Disruption of the anaphase-promoting complex confers resistance to TTK inhibitors in triple-negative breast cancer.

TTK protein kinase (TTK), also known as Monopolar spindle 1 (MPS1), is a key regulator of the spindle assembly checkpoint (SAC), which functions to maintain genomic integrity. TTK has emerged as a promising therapeutic target in human cancers, including triple-negative breast cancer (TNBC). Several TTK inhibitors (TTKis) are being evaluated in clinical trials, and an understanding of the mechanisms mediating TTKi sensitivity and resistance could inform the successful development of this class of agents. We evaluated the cellular effects of the potent clinical TTKi CFI-402257 in TNBC models. CFI-402257 induced apoptosis and potentiated aneuploidy in TNBC lines by accelerating progression through mitosis and inducing mitotic segregation errors. We used genome-wide CRISPR/Cas9 screens in multiple TNBC cell lines to identify mechanisms of resistance to CFI-402257. Our functional genomic screens identified members of the anaphase-promoting complex/cyclosome (APC/C) complex, which promotes mitotic progression following inactivation of the SAC. Several screen candidates were validated to confer resistance to CFI-402257 and other TTKis using CRISPR/Cas9 and siRNA methods. These findings extend the observation that impairment of the APC/C enables cells to tolerate genomic instability caused by SAC inactivation, and support the notion that a measure of APC/C function could predict the response to TTK inhibition. Indeed, an APC/C gene expression signature is significantly associated with CFI-402257 response in breast and lung adenocarcinoma cell line panels. This expression signature, along with somatic alterations in genes involved in mitotic progression, represent potential biomarkers that could be evaluated in ongoing clinical trials of CFI-402257 or other TTKis.

The single-patient room in the NICU: maternal and family effects.

OBJECTIVE: To explore differences in maternal factors, including visitation and holding, among premature infants cared for in single-patient rooms (SPR) compared with open-bay in the neonatal intensive care unit (NICU). STUDY DESIGN: A total of 81 premature infants were assigned to a bed space in either the open-bay area or in a SPR upon NICU admission, based on bed space and staffing availability in each area. Parent visitation and holding were tracked through term equivalent, and parents completed a comprehensive questionnaire at discharge to describe maternal health. Additional maternal and medical factors were collected from the medical record. Differences in outcome variables were investigated using linear regression. RESULT: No significant differences in gestational age at birth, initial medical severity, hours of intubation or other factors that could affect the outcome were observed across room type. Significantly more hours of visitation were observed in the first 2 weeks of life (P=0.02) and in weeks 3 and 4 (P=0.02) among infants in the SPR. More NICU stress was reported by mothers in the SPR after controlling for social support (P=0.04). CONCLUSION: Increased parent visitation is an important benefit of the SPR, however, mothers with infants in the SPR reported more stress.

Differences among raters evaluating the success of EMLA cream in alleviating procedure-related pain in children with cancer.

We evaluated the analgesic efficacy of EMLA cream after repeated bone marrow aspirations or lumbar punctures (LPs) in children with cancer, and compared the ratings among patients, their parents, physicians, and nurses. Data from LPs were analyzed at the last procedure without EMLA (T1) and the first and last procedures with EMLA (T2 and T3). Friedman's nonparametric analysis of variance was used for statistical analysis. A total of 272 procedures in 29 children were analyzed. For 179 procedures without EMLA, physicians rated pain lower than other raters, and for the 93 with EMLA physicians rated pain less than the children. Children rated pain at T2 lower than at T1 or T3. Physicians rated pain at T2 less than at T3. Both children and physicians rated pain at T3 as not different from that at T1. No differences were noted at these time points for other raters in LP distress ratings, or in bone marrow aspiration pain or distress ratings. Thus EMLA was associated with decreased pain ratings for LPs, but this effect was not sustainable with repeated procedures. The cream alone should not be relied on to control pain of bone marrow aspiration or repeated LPs in children. Physicians underestimated pain, which may have implications for undertreatment in this patient population.

Economic impact with home delivery of chemotherapy to pediatric oncology patients.

