Chemotherapy or allogeneic transplantation in high-risk Philadelphia chromosome-negative adult lymphoblastic leukemia.

The need for allogeneic hematopoietic stem cell transplantation (allo-HSCT) in adults with Philadelphia chromosome-negative (Ph-) acute lymphoblastic leukemia (ALL) with high-risk (HR) features and adequate measurable residual disease (MRD) clearance remains unclear. The aim of the ALL-HR-11 trial was to evaluate the outcomes of HR Ph- adult ALL patients following chemotherapy or allo-HSCT administered based on end-induction and consolidation MRD levels. Patients aged 15 to 60 years with HR-ALL in complete response (CR) and MRD levels (centrally assessed by 8-color flow cytometry) <0.1% after induction and <0.01% after early consolidation were assigned to receive delayed consolidation and maintenance therapy up to 2 years in CR. The remaining patients were allocated to allo-HSCT. CR was attained in 315/348 patients (91%), with MRD <0.1% after induction in 220/289 patients (76%). By intention-to-treat, 218 patients were assigned to chemotherapy and 106 to allo-HSCT. The 5-year (±95% confidence interval) cumulative incidence of relapse (CIR), overall survival (OS), and event-free survival probabilities for the whole series were 43% ± 7%, 49% ± 7%, and 40% ± 6%, respectively, with CIR and OS rates of 45% ± 8% and 59% ± 9% for patients assigned to chemotherapy and of 40% ± 12% and 38% ± 11% for those assigned to allo-HSCT, respectively. Our results show that avoiding allo-HSCT does not hamper the outcomes of HR Ph- adult ALL patients up to 60 years with adequate MRD response after induction and consolidation. Better postremission alternative therapies are especially needed for patients with poor MRD clearance. This trial was registered at www.clinicaltrials.gov as # NCT01540812.

Genomics improves risk stratification of adults with T-cell acute lymphoblastic leukemia enrolled in measurable residual disease-oriented trials.

Genetic information has been crucial to understand the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) at diagnosis and at relapse, but still nowadays has a limited value in a clinical context. Few genetic markers are associated with the outcome of T-ALL patients, independently of measurable residual disease (MRD) status after therapy. In addition, the prognostic relevance of genetic features may be modulated by the specific treatment used. We analyzed the genetic profile of 145 T-ALL patients by targeted deep sequencing. Genomic information was integrated with the clinicalbiological and survival data of a subset of 116 adult patients enrolled in two consecutive MRD-oriented trials of the Spanish PETHEMA (Programa Español de Tratamientos en Hematología) group. Genetic analysis revealed a mutational profile defined by DNMT3A/ N/KRAS/ MSH2/ U2AF1 gene mutations that identified refractory/resistant patients. Mutations in the DMNT3A gene were also found in the non-leukemic cell fraction of patients with T-ALL, revealing a possible mutational-driven clonal hematopoiesis event to prime T-ALL in elderly. The prognostic impact of this adverse genetic profile was independent of MRD status on day +35 of induction therapy. The combined worse-outcome genetic signature and MRD on day +35 allowed risk stratification of T-ALL into standard or high-risk groups with significantly different 5- year overall survival (OS) of 52% (95% confidence interval: 37-67) and 17% (95% confidence interval: 1-33), respectively. These results confirm the relevance of the tumor genetic profile in predicting patient outcome in adult T-ALL and highlight the need for novel gene-targeted chemotherapeutic schedules to improve the OS of poor-prognosis T-ALL patients.

Outcomes and prognostic factors of adults with refractory or relapsed T-cell acute lymphoblastic leukemia included in measurable residual disease-oriented trials.

Despite high complete remission (CR) rates with frontline therapy, relapses are frequent in adults with T-cell acute lymphoblastic leukemia (T-ALL) with limited salvage options. We analyzed the outcomes and prognostic factors for CR to salvage therapy and overall survival (OS) of patients with R/R T-ALL included in two prospective measurable residual disease-oriented trials. Seventy-five patients (70 relapsed, 5 refractory) were identified. Relapses occurred in bone marrow, isolated or combined in 50 patients, and in the central nervous system (CNS; isolated or combined) in 20. Second CR was attained in 30/75 patients (40%). Treatment with FLAG-Ida and isolated CNS relapse were independently associated with a higher CR rate after first salvage therapy. The median OS was 6.2 (95% confidence interval [CI], 3.9-8.6) months, with a 4-year OS probability of 18% (95% CI, 9%-27%). No differences in survival were observed according to the treatment with hematopoietic stem cell transplantation in patients in CR after first salvage therapy. Multivariable analysis showed a ≥12-month interval between first CR and relapse, CR after first salvage therapy and isolated CNS relapse as favorable prognostic factors for OS with hazard ratios (HR) (95% CI) of 1.931 (1.109-3.362), 2.958 (1.640-5.334), and 2.976 (1.157-7.655), respectively. This study confirms the poor outcomes of adults with R/R T-ALL among whom FLAG-Ida was the best of the rescue therapies evaluated. Late relapse, CR after first rescue therapy and isolated CNS relapse showed prognostic impact on survival. More effective rescue therapies are needed in adults with R/R T-ALL.

Genetics and epigenetics of leukemia and lymphoma: from knowledge to applications, meeting report of the Josep Carreras Leukaemia Research Institute.

The meeting, which brought together leading scientists and clinicians in the field of leukemia and lymphoma, was held at the new headquarters of the Josep Carreras Leukaemia Research Institute (IJC) in Badalona, Catalonia, Spain, September 19-20, 2019. Its purpose was to highlight the latest advances in our understanding of the molecular mechanisms driving blood cancers, and to discuss how this knowledge can be translated into an improved management of the disease. Special emphasis was placed on the role of genetic and epigenetic heterogeneity, and the exploitation of epigenetic regulation for developing biomarkers and novel treatment approaches.

Molecular profiling refines minimal residual disease-based prognostic assessment in adults with Philadelphia chromosome-negative B-cell precursor acute lymphoblastic leukemia.

Minimal residual disease (MRD) assessment is an essential tool in contemporary acute lymphoblastic leukemia (ALL) protocols, being used for therapeutic decisions such as hematopoietic stem cell transplantation in high-risk patients. However, a significant proportion of adult ALL patients with negative MRD still relapse suggesting that other factors (ie, molecular alterations) must be considered in order to identify those patients with high risk of disease progression. We have identified partial IKZF1 gene deletions and CDKN2A/B deletions as markers of disease recurrence and poor survival in a series of uniformly treated adolescent and adult Philadelphia chromosome-negative B-cell progenitor ALL patients treated according to the Programa Español de Tratamientos en Hematología protocols. Importantly, CDKN2A/B deletions showed independent significance of MRD at the end of induction, which points out the need for treatment intensification in these patients despite being MRD-negative after induction therapy.

Incidence and outcome after first molecular versus overt recurrence in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia included in the ALL Ph08 trial from the Spanish PETHEMA Group.

BACKGROUND: Disease recurrence occurs in 20% to 40% of adults with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) who are treated with chemotherapy and tyrosine kinase inhibitors (TKIs). In the current study, the authors report the incidence, treatment, and outcome after first disease recurrence in young and older adults treated in the ALL Ph08 trial (ClinicalTrials.gov identifier NCT01491763). METHODS: Patients aged 18 to 55 years with de novo Ph+ ALL were treated with imatinib concurrently with standard-dose induction and consolidation therapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) when possible. In patients with first disease recurrence, the authors analyzed the type of recurrence, timing, location, presence of kinase domain mutations, type of treatment, and outcomes. RESULTS: Of the 125 patients, 28 patients (22%) developed disease recurrence before (4 patients) or after (24 patients) HSCT, with the recurrences being molecular in 11 patients (39%) and overt in 17 patients (61%). T315I was the most common mutation noted at the time of disease recurrence. Change in TKI was the most frequent treatment for patients with molecular disease recurrence whereas rescue chemotherapy and TKI change followed by second allo-HSCT when possible were performed for the most part in patients with overt disease recurrence. A total of 20 patients (71%) achieved response. The median disease-free survival (DFS) and overall survival (OS) were 8.5 months and 15.3 months, respectively. A trend for better DFS and OS was observed in patients with molecular recurrence compared with those with overt recurrence (median of 16.9 months vs 6.3 months [P = .05] and 28.7 months vs 11.5 months [P = .05] for DFS and OS, respectively). CONCLUSIONS: Disease recurrence was frequent in young and older adults with Ph+ ALL who were treated with imatinib and chemotherapy with HSCT. Although the majority of patients responded to rescue therapy, their outcomes were poor, especially with regard to overt disease recurrence.

