact on telomerase activity in a subgroup of patients with B-cell chronic lymphocytic leukemia and therefore is assumed to play a role in regulating hTERT gene expression in these patients.

Inter-telomeric recombination is present in telomerase-positive human cells.

Immortal cells require a mechanism of telomere length control in order to divide infinitely. One mechanism is telomerase, an enzyme that compensates the loss of telomeric DNA. The second mechanism is the alternative lengthening of telomeres (ALT) pathway. In ALT pathway cells, homologous recombination between telomeric DNA is the mechanism by which telomere homeostasis is achieved. We developed a novel homologous recombination reporter system that is able to measure inter-telomeric recombination in a sensitive manner. We asked the fundamental question if homologous recombination between different telomeres is present in telomerase-positive cells. In this in vitro study, we showed that homologous recombination between telomeres is detectable in ALT cells with the same frequency as in cells that utilize the telomerase pathway. We further described an ALT cell clone that showed peaks of recombination which were not detected in telomerase-positive clones. In telomerase-positive cells the frequency of inter-telomeric recombination was not increased by shortened telomeres or by a fragile telomere phenotype induced with aphidicolin. ALT cells, in contrast, responded to aphidicolin with an increase in the frequency of recombination. Our results indicate that inter-telomeric recombination is present in both pathways of telomere length control, but the factors that increase recombination are different in ALT and telomerase-positive cells.

Detection of circular telomeric DNA without 2D gel electrophoresis.

The end of linear chromosomes forms a lasso-like structure called the t-loop. Such t-loops resemble a DNA recombination intermediate, where the single-stranded 3' overhang is arrested in a stretch of duplex DNA. Presumably, such a t-loop can also be deleted via a recombination process. This would result in the occurrence of circular extrachromosomal telomeric DNA (t-circles), which are known to be abundantly present in immortal cells engaging the recombination-based alternative lengthening of telomeres pathway (ALT pathway). Little is known about the basic mechanism of telomeric recombination in these cells and what ultimately causes the generation of such t-circles. Current standard procedures for detecting these molecules involve 2D gel electrophoresis or electron microscopy. However, both methods are labor intense and sophisticated to perform. Here, we present a simpler, faster, and equally sensitive method for detecting t-circles. Our approach is a telomere restriction fragment assay that involves the enzymatic preservation of circular DNA with Klenow enzyme followed by Bal31 degradation of the remaining linear DNA molecules. We show that with this approach t-circles can be detected in ALT cell lines, whereas no t-circles are present in telomerase-positive cell lines. We consider our approach a valid method in which t-circle generation is the experimental readout.

Homologous recombination in human telomerase-positive and ALT cells occurs with the same frequency.

Homologous recombination is thought to be the molecular mechanism for maintaining telomere length in alternative lengthening of telomeres (ALT) cells. We used a recombination reporter system to show that the frequency of homologous recombination is the same for ALT- and telomerase-positive cells, suggesting that if ALT cells have a recombination defect it specifically involves telomeric sequences. We compared internal and telomere-adjacent positions of our reporter construct to investigate if telomeric sequences near an induced double-strand break alter the frequency of recombination between nontelomeric sequences, and found no differences among the different cell lines analysed. Our results indicate that the underlying defect in homologous recombination in ALT cells does not affect sequences independent of their chromosomal location but is likely to be primarily a specific telomeric defect.