OBJECTIVE: To examine the economic impact of a home chemotherapy program (HCP) for pediatric oncology patients. RATIONALE: Factors that led to initiation of an HCP included availability of specially trained nurses and programmable ambulatory infusion devices at local home care agencies, routine central venous catheter placement, inpatient bed space shortages, and the availability of ondansetron. SETTING: Chemotherapy delivery in the home setting from June 1991 through June 1994. DESIGN: Charge data and nausea and vomiting severity data were collected for patients treated through the HCP. METHODS: Economic impact was calculated by incorporating and summing all charge categories associated with hospital admission for chemotherapy (HAC) versus delivery by the HCP. All data were adjusted for 1993 dollars, and reflect changes for the average patient size (1 m2). Charge data for each chemotherapy protocol delivered in the home were analyzed by calculating the differences between HAC and HCP charges using the following formula: charge difference (HAC - HCP) per protocol times the number of courses. Total economic impact was calculated by summing the differences in charges for each protocol. RESULTS: A total of 262 chemotherapy courses were given to 44 patients (mean age 9.5 +/- 5.1 y) through the HCP, which represented 1012 patient care days and 24 different chemotherapy protocols. Monetary savings from the HCP ranged from $5180 per course of ifosfamide plus etoposide to $367 per course for high-dose methotrexate. Total monetary savings from the HCP during the 3-year period was $640,793. Successful control of nausea and vomiting with a combination of ondansetron plus methylprednisolone was achieved in approximately 80% of the patients receiving highly emetogenic chemotherapy protocols. CONCLUSIONS: HCP for pediatric oncology patients results in substantial monetary savings to payors. Effective control of nausea and vomiting can be accomplished at home in the majority of patients with an ondansetron-based antiemetic regimen.

Visual and verbal short-term memory deficits in childhood leukemia survivors after intrathecal chemotherapy.

Assessed survivors of childhood lymphoblastic leukemia (ALL) treated with intrathecal chemotherapy, using the Wide Range Assessment of Memory and Learning (WRAML), compared to controls without cancer, matched as closely as possible in age, SES, and gender. Mild, but consistent, deficits were found in both visual-spatial and verbal single-trial memory tasks. In multitrial learning, only visual-spatial tasks resulted in deficient scores, while verbal learning was within the normal range. IQ results indicated scores 10-20 points lower in the ALL group. Memory results are related to deficits in strategic planning and attentional distractiveness. The WRAML may be a useful clinical tool to evaluate differential memory deficits in children with ALL.

Multidrug resistance-1 (MDR1) expression and functional dye/drug efflux is highly correlated with the t(8;21) chromosomal translocation in pediatric acute myeloid leukemia.

Resistance to chemotherapy is a major problem in acute myeloid leukemia (AML). An important resistance mechanism in adult AML is active drug efflux mediated by the multidrug resistance protein-1 (MDR1). To determine if MDR1 is important in childhood AML, we examined MDR1 expression and functional dye/drug efflux in 20 pediatric/adolescent AML patients; results were correlated with cytogenetics and clinical outcome. Using flow cytometry, MDR1 protein expression on the leukemic blasts was measured with the antibody MRK16, while efflux was measured by extrusion of the fluorescent dye DiO(C2)3 in the presence/absence of cyclosporin A (CsA). Six of 20 cases expressed MDR1. While all six MDR1+ cases were efflux+, three of 14 MDR1- cases also demonstrated efflux. Both MDR1 and efflux were strongly correlated with the t(8;21). All six MDR1 +/efflux+ cases and 2/3 MDR1 -/efflux+ cases had a t(8;21), while no MDR1-/efflux- cases had a t(8;21) (P < 0.0005). This correlation between MDR1, efflux, and the t(8;21) in pediatric AML was not found in 11 adult t(8;21) cases similarly studied. Although the clinical relevance of MDR1 in pediatric AML awaits larger studies, our results suggest a biologic subset of pediatric AML patients may benefit from regimens which include MDR1-reversing agents or non-MDR1 substrates.

EWS and WT-1 gene fusion in desmoplastic small round cell tumor of the abdomen.

Chromosome translocations found in neoplasms often result in the creation of hybrid genes encoding chimeric proteins. This case study describes a patient with desmoplastic small round cell tumor (DSRCT) of the abdomen, an aggressive neoplasm characterized by translocation of chromosomes 11 and 22. Southern hybridization showed that the Ewing sarcoma gene (EWS) gene was rearranged in the DSRCT. Reverse transcriptase-polymerase chain reaction analysis of tumor cell RNA revealed that exons 1 to 7 of the EWS gene were joined to exons 8 to 10 of the Wilms' Tumor-1 (WT-1) gene resulting in the production of a chimeric message. The WT-1 and EWS genes encode DNA and RNA binding proteins involved in Wilms' tumor and Ewing sarcoma pathogenesis, respectively. The fusion of these two genes in DSRCT results in the production of a putatively oncogenic protein composed of the zinc finger DNA binding domains of WT-1 linked to potential transcriptional regulatory domains of EWS. DNA sequencing revealed the genomic breakpoints of translocation on chromosomes 11 and 22. The genomic breakpoint on chromosome 22 occurred in EWS intron 7 just 2 nucleotides 3' of exon 7. Polymerase chain reaction-based assays were developed that could detect the fused genes in the DSRCT tumor using either RNA or genomic DNA. The potential diagnostic use of these assays is discussed.