The poor prognosis of low hypodiploidy in adults with B-cell precursor acute lymphoblastic leukaemia is restricted to older adults and elderly patients.

The prognostic significance of low-hypodiploidy has not been extensively evaluated in minimal residual disease (MRD)-oriented protocols for adult acute lymphoblastic leukaemia (ALL). We analysed the outcome of hypodiploid adult ALL patients treated within Programa Español de Tratamientos en Hematología (PETHEMA) protocols. The 5-year cumulative incidence of relapse (CIR) of low-hypodiploid B-cell precursor (BCP)-ALL was significantly higher than that of high-hypodiploids (52% vs. 12%, P = 0.013). Low-hypodiploid BCP-ALL patients aged ≤35 years showed superior survival (71% vs. 21%, P = 0.026) and lower 5-year CIR (17% vs. 66%, P = 0.090) than low-hypodiploids aged >35 years. Older adults and elderly low-hypodiploid BCP-ALL patients show dismal prognosis although achieving an end-induction good MRD response.

Increased survival due to lower toxicity for high-risk T-cell acute lymphoblastic leukemia patients in two consecutive pediatric-inspired PETHEMA trials.

OBJECTIVE AND METHODS: Pediatric-inspired regimens have been adopted by several groups as the treatment strategy for adult patients with acute lymphoblastic leukemia (ALL). Whether subsequent modifications of these protocols have led to an improvement in the outcome of patients is uncertain, especially in T-cell ALL. We analyzed 169 patients with high-risk T-cell ALL included in two consecutive trials of the PETHEMA Group (HR-ALL03 [n = 104] and the more contemporary HR-ALL11 [n = 65]). RESULTS: Patients and disease characteristics were balanced between both groups. Regarding efficacy, we observed a similar complete remission (CR) rate, relapse and disease-free survival (DFS) between both protocols. Patients included in the HR-ALL11 trial had better 2-year overall survival (OS) compared with the HR-ALL03 (65% [95% CI 51%-79%] vs 44% [95% CI 34%-54%], P = 0.026). Regarding toxicity, we observed a better safety profile in the HR-11 protocol. Irrespective of the protocol, patients with good measurable residual disease (MRD) clearance had a promising outcome without allogeneic hematopoietic stem cell transplantation (allo-HSCT) in CR1, with 2-year OS of 67%. CONCLUSION: Patients with T-cell ALL included in the HR-11 trial showed better OS than patients in the HR-03, mostly driven by a reduction of NRM.

Genome-wide identification of microRNA signatures associated with stem/progenitor cells in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a malignant transformation with uncontrolled proliferation of lymphoid precursor cells within bone marrow including a dismal prognosis after relapse. Survival of a population of quiescent leukemia stem cells (LSCs, also termed leukemia-initiating cells (LICs)) after treatment is one of the relapse reasons in Ph+ ALL patient. MicroRNAs (miRNAs) are known as highly conserved 19-24 nucleotides non-protein-coding small RNAs that regulate the expression of human genes. miRNAs are often involved in the tuning of hematopoiesis. Therefore, the deregulation of miRNA expression and function in hematopoietic cells can cause cancer and promote its progression. This is the first comprehensive analysis of miRNA expression differences between CD34+CD38- LSCs and CD34+CD38+ leukemic progenitors (LPs) from the same Ph+ B-ALL bone marrow samples using high-throughput sequencing technologies. We identified multiple differentially expressed miRNAs including hsa-miR-3143, hsa-miR-6503-3p, hsa-miR-744-3p, hsa-miR-1226-3p, hsa-miR-10a-5p, hsa-miR-4658 and hsa-miR-493-3p related to LSC and LP populations which have regulatory functions in stem-cell associated biological processes. The deregulation of these miRNAs could affect leukemogenesis, clonogenic and stemness capacities in these subpopulations of Ph+ B-ALL. Therefore, identification of these LSC associated miRNAs may improve the diagnosis and management of B-ALL. These findings may also lead to future strategies to eliminate the presence of resistant LSCs, either by induction of apoptosis or by sensitizing these cells to chemotherapy.

