Advances In Cancer Chemotherapy Research Articles

Kinase Inhibitor Screening Displayed ALK as a Possible Therapeutic Biomarker for Gastric Cancer.

Despite advances in cancer chemotherapy, gastric cancer (GC) continues to have high recurrence rates and poor prognosis with limited treatment options. Understanding the etiology of GC and developing more effective, less harmful therapeutic approaches are vital and urgent. Therefore, this work describes a novel kinase target in malignant gastric cells as a potential therapeutic strategy. Our results demonstrate that among 147 kinase inhibitors (KI), only three molecules were significantly cytotoxic for the AGP-01 cell line. Hence, these three molecules were further characterized in their cellular mode of action. There was significant cell cycle impairment due to the expression modulation of genes such as TP53, CDKN1A, CDC25A, MYC, and CDK2 with subsequent induction of apoptosis. In fact, the Gene Ontology analysis revealed a significant enrichment of pathways related to cell cycle regulation (GO:1902749 and GO:1903047). Moreover, the three selected KIs significantly reduced cell migration and Vimentin mRNA expression after treatment. Surprisingly, the three KIs share the same target, ALK and INSR, but only the ALK gene was found to have a high expression level in the gastric cancer cell line. Additionally, lower survival rates were observed for patients with high ALK expression in TCGA-STAD analysis. In summary, we hypothesize that ALK gene overexpression can be a promising biomarker for prognosis and therapeutic management of gastric adenocarcinoma.

Considerations in cardio-oncology: Multiple mobile left-sided cardiac thrombi in chemotherapy-induced cardiomyopathy

With advances in cancer chemotherapy, the importance of the new clinical discipline of cardio-oncology, which is concerned with the cardiac effects of chemotherapy, is increasing. Herein we describe the case of a 48-year-old woman with a history of breast cancer who presented with symptoms of heart failure due to chemotherapy-induced cardiomyopathy. Treatment for the patient's breast cancer had included surgery and chemotherapy with anthracyclines and trastuzumab. Echocardiography revealed multiple mobile thrombi in the left ventricle and atrium. In addition, brain magnetic resonance imaging revealed small acute cerebral infarctions due to embolization. Given the high risk of re-embolization, surgical thrombectomy was performed. Thus far, there are no standardized therapeutic guidelines for left-sided cardiac thrombi and the optimal treatment remains contentious. Although this patient was managed successfully with surgical thrombectomy, patients should be managed individually, taking into consideration embolization, bleeding, and surgical risks. With further improvements in cancer chemotherapy, there may be an increase in the incidence of complications such as multiple cardiac thrombi. From the cardio-oncology standpoint, we propose close interactions between cardiologists and oncologists for the optimal care of cancer patients.

PH- and NIR Light-Responsive Polymeric Prodrug Micelles for Hyperthermia-Assisted Site-Specific Chemotherapy to Reverse Drug Resistance in Cancer Treatment.

Despite the exciting advances in cancer chemotherapy over past decades, drug resistance in cancer treatment remains one of the primary reasons for therapeutic failure. IR-780 loaded pH-responsive polymeric prodrug micelles with near infrared (NIR) photothermal effect are developed to circumvent the drug resistance in cancer treatment. The polymeric prodrug micelles are stable in physiological environment, while exhibit fast doxorubicin (DOX) release in acidic condition and significant temperature elevation under NIR laser irradiation. Phosphorylcholine-based biomimetic micellar shell and acid-sensitive drug conjugation endow them with prolonged circulation time and reduced premature drug release during circulation to conduct tumor site-specific chemotherapy. The polymeric prodrug micelles combined with NIR laser irradiation could significantly enhance intracellular DOX accumulation and synergistically induce the cell apoptosis in DOX-resistant MCF-7/ADR cells. Meanwhile, the tumor site-specific chemotherapy combined with hyperthermia effect induces significant inhibition of MCF-7/ADR tumor growth in tumor-bearing mice. These results demonstrate that the well-designed IR-780 loaded polymeric prodrug micelles for hyperthermia-assisted site-specific chemotherapy present an effective approach to reverse drug resistance.

New Indings in Methotrexate Pharmacology - Diagnostic Possibilities and Impact on Clinical Care

Methotrexate is an anti-cancer drug used to treat several malignancies including pediatric acute lymphoblastic leukemia and choriocarcinoma. Despite recent advances in cancer chemotherapy, it remains a mainstay of therapy since its discovery in the early second half of the previous century. Moreover, low-dose methotrexate is a gold standard antirheumatic drug in the treatment of rheumatoid arthritis, psoriasis, systemic scleroderma and other autoimmune disorders. Side effects of methotrexate treatment are well known and described; however, their occurrence may often be unpredictable due to lack of specific biomarkers of toxicity. Methotrexate plasma levels are routinely monitored by therapeutic drug monitoring, nevertheless, occurrence and concentrations of its metabolites are not measured. During methotrexate treatment 7- hydroxymethotrexate and 2,4- diamino- N10- mehylpteroic acid appear in plasma. The latter can further be hydroxylated and glucuronidated resulting in five possible extracellular methotrexate metabolites. In addition, methotrexate is intracellularly converted to its active polyglutamylated forms. Therapeutic efficacy is dependent on formation of methotrexate polyglutamates as it keeps intracellular pool of the drug and enhances its affinity towards various target enzymes. In this study, we describe pharmacokinetic and pharmacodynamic characteristics of methotrexate metabolites. We also review methotrexate blood brain barrier transport to cerebrospinal fluid regarding its use in the prevention of leukemic central nervous system involvement and management of methotrexate toxicity with the use of carboxypeptidase- G2. Finally, we discuss laboratory methods for monitoring methotrexate metabolites and benefits of simultaneous determination of methotrexate and metabolites as possible biomarkers of therapeutic efficacy and clinical toxicity.

CYTOTOXICITY OF SORAFENIB IN HUMAN HEPATOCELLULAR CARCINOMA CELLS

Hepatocellular carcinoma (HCC) is the predominant type of primary liver malignancy with high rates of mortality worldwide. Most HCC tumors are inherently resistant to chemotherapy and despite the tremendous advances in cancer chemotherapy, their treatment remains quite challenging. Sorafenib, a multikinase inhibitor, has recently been approved for the treatment of advanced HCC. The present study aimed to further explore the potential cytotoxic activities of sorafenib in HepG2 cells as well as the possible underlying mechanisms. Thus, HepG2 cells were treated with different concentrations of sorafenib. The concentration that inhibited the growth of the cells by 50% was calculated from the fitted survival curves. The effect of sorafenib on cell cycle, apoptosis and proliferation was investigated. Sorafenib- induced cytotoxicity in HepG2 cells. This could be partially attributed to increased apoptosis by augmenting the level of active caspase-3. Moreover, sorafenib induced cell cycle arrest and had anti-proliferative effects by decreasing the level of p-Akt.

Pitfalls in Oncologic Imaging: Complications of Chemotherapy and Radiotherapy in the Chest

Advances in cancer chemotherapy and radiation therapy continue to improve patients' lives and affect patient care. Chemotherapy has progressed from cytotoxic agents to newer targeted agents that are geared to attack specific mutations in cancer cells. The toxicities of these newer cytotoxic drugs and targeted agents have specific imaging appearances depending on the mechanism of action. Similarly, advances in radiation therapy allow for better tumor, with less effect on normal adjacent structures. Knowledge of the imaging appearance of toxicities of therapy is crucial for radiologists to accurately diagnose complications of therapy and to avoid imaging pitfalls. Radiologists must use the clinical history and the mechanism of action of the drugs to affect patient care and prevent misdiagnosis. In this article, we review some of the common pitfalls of imaging that arise because of complications of therapy.

EFFECT OF GEMCITABINE ON FEMALE FERTILTY – AN ANIMAL MODEL STUDY

Background and objectives: Advances in cancer chemotherapy have led to improved survival rates and poses greater emphasis on preserving quality of life post-treatment. Gonadotoxicity is a well-recognized side effect of many cancer chemotherapeutic agents. This study was designed to evaluate the effects of gemcitabine, an antimetabolite anticancer drug, on oogenesis in Swiss albino mice and its reversibility. Methods: Thirty six inbred female Swiss albino mice in diestrous phase were selected and divided into three groups of twelve each. Groups were labelled as A, B and Control. Groups A and B received 40 mg/kg and 80 mg/kg of gemcitabine intraperitoneally. The control group received saline intraperitoneally. At the end of two weeks 6 mice were sacrificed from each group and the rest at the end of 2 months. Ovaries were studied histologically. Results: After 2 weeks, the ovaries of experimental group mice showed more number of atretic follicles as compared to control group (p<0.01). The diameter of corpus lutea was more, though a reduction in number was recorded in experimental group (p<0.05). Whereas after 2 months, both the experimental groups showed no difference in terms of atretic follicles, diameter and number of corpus lutea (p>0.05). Conclusions: These findings suggest that administration of gemcitabine may have profound effects on oogenesis and hence female fertility. This study also suggests that the effects are reversible. Keywords: Gemcitabine; Oogenesis; Swiss albino mice; atretic follicles; corpus luteum.

Comprehensive analysis of Excision repair complementation group 1, Glutathione S-transferase, Thymidylate synthase, and Uridine diphosphate glucuronosyltransferase 1A1 polymorphisms predictive for treatment outcome in patients with advanced gastric cancer treated with FOLFOX or FOLFIRI

e15580 Background: Pharmacogenetic advances in cancer chemotherapy have the potential to predict clinical benefit to particular regimens. Oxaliplatin and irinotecan have shown to be effective in the treatment of gastric cancer. We assessed whether single nucleotide polymorphisms (SNPs) in ERCC1, GST, TS, and UGT1A1predicted overall survival in gastric cancer patients receiving FOLFOX and/or FOLFIRI chemotherapy. Methods: Total genomic DNA was extracted from whole blood of patient. The PCR- restriction fragment length polymorphism (RFLP) method was applied to detect the known variant sites of ERCC1, GST, TS, and UGT1A1. Results: Response rate of FOLFOX (N=75) was 24%. Grade 3–4 neutropenia and neurotoxicity were observed 34.7% and 16%, respectively. TTP and OS of 1st line administration of FOLFOX (N=35) was 3.1 months (95% CI, 0.1–6.1 months) and 13.9 months (95% CI, 12.2–15.6 months). Only the GSTM1 positive genotype showed a significantly better time to progression (P=0.023). But significant genotype variation of TS, GST and ERCC1,which assumed to affect to activity of oxaliplatin was not observed to RR, toxicity, and overall survival. Response rate of FOLFIRI (N=74) was 23%. Grade 3–4 neutropenia and diarrhea were observed 55.4% and 9.5%, respectively. TTP and OS of 1st line administration of FOLFIRI (N=33) was 4.9 months (95% CI, 3.5–6.4 months) and 19.0 months (95% CI, 8.5–29.5months). Low expression type (2R/2R, 2R/3C, and 3C/3C) of TS was associated with high incidence of grade ≥3 neutropenia. But significant genotype variation of UGT1A1,which assumed to affect to toxicity of irinotecan was not observed to RR, toxicity, and survival. Conclusions: In this study, GSTM1 positive genotype showed a significantly better time to progression in the advanced gastric cancer treated with FOLFOX. Low expression type (2R/2R, 2R/3C, and 3C/3C) of TS was associated with high incidence of grade ≥3 neutropenia in the advanced gastric cancer treated with FOLFIRI. Well designed prospective trial will be clearly identifying relations between chemotherapy and genetic variations. No significant financial relationships to disclose.

Editorial [ Recent Advances in Cancer Chemotherapy- Part II Guest Editor: Diwan S. Rawat

Editorial [ Recent Advances in Cancer Chemotherapy- Part II Guest Editor: Diwan S. Rawat

Polymorphism of XRCC1 predicts overall survival of gastric cancer patients receiving oxaliplatin-based chemotherapy in Chinese population

Pharmacogenetic advances in cancer chemotherapy have the potential to predict clinical benefit to particular regimens. Platinum agents have shown to be effective in the treatment of gastric cancer. We assessed whether single nucleotide polymorphisms (SNPs) in xeroderma pigmentosum group D (XPD), X-ray repair cross complementing group 1 (XRCC1) and glutathione S-transferase P1 (GSTP1) predicted overall survival in gastric cancer patients receiving oxaliplatin-based chemotherapy in Chinese population. SNPs of XPD-751, XRCC1-399 and GSTP1-105 in 62 gastric cancer patients were evaluated using the TaqMan 5' nuclease assay. Genotypes were correlated to survival. The median overall survival time was 322 days (range: 56-2058 days). The median survival times for patients with Arg/Arg or Arg/Gln genotypes of XRCC1 gene were significantly longer than others (P=0.03). For 58 patients with ECOG PS (Eastern Cooperative Oncology Group performance status)<or=2, more obvious significance was demonstrated (P=0.002). Patients with XRCC1-399 Gln/Gln genotype demonstrated a significant worse survival. No significant association was found between SNPs of XPD-751, GSTP1-105 and survival (P=0.125, 0.475, respectively). XRCC1 genotyping might make tailor chemotherapy possible for gastric cancer patients treated with oxaliplatin-based chemotherapy.

The NCI60 human tumour cell line anticancer drug screen

The US National Cancer Institute (NCI) 60 human tumour cell line anticancer drug screen (NCI60) was developed in the late 1980s as an in vitro drug-discovery tool intended to supplant the use of transplantable animal tumours in anticancer drug screening. This screening model was rapidly recognized as a rich source of information about the mechanisms of growth inhibition and tumour-cell kill. Recently, its role has changed to that of a service screen supporting the cancer research community. Here I review the development, use and productivity of the screen, highlighting several outcomes that have contributed to advances in cancer chemotherapy.

Treatment Cost of Pancreatic Cancer in Japan: Analysis of the Difference after the Introduction of Gemcitabine

Recent advances in cancer chemotherapy have increased not only the survival rate but also the treatment cost, although there has been little interest in cost analyses in Japan. The actual cost of pancreatic cancer treatment was surveyed especially with respect to the difference after April 2001, which was the date that gemcitabine was introduced in Japan. A total of 113 patients were admitted consecutively from April 2000 to March 2002. Among the 113 patients, the total treatment cost over a lifetime was calculated in 54. In those 54 patients, the median treatment cost and survival time were $43,865 and 26.2 months for resectable disease (n = 14), $30,676 and 10.0 months for locally advanced disease (n = 21), and $29,255 and 4.8 months for metastatic disease (n = 19), respectively. Of the 54, 26 patients were admitted before April 2001 (Group A) and the remaining 28 were admitted thereafter (Group B). There were significantly more patients who received gemcitabine chemotherapy in Group B (19 of the 28) than in Group A (none of the 26). The median treatment cost and survival times were $35,744 and 7.4 months in Group A, and $35,226 and 8.8 months in Group B, respectively, whereas the total cost of anticancer agents was significantly higher in Group B than in Group A. Although cost of gemcitabine is about 18-fold higher than 5-fluorouracil in Japan, the total costs after gemcitabine introduction did not tend to become higher in our hospital, probably because of simplification in examinations and shorter hospitalization.

Cell death in irradiated prostate cancer cells assessed by flow cytometry

Despite the significant advances in cancer chemotherapy, radiotherapy still remains a method of choice for treatment of metastatic human prostate cancer. This study presents quantitative analysis of 60Co gamma-radiation effects on cell growth and cell death of metastatic human prostate cancer PC-3 cell line, performed in time (24-72h) and dose (2-20 Gy) dependent manner. The irradiated PC-3 cells were mostly dying by necrosis at late time intervals (72h), while apoptotic cell death was negligible. The EC50 or 50% of cytotoxicity was not achieved within the radiation doses used (2-20 Gy), but significant cell growth inhibition with IC50 of 10.4 Gy was observed. It is concluded that the increase in the radiation dose may have an important cytostatic effect, but for the complete eradication of metastatic prostate cancer novel cytotoxic drugs and radiosensitizers should be introduced as adjuvant.

Association between overexpression of cyclooxygenase-2 and suppression of apoptosis in advanced cancer of the uterine cervix after cyclic balloon-occluded arterial infusion.