Health care outcomes case study: Febrile neutropenia.

A case study of the clinical and economic impact of filgrastim in cancer patients in the home care setting is described. The risk of febrile neutropenia is greatly reduced when filgrastim therapy is begun prophylactically after a course of antineoplastic drug therapy. Such use of filgrastim may also reduce the need for hospitalization, prevent mucositis, and enable the next course of chemotherapy to be administered on schedule. Investigators at the University of New Mexico, noting studies showing merit in early hospital discharge of pediatric cancer patients with febrile neutropenia, decided to take the approach a step further by treatment febrile neutropenia entirely at home. Patients seen in the pediatric oncology clinic at the university hospital were considered candidates for home treatment if they had stable vital signs, were already receiving filgrastim after the completion of cancer chemotherapy, and lived within one hour of the hospital. Six cancer patients 2-17 years of age were studied between August 1992 and February 1994. These patients had 16 episodes of febrile neutropenia that were treated at home. The drug regimen consisted of prophylactic filgrastim (begun the day after completion of a course of antineoplastic therapy) and ceftazidime (when febrile neutropenia developed). None of the children were readmitted to the hospital. Infections resolved within 12 days in all cases. The total charge for treating the 16 episodes at home was $22,400 (drug charge for ceftazidime, visits by nurses, infusion-pump rental, and ancillary charges). The total charge for hospital treatment of these episodes would be $112,924 (bed charge and pharmacy charge). The difference suggests a potential savings of $90,524 from home care for this small group of patients. Treating febrile neutropenia in pediatric cancer patients at home is effective and cost-efficient if the patients are clinically stable and prophylactic filgrastim therapy has been started after the completion of cancer chemotherapy.

Assessment of chemotherapy-induced emesis and evaluation of a reduced-dose intravenous ondansetron regimen in pediatric outpatients with leukemia.

OBJECTIVE: To measure the severity of nausea and vomiting in pediatric patients receiving intravenous or intrathecal chemotherapy for acute lymphoblastic leukemia and to evaluate the effectiveness of 2 intravenous doses of ondansetron for this condition. DESIGN: Patients were surveyed during repeated treatments of maintenance chemotherapy, given with or without ondansetron, using a repeated measures pretest/posttest design. SETTING: Outpatient pediatric oncology clinic. PATIENT POPULATION: Sixteen pediatric patients (aged 2-15 years, mean 6.2) with acute lymphoblastic leukemia. METHODS: Surveys to assess nausea and vomiting and the extent of interference with daily activities were administered following emetogenic chemotherapy with or without ondansetron. RESULTS: A total of 255 surveys following emetogenic chemotherapy with daunorubicin, cyclophosphamide, carmustine, and etoposide and cytarabine combined, as well as intrathecal therapy with methotrexate, hydrocortisone, and cytarabine, were analyzed. Analysis was performed on surveys of 149 courses without antiemetic therapy and 106 courses after 2 doses of ondansetron 0.15 mg/kg iv. The most emetogenic chemotherapy treatment was the etoposide/cytarabine combination (p < 0.05). Ondansetron completely protected patients (defined as no nausea or no vomiting) during most (> 50%) of the chemotherapy treatments, except for those in which cyclophosphamide was used. Ondansetron provided greater control of nausea and vomiting, a higher percentage of complete protection, and decreased the daily activity interference rating for carmustine and etoposide/cytarabine compared with courses of chemotherapy without antiemetics (p < 0.05). Two intravenous doses of ondansetron also provided durable antiemetic efficacy over time for the most emetogenic chemotherapy treatment (etoposide/cytarabine). CONCLUSIONS: Etoposide/cytarabine proved to be the most emetogenic of the chemotherapy treatments studied. A reduced-dose regimen of intravenous ondansetron was shown to be an effective antiemetic for the outpatient treatments with etoposide/cytarabine and carmustine, but not with cyclophosphamide.

Continuous midazolam infusion for the management of morphine-induced myoclonus.

OBJECTIVE: To describe a patient with morphine-induced myoclonus treated with a continuous infusion of midazolam and continued morphine dose escalation. DESIGN: Single case report. SETTING: Delivery, monitoring, and titration of morphine and midazolam in the patient's home by a homecare agency. RESULTS: The use of high dosages of morphine (i.e., 500 mg/h) produced myoclonic spasms in this patient, which in turn resulted in increasing pain. To allow for continuation of effective analgesia and to control the myoclonic spasms, an infusion of midazolam was initiated and titrated. The midazolam infusion allowed for continuation of the morphine dosage and also permitted further dosage escalation. As morphine dosages were further escalated, it was also necessary to increase the midazolam infusion to control additional myoclonic spasms. CONCLUSIONS: Use of a concomitant midazolam infusion with high doses of morphine appears to be safe and is an effective means of controlling morphine-induced myoclonus. If further dosage increase of morphine are necessary in this setting, increases in the midazolam infusion also may be required.

Langerhans'-cell histiocytosis (histiocytosis X)--a clonal proliferative disease.

BACKGROUND: The lesions of Langerhans'-cell histiocytosis (histiocytosis X), a proliferative histiocytic disorder of unknown cause, contain histiocytes similar in phenotype to dendritic Langerhans' cells. The disease ranges in severity from a fatal leukemia-like disorder to an isolated lytic lesion of bone. Intermediate forms of the disease are usually characterized by multiorgan involvement, diabetes insipidus, and a chronic course. METHODS: To determine whether Langerhans' histiocytosis is a polyclonal reactive disease or a clonal disorder, we used X-linked polymorphic DNA probes (HUMARA, PGK, M27 beta[DXS255], and HPRT) to assess clonality in lesional tissues and control leukocytes from 10 female patients with various forms of the disease. Lymphoid clonality was also assessed by analysis of rearrangements at immunoglobulin and T-cell-receptor gene loci. RESULTS: The HUMARA assay detected clonal cells in the lesions of 9 of the 10 patients: 3 patients had acute disseminated disease, 3 had unifocal disease, and 3 had intermediate forms. The percentage of clonal cells closely approximated the percentage of CD1a-positive histiocytes in each lesion. Clonality was also confirmed in two of nine cases with the PGK or M27 beta probe. Extreme constitutional lyonization precluded assessment of clonality in the 10th case. Lymphoid clonality was ruled out in all cases. CONCLUSIONS: The detection of clonal histiocytes in all forms of Langerhans'-cell histiocytosis indicates that this disease is probably a clonal neoplastic disorder with highly variable biologic behavior. Thus, genetic mutations that promote clonal expansion of Langerhans' cells or their precursors may now be identified.

Practical approach to the investigation of neutropenia.

This article discusses the evaluation of a patient for possible neutropenia. An evaluation must include an integration of complex factors such as age- and race-related normal ranges for absolute neutrophil count, an assessment of other hematopoietic elements in blood, an investigation for possible underlying infections, an examination for splenomegaly, and an evaluation for evidence of recurrent infections or family history suggestive of a constitutional neutropenic disorder.

DNA content measurement can be obtained using archival material for DNA flow cytometry. A comparison with cytogenetic analysis in 56 pediatric solid tumors.

BACKGROUND: Although flow cytometry (FCM) has become a widely used technique for the measurement of DNA content in solid tumors, the correlation of ploidy analysis by FCM with cytogenetic analysis (CGA) is not well described. The sensitivities of G-banded CGA and FCM were compared to determine the accuracy of the DNA index value (DI) as a measurement of chromosome number. METHODS: Tumor specimens from 56 pediatric cases were analyzed for DNA content by both FCM and CGA. Nuclei for FCM were prepared from archival tissue in 53 specimens using a modification of the Hedley technique and from fresh tissue in 3 specimens. Metaphase chromosomes for CGA were prepared from standard solid tumor harvests and Giemsa-trypsin banding procedures. Ploidy status for this study was defined as (1) diploid--DI between 0.97 and 1.03 by FCM or chromosome number +/- 2 from normal by CGA (44-48); and (2) aneuploid--DI < 0.97 or > 1.03 by FCM or total chromosomes < 44 or > 48 by CGA. RESULTS: Forty-nine of the 56 pediatric specimens were evaluable by both techniques. Concordance was observed in 34 cases (69%) between the two techniques in assigning similar ploidy status to a tumor (22 diploid and 12 aneuploid). It also was observed that among the aneuploid concordant cases, the actual DI obtained from archival material could predict total chromosome number with 95% accuracy. The 15 discordant cases showed a distinct aneuploid population by FCM, but were diploid by CGA. CONCLUSIONS: A correlation of 69% was obtained between both techniques to assign a similar ploidy status (diploid versus aneuploid) in 56 pediatric solid tumors. These results support the combined use of CGA and FCM to obtain the most complete analysis of DNA content and chromosome abnormalities in pediatric solid tumors. FCM on formalin-fixed, paraffin-embedded tissue can be used to measure total DNA content.