Copy number profiling of adult relapsed B-cell precursor acute lymphoblastic leukemia reveals potential leukemia progression mechanisms.

The outcome of relapsed adult acute lymphoblastic leukemia (ALL) remains dismal despite new therapeutic approaches. Previous studies analyzing relapse samples have shown a high degree of heterogeneity regarding gene alterations without an evident relapse signature. Bone marrow or peripheral blood samples from 31 adult B-cell precursor ALL patients at first relapse, and 21 paired diagnostic samples were analyzed by multiplex ligation probe-dependent amplification (MLPA). Nineteen paired diagnostic and relapse samples of these 21 patients were also analyzed by SNP arrays. A trend to acquire homozygous CDKN2A/B deletions and a significant increase in the number of copy number alterations (CNA) was observed from diagnosis to first relapse. Evolution from an ancestral clone was the main pattern of clonal evolution. Relapse samples were extremely heterogeneous regarding CNA frequencies. However, CDKN2A/B, PAX5, ETV6, ATM, IKZF1, VPREB1, and TP53 deletions and duplications of 1q, 8q, 17q, 21, X/Y PAR1, and Xp were frequently detected at relapse. Duplications of genes involved in cell proliferation, drug resistance and stem cell homeostasis regulation, as well as deletions of KDM6A and STAG2 genes emerged as specific alterations at relapse. Genomics of relapsed adult B-cell precursor ALL is highly heterogeneous, although some recurrent lesions involved in essential pathways deregulation were frequently observed. Selective and simultaneous targeting of these deregulated pathways may improve the results of current salvage therapies.

Prognostic significance of copy number alterations in adolescent and adult patients with precursor B acute lymphoblastic leukemia enrolled in PETHEMA protocols.

BACKGROUND: Some copy number alterations (CNAs) have independent prognostic significance for adults with acute lymphoblastic leukemia (ALL). METHODS: This study analyzed via multiplex ligation-dependent probe amplification the frequency and prognostic impact of CNAs of 12 genetic regions in 142 adolescents and adults with de novo precursor B-cell ALL. RESULTS: The cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion (59 of 142 or 42%) was the most frequent CNA, and it was followed by Ikaros family zinc finger 1 (IKZF1) losses (49 of 142 or 35%). IKZF1 deletions were more prevalent in Philadelphia chromosome (Ph)-positive ALL and were associated with advanced age and high white blood cell (WBC) counts. The multivariate analysis showed that advanced age and early B-cell factor 1 (EBF1) deletions were associated with chemotherapy resistance in both the whole series (hazard ratios, 0.949 and 0.135, respectively) and the Ph-negative subgroup (hazard ratios, 0.946 and 0.118, respectively). High WBC counts and focal IKZF1 deletions correlated with disease recurrence (hazard ratios, 1.005 and 1.869, respectively), whereas advanced age and CDKN2A/B losses influenced overall survival in both the whole series (hazard ratios, 1.038 and 2.545, respectively) and the Ph-negative subgroup (hazard ratios, 1.044 and 2.105, respectively). CONCLUSIONS: Deletions of EBF1, IKZF1, and CDKN2A/B have an independent adverse prognosis for adolescents and adults with B-precursor ALL, and this suggests that these CNAs should be included in the initial risk assessment of ALL.

Latest Contributions of Genomics to T-Cell Acute Lymphoblastic Leukemia (T-ALL).

As for many neoplasms, initial genetic data about T-cell acute lymphoblastic leukemia (T-ALL) came from the application of cytogenetics. This information helped identify some recurrent chromosomal alterations in T-ALL at the time of diagnosis, although it was difficult to determine their prognostic impact because of their low incidence in the specific T-ALL cohort analyzed. Genetic knowledge accumulated rapidly following the application of genomic techniques, drawing attention to the importance of using high-resolution genetic techniques to detect cryptic aberrations present in T-ALL, which are not usually detected by cytogenetics. We now have a clearer appreciation of the genetic landscape of the different T-ALL subtypes at diagnosis, explaining the particular oncogenetic processes taking place in each T-ALL, and we have begun to understand relapse-specific mechanisms. This review aims to summarize the latest advances in our knowledge of the genome in T-ALL. We highlight areas where the research in this subtype of ALL is progressing with the aim of identifying key questions that need to be answered in the medium-long term if this knowledge is to be applied in clinics.

Early T-Cell Precursor ALL and Beyond: Immature and Ambiguous Lineage T-ALL Subsets.

A wide range of immature acute leukemias (AL), ranging from acute myeloid leukemias with minimal differentiation to acute leukemias with an ambiguous lineage, i.e., acute undifferentiated leukemias and mixed phenotype acute leukemia with T- or B-plus myeloid markers, cannot be definitely assigned to a single cell lineage. This somewhat "grey zone" of AL expresses partly overlapping features with the most immature forms of T-cell acute lymphoblastic leukemia (T-ALL), i.e., early T-cell precursor ALL (ETP-ALL), near-ETP-ALL, and pro-T ALL. These are troublesome cases in terms of precise diagnosis because of their similarities and overlapping phenotypic features. Moreover, it has become evident that they share several genomic alterations, raising the question of how their phenotypes reflect distinct AL entities. The aim of this review was to provide a systematic overview of the genetic events associated with immature T-ALL and outline their relationship with treatment choices and outcomes, especially looking at the most recent preclinical and clinical studies. We wish to offer a basis for using the genetic information for new diagnostic algorithms, in order to better stratify patients and improve their management with more efficient and personalized therapeutic options. Understanding the genetic profile of this high-risk T-ALL subset is a prerequisite for changing the current clinical scenario.

Ponatinib, chemotherapy, and transplant in adults with Philadelphia chromosome-positive acute lymphoblastic leukemia.

Promising results have been shown with the combination of ponatinib and chemotherapy in adults with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The PONALFIL (Ponatinib With Chemotherapy for Young Adults Ph Positive Acute Lymphoblastic Leukemia) trial combined ponatinib (30 mg/d) with standard induction and consolidation chemotherapy followed by allogeneic hematopoietic stem cell transplant (alloHSCT) in newly diagnosed Ph+ ALL patients aged 18 to 60 years. Ponatinib was only given pre-emptively after alloHSCT. Primary end points were hematologic and molecular response before alloHSCT and event-free survival (EFS), including molecular relapse as event. Thirty patients (median age, 49 years; range, 19-59 years) entered the trial. All exhibited hematologic response, and alloHSCT was performed in 26 patients (20 in complete molecular response and 6 in major molecular response). Only 1 patient died (of graft-versus-host disease), and 5 patients exhibited molecular relapse after alloHSCT. No tyrosine kinase inhibitor was given after HSCT in 18 of 26 patients. Twenty-nine patients are alive (median follow-up, 2.1 years; range, 0.2-4.0 years), with 3-year EFS and overall survival (OS) of 70% (95% confidence interval, 51-89) and 96% (95% confidence interval, 89-100), respectively. Comparison of the PONALFIL and the ALLPh08 (Chemotherapy and Imatinib in Young Adults With Acute Lymphoblastic Leukemia Ph [BCR-ABL] Positive; same schedule, using imatinib as the tyrosine kinase inhibitor) trials by propensity score showed significant improvement in OS for patients in PONALFIL (3-year OS, 96% vs 53%; P = .002). The most frequent grade 3 to 4 adverse events were hematologic (42%), infectious (17%), and hepatic (22%), with only one vascular occlusive event. The combination of chemotherapy with ponatinib followed by alloHSCT is well tolerated, with encouraging EFS in adults with newly diagnosed Ph+ ALL. Cross-trial comparison suggests improvement vs imatinib (clinicaltrials.gov identifier #NCT02776605).

The Yin and Yang-Like Clinical Implications of the CDKN2A/ARF/CDKN2B Gene Cluster in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a malignant clonal expansion of lymphoid hematopoietic precursors that exhibit developmental arrest at varying stages of differentiation. Similar to what occurs in solid cancers, transformation of normal hematopoietic precursors is governed by a multistep oncogenic process that drives initiation, clonal expansion and metastasis. In this process, alterations in genes encoding proteins that govern processes such as cell proliferation, differentiation, and growth provide us with some of the clearest mechanistic insights into how and why cancer arises. In such a scenario, deletions in the 9p21.3 cluster involving CDKN2A/ARF/CDKN2B genes arise as one of the oncogenic hallmarks of ALL. Deletions in this region are the most frequent structural alteration in T-cell acute lymphoblastic leukemia (T-ALL) and account for roughly 30% of copy number alterations found in B-cell-precursor acute lymphoblastic leukemia (BCP-ALL). Here, we review the literature concerning the involvement of the CDKN2A/B genes as a prognosis marker of good or bad response in the two ALL subtypes (BCP-ALL and T-ALL). We compare frequencies observed in studies performed on several ALL cohorts (adult and child), which mainly consider genetic data produced by genomic techniques. We also summarize what we have learned from mouse models designed to evaluate the functional involvement of the gene cluster in ALL development and in relapse/resistance to treatment. Finally, we examine the range of possibilities for targeting the abnormal function of the protein-coding genes of this cluster and their potential to act as anti-leukemic agents in patients.

Adverse prognostic impact of complex karyotype (≥3 cytogenetic alterations) in adult T-cell acute lymphoblastic leukemia (T-ALL).

The potential prognostic value of conventional karyotyping in adult T-cell acute lymphoblastic leukemia (T-ALL) remains an open question. We hypothesized that a modified cytogenetic classification, based on the number and type of cytogenetic abnormalities, would allow the identification of high-risk adult T-ALL patients. Complex karyotype defined by the presence of ≥3 cytogenetic alterations identified T-ALL patients with poor prognosis in this study. Karyotypes with ≥3 abnormalities accounted for 16 % (22/139) of all evaluable karyotypes, corresponding to the largest poor prognosis cytogenetic subgroup of T-ALL identified so far. Patients carrying karyotypes with ≥3 cytogenetic alterations showed a significantly inferior response to therapy, and a poor outcome in terms of event-free survival (EFS), overall survival (OS) and cumulative incidence of relapse (CIR), independently of other baseline characteristics and the end-induction minimal residual disease (MRD) level. Additional molecular analyses of patients carrying ≥3 cytogenetic alterations showed a unique molecular profile that could contribute to understand the underlying molecular mechanisms of resistance and to evaluate novel targeted therapies (e.g. IL7R directed) with potential impact on outcome of adult T-ALL patients.

Who Should Receive an Allogeneic Transplant in First Complete Remission?

The decision to incorporate allogeneic hematopoietic stem cell transplant (allo-HSCT) into front-line therapy in adult acute lymphoblastic leukemia (ALL) should be primarily guided by measurable residual disease (MRD) status and the ALL regimen utilized. While there is no doubt that allo-HSCT benefits patients with poor MRD response after induction or consolidation, the indication of allo-HSCT in cases of good MRD clearance is not clear. As targeted immunotherapies result in high MRD-negative CR rates, early incorporation of these therapies may also prove valuable in reducing the need for HCT in the front-line setting. This review discusses the data and controversies related to allo-HSCT in the front-line therapy of Philadelphia chromosome-negative and -positive ALL.

Bispecific T-cell engaging antibodies in B-cell precursor acute lymphoblastic leukemias: focus on blinatumomab.

Bispecific T-cell engaging antibodies are constructs engineered to bind to two different antigens, one to a tumor-specific target and the other to CD3-positive T cells or natural killer (NK) cells. Blinatumomab engages CD19 and CD3, performing effective serial lysis. The clinical development program in acute lymphoblastic leukemia (ALL) includes clinical trials in relapsed or refractory (R/R) patients and in B-cell precursor (BCP) ALL patients with measurable residual disease. Several trials are currently being conducted in de novo BCP-ALL, either in induction, consolidation, or before or after hematopoietic stem cell transplant. Combination with other targeted therapies or with other immunotherapeutic approaches are also underway. Several strategies are aimed to optimize the use of blinatumomab either by overcoming the mechanisms of resistance (e.g. inhibition of PD-1/PD-L1) or by improvements in the route of application, among others.