Recent advances in cancer chemotherapy have drawn the attention of investigators to the usefulness of chemotherapy for cancer of the uterine cervix in general, and we ourselves have previously reported satisfactory therapeutic results of cisplatin-based cyclic balloon-occluded arterial infusion chemotherapy (BOAI), which enabled treatment by hysterectomy in patients with advanced cervical cancer. Nevertheless, there have been some patients in whom CDDP therapy by BOAI was ineffective, and hysterectomy was impracticable. In the present study we investigated the expression of cyclooxygenase-2 (COX-2) in these cases in an attempt to identify the cause of the lack of efficacy. The subjects of the study were 38 women with advanced cervical cancer classified as FIGO (International Federation of Gynecology and Obstetrics) stage III or higher who were admitted to the Department of Gynecology of Osaka City University Medical School Hospital between July 1993 and April 2000. All of these patients were treated by BOAI, and expression of COX-2, angiogenic factors, and cancer cell apoptosis, before and after BOAI were examined, and survival rates were compared. The 18 patients in whom BOAI was ineffective showed significantly higher expression of COX-2 after BOAI, and cancer cell apoptosis was inhibited. The 5-year survival rate of these patients was 16.8%, as opposed to 54.1% in the 20 patients in whom BOAI was effective. These results suggest that overexpression of COX-2 inhibits cancer cell apoptosis and adversely influences the prognosis.

Emerging fungal infections in the immunocompromised host

Over the last 2 decades, numerous factors, including advances in cancer chemotherapy and transplant medicine and the emergence of immunocompromising illnesses such as acquired immunodeficiency syndrome, have led to the emergence of a population of vulnerable hosts with special needs. Invasive fungal infections caused by long-recognized pathogens such as Candida species and Aspergillus species, as well as emerging fungi such as Pseudallescheria species, Alternaria species, and Fusarium species, have been noted to be an important cause of mortality and morbidity. This article reviews the existing experience in diagnosis and treatment of many of such fungal infections, with a focus on immunocompromised children. A high index of suspicion for fungal infection in an immunocompromised child on broad spectrum antibiotics, especially with pneumonia, sinusitis, or new skin lesions; early and aggressive use of currently available antifungal agents; and measures directed toward reversal of the underlying predisposing conditions are key factors for a favorable outcome. Copyright © 2001 by W.B. Saunders Company

Information needs of cancer patients receiving chemotherapy at a day‐case unit in Northern Ireland

• Recent restructuring of cancer services in the United Kingdom and advances in cancer chemotherapy have resulted in the majority of chemotherapy being delivered on an outpatient basis.• Patients receiving chemotherapy are at risk of developing multiple problems and increased anxiety levels and are now more likely to encounter these problems outside the hospital setting. Therefore, providing all the necessary information at chemotherapy sessions is crucial to self‐care activities and coping.• A study was carried out to investigate the information needs of patients at various stages of chemotherapy treatment and this article reports these needs at the beginning of treatment.• Most patients wanted to receive all possible information about their condition and reported satisfaction with the information provided.• The overwhelming finding was lack of information given to patients regarding family relationships. Whilst almost all patients wanted this information, more than half reported that it had not been given.

Plant Secondary Metabolites as Potential Anticancer Agents and Cancer Chemopreventives

A. Douglas KinghornProgram for Collaborative Research in the Pharmaceutical Sciences and Department of MedicinalChemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 SouthWood Street, Chicago, IL 60612, U.S.ATel.: (312) 996-0914, Fax: (312) 996-7107, E-mail: kinghorn@uic.eduThere is considerable interest in the screening of plant and other natural product extracts in moderndrug discovery programs, since structurally novel chemotypes with potent and selective biological ac-tivity may be obtained [1-4]. A consideration of biological activity in addition to the isolation andstructure elucidation stages in a phytochemical investigation may add a great deal to the overall scien-tific significance of the work. Phytochemists may gain considerable information by using panels ofsimple bioassays and/or more specialized in vitro bioassays to follow each step of a purification proce-dure [3]. In the following paragraphs, recent examples of bioactive compounds obtained in the author’slaboratory in projects directed towards the search for novel anticancer agents and cancer chemopre-ventives from higher plants will be presented.In the United States in 1999, it is estimated that over 1.2 million persons will be diagnosed with in-vasive forms of cancer, and over 1,500 people will die as a result of cancer each day [5]. Among manyrecent advances in cancer chemotherapy, plant natural products have played an important role in con-tributing to the arsenal of the approximately 60 cancer chemotherapeutic drugs on the market. For in-stance, in the United States, there are now four structural classes of plant anticancer agents available,constituted by the Catharanthus (Vinca) alkaloids (vinblastine, vincristine, vinorelbine), the epipodo-phyllotoxins (etoposide, etoposide phosphate, teniposide), the taxanes (paclitaxel and docetaxel), andthe camptothecin derivatives (irinotecan and topotecan) [6]. Several other plant-derived compoundsare currently in preclinical and clinical trials [6,7].As part of a National Cooperative Natural Products Drug Discovery Group (NCNPDDG) researchproject funded by the United States National Cancer Institute (1995-2000), our collaborative team atthe College of Pharmacy, University of Illinois at Chicago (Chicago, Illinois), and Research TriangleInstitute (Research Triangle Park, North Carolina), and Bristol-Myers Squibb (Princeton, New Jersey)is evaluating about 400 plant samples per year, with the aim of discovering and evaluating novel plant-derived anticancer agents. During the funding period 1990-1995, the industrial partner was GlaxoWellcome Medicines Research Centre (Sevenage, U.K.), and past progress made in the project hasbeen reviewed [8]. Since 1995, the primary plant samples have been collected in the Dominican Re-public, Peru, and Indonesia. Plant recollections have taken place mainly in Thailand and Zimbabwe in

Recent advances in cancer chemotherapy

Recent advances in cancer chemotherapy

Cytogenetic and molecular characterization of random chromosomal rearrangements activating the drug resistance gene, MDR1/P-glycoprotein, in drug-selected cell lines and patients with drug refractory ALL.

Drug resistance, both primary and acquired, is a major obstacle to advances in cancer chemotherapy. In vitro, multidrug resistance can be mediated by P-glycoprotein (PGY1), a cell surface phosphoglycoprotein that acts to efflux natural products from cells. PGY1 is encoded by the MDR1 gene located at 7q21.1. Overexpression of MDR1 has been demonstrated in many cancers, both in patient tumors and in cell lines selected with a variety of chemotherapeutic agents. Recent studies in drug-selected cell lines and patients samples have identified hybrid mRNAs comprised of an active, but apparently random, gene fused 5' to MDR1. This observation indicates that random chromosomal rearrangements, such as translocations and inversions, leading to "capture" of MDR1 by constitutively expressed genes may be a mechanism for activation of this gene following drug exposure. In this study, fluorescence in situ hybridization (FISH) using whole chromosome paints (WCP) and bacterial artificial chromosome (BAC)-derived probes showed structural rearrangements involving 7q in metaphase and interphase cells, and comparative genomic hybridization (CGH) revealed high levels of amplification at chromosomal breakpoints. In an adriamycin-selected resistant colon cancer line (S48-3s/Adr), WCP4/WCP7 revealed t(4;7)(q31;q21) and BAC-derived probes demonstrated that the breakpoint lay between MDR1 and sequences 500-1000 KB telomeric to it. Similarly, in a subline isolated following exposure to actinomycin D (S48-3s/ActD), a hybrid MDR1 gene composed of heme oxygenase-2 sequences (at 16p13) fused to MDR1 was identified and a rearrangement confirmed with WCP7 and a subtelomeric 16p probe. Likewise, in a paclitaxel-selected MCF-7 subline where CASP sequences (at 7q22) were shown to be fused to MDR1, WCP7 showed an elongated chromosome 7 with a homogeneously staining regions (hsr); BAC-derived probes demonstrated that the hsr was composed of highly amplified MDR1 and CASP sequences. In all three selected cell lines, CGH demonstrated amplification at breakpoints involving MDR1 (at 7q21) and genes fused to MDR1 at 4q31, 7q22, and 16p13.3. Finally, in samples obtained from two patients with drug refractory ALL, BAC-derived probes applied to archived marrow cells demonstrated that a breakpoint occurred between MDR1 and sequences 500-1000 KB telomeric to MDR1, consistent with a random chromosomal rearrangement. These results support the proposal that random chromosomal rearrangement leading to capture and activation of MDR1 is a mechanism of acquired drug resistance.

Mechanisms of Bacterial Resistance

Antibiotic resistance is a growing problem in clinical pediatrics. Many of the agents traditionally used to treat pediatric pathogens are becoming less effective because of increasing bacterial resistance. In addition, many more children are immunocompromised because of primary or acquired immunodeficiencies and because of advances in cancer chemotherapy and transplantation. These children are being admitted to hospitals where they may be exposed to multiply resistant nosocomial pathogens. An improved understanding of the mechanisms of antibiotic resistance and the development of treatment strategies to prevent the emergence of resistance will be increasingly required in pediatrics.