Expression of group B protective surface protein (BPS) by invasive and colonizing isolates of group B streptococci.

Group B protective surface protein (BPS) is expressed on the cell surface of some group B streptococcal (GBS) (Streptococcus agalactiae) strains and adds to the identification by capsular polysaccharide (CPS), and c or R proteins. We investigated the prevalence of BPS among GBS clinical isolates (303 invasive, 4122 colonizing) collected over 11 years in four American cities. Hot HCl cell extracts were tested by immunoprecipitation in agarose with rabbit antisera to BPS; the alpha (α) and beta (ß) components of c protein; R1, R3, and R4 species of R protein; and CPS serotypes Ia-VIII. BPS was found in 155 isolates (seven invasive, 148 colonizing). Of these, 87 were Ia, 37 II, 20 V; none were III. BPS was expressed usually with another protein: a species of R by 87 or a component of c by 39. The predominant CPS/protein profiles with BPS were Ia/R1,BPS and II/c(α + ß),BPS. Thus, along with CPS serotype and other surface proteins, BPS can be a valuable marker for precise strain characterization of unique GBS clinical isolates with complex surface protein profiles.

Serotype IV and invasive group B Streptococcus disease in neonates, Minnesota, USA, 2000-2010.

Group B Streptococcus (GBS) is a major cause of invasive disease in neonates in the United States. Surveillance of invasive GBS disease in Minnesota, USA, during 2000-2010 yielded 449 isolates from 449 infants; 257 had early-onset (EO) disease (by age 6 days) and 192 late-onset (LO) disease (180 at age 7-89 days, 12 at age 90-180 days). Isolates were characterized by capsular polysaccharide serotype and surface-protein profile; types III and Ia predominated. However, because previously uncommon serotype IV constitutes 5/31 EO isolates in 2010, twelve type IV isolates collected during 2000-2010 were studied further. By pulsed-field gel electrophoresis, they were classified into 3 profiles; by multilocus sequence typing, representative isolates included new sequence type 468. Resistance to clindamycin or erythromycin was detected in 4/5 serotype IV isolates. Emergence of serotype IV GBS in Minnesota highlights the need for serotype prevalence monitoring to detect trends that could affect prevention strategies.

A phase 1 trial of E7974 administered on day 1 of a 21-day cycle in patients with advanced solid tumors.

BACKGROUND: E7974, a synthetic analog of hemiasterlin, interacts with the tubulin molecule and overcomes resistance to other antitubulin drugs (taxanes and vinca alkaloids). METHODS: In a phase 1 study, E7974 was given intravenously over a 2- to 5-minute infusion on day 1 of every 21-day cycle. Adult patients with advanced refractory solid tumors who had adequate organ function and Eastern Cooperative Oncology Group performance status 0 to 2 were eligible for this study. A modified Fibonacci schema was used. The maximal tolerated dose (MTD) was the dose where <2 of 6 patients developed a dose-limiting toxicity (DLT). RESULTS: Twenty-eight patients (19 men and 9 women; median age, 64 years) treated at different cohort dose levels (0.18 mg/m(2) , 0.27 mg/m(2) , 0.36 mg/m(2) , 0.45 mg/m(2) , and 0.56 mg/m(2) ) received a total of 66 courses of E7974. The MTD was established at 0.45 mg/m(2) , where 1 of 6 patients experienced DLT (grade 4 febrile neutropenia). Of the 17 refractory colon cancer patients with a median of 3 prior treatments, stable disease was seen in 7 patients (41%). There were no tumor responses. Median progression-free survival was 1.2 months, and median overall survival was 6.7 months. In pharmacokinetic analysis, E7974 was characterized by a fast and moderately large distribution (37.95-147.93 L), slow clearance (2.23-7.15 L/h), and moderate to slow elimination (time to half-life, 10.4-30.5 hours). CONCLUSIONS: This study shows that E7974 once every 21-day cycle shows antitumor activity in patients with refractory solid tumors. The recommended phase 2 dose is 0.45 mg/m(2).

Phase Ib study of drozitumab combined with first-line mFOLFOX6 plus bevacizumab in patients with metastatic colorectal cancer.

In this multicenter phase Ib study, drozitumab was given in combination with the mFOLFOX6 regimen and bevacizumab in patients with previously untreated, locally advanced recurrent or metastatic colorectal cancer on day 1 of every 14-day cycle. Nine patients were treated at 2 different cohort dose levels of drozitumab. No dose-limiting toxicities occurred at either dose level and the maximum tolerated dose was not reached. Two patients had a partial response of 4.93 and 4.96 months duration. Cohort 2 dose level is the recommended starting dose level for future trials.

Clonal analysis of colonizing group B Streptococcus, serotype IV, an emerging pathogen in the United States.

Colonizing group B Streptococcus (GBS) capsular polysaccharide (CPS) type IV isolates were recovered from vaginal and rectal samples obtained from 97 (8.4%) nonpregnant women of 1,160 women enrolled in a U.S. multicenter GBS vaccine study from 2004 to 2008. Since this rate was much higher than the rate of prevalence of 0.4 to 0.6% that we found in previous studies, the isolates were analyzed by using surface protein profile identification, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) to characterize them and identify trends in DNA clonality and divergence. Of the 101 type IV isolates studied, 53 expressed alpha and group B protective surface (BPS) proteins, 27 expressed BPS only, 20 expressed alpha only, and 1 had no detectable surface proteins. The isolates spanned three PFGE macrorestriction profile groups, groups 37, 38, and 39, of which group 37 was predominant. The isolates in group 37 expressed the alpha and BPS proteins, while those in groups 38 and 39 expressed the alpha protein only, with two exceptions. MLST studies of selective isolates from the four protein profile groups showed that isolates expressing alpha,BPS or BPS only were of a new sequence type, sequence type 452, while those expressing alpha only or no proteins were mainly of a new sequence type, sequence type 459. Overall, our study revealed a limited diversity in surface proteins, MLST types, and DNA macrorestriction profiles for type IV GBS. There appeared to be an association between the MLST types and protein expression profiles. The increased prevalence of type IV GBS colonization suggested the possibility that this serotype may emerge as a GBS pathogen.

Oxaliplatin and fixed-rate infusional gemcitabine in the second-line treatment of patients with metastatic colon cancer: final results of a Phase II trial prematurely closed as a result of poor accrual.

BACKGROUND: Second-line therapy of advanced colorectal cancer (CRC) after failure of a combination of irinotecan, a fluoropyrimidine, and bevacizumab includes the use of oxaliplatin and a fluoropyrimidine. In animal models, synergistic effects of gemcitabine and platinum agents have been established. Additionally, superior antitumor activity of prolonged administration of gemcitabine compared with bolus administration has been demonstrated in vivo against murine colon tumors. PATIENTS AND METHODS: A 2-stage phase II trial was developed to assess the efficacy (primary endpoint: response rate) and safety of gemcitabine 1000 mg/m(2) over 100 minutes on days 1 and 15 in combination with oxaliplatin 100 mg/m(2) over 2 hours on days 2 and 16, every 4 weeks. Patients with metastatic CRC in whom irinotecan and a fluoropyrimidine treatment had failed were enrolled. Calcium and magnesium infusion was routinely given before and after oxaliplatin administration. RESULTS: Because of slow accrual as a result of oxaliplatin becoming more commonly used in first-line treatment, the trial was stopped with only 10 patients enrolled. Eight were men and 2 were women. Median age was 58.5 years (range, 47-72 years). Nine patients had an Eastern Cooperative Oncology Group performance status of 0/1. A median of 3.5 cycles was administered (range, 1-9; total, 42). Six patients had stable disease and 1 had progressive disease. Two patients had confirmed partial responses, and 1 patient had a partial response but developed necrotizing fasciitis, declined surgical treatment, and died before a confirmatory scan could be performed. The regimen was otherwise well tolerated: 1 patient developed grade 3 neutropenia. With a median follow-up of 5.5 months, 4 patients have died. The time to treatment failure was 3.7 months. CONCLUSION: Despite premature study closure because of poor accrual, oxaliplatin in combination with fixed-rate infusional gemcitabine seems to be a safe and potentially effective regimen in the treatment of CRC. Further studies should be considered with the addition of targeted therapy.

Identification of novel cps locus polymorphisms in nontypable group B Streptococcus.

Group B Streptococcus (GBS) is an important pathogen responsible for a variety of diseases in newborns and the elderly. A clinical GBS isolate is considered nontypable (NT) when serological methods fail to identify it as one of nine known GBS serotypes. Eight clinical isolates (designated A1-A4, B1-B4) showed PFGE profiles similar to that of a GBS serotype V strain expressing R1, R4 surface proteins. These unique isolates were further characterized by immunologic and genetic methods. Rabbit sera to isolates A1 and A2 reacted weakly with concentrated HCl extracts of A1-A4 isolates, but not with those of B1-B4 isolates. In addition, a type V capsular polysaccharide (CPS) inhibition ELISA revealed that cell wall extracts from isolates A1-A4, but not from B1-B4, expressed low but measurable amounts of type V CPS. Molecular serotyping with PCR analysis showed that all eight isolates contained a type V-specific CPS gene (cpsO) and harboured the gene encoding the surface protein Alp3. Multilocus sequence typing identified isolate A1 as belonging to a new sequence type (ST) designated ST-173, whereas the other seven isolates keyed to ST-1. Sequencing of the 18 genes (17 736 bp) in the cps locus showed that each NT isolate harboured one to three unique polymorphisms, and also identified an IS1381 element in cpsE of the B4 isolate. Collectively, genetic and immunologic analyses revealed that these NT isolates expressing R1, R4 proteins have a genetic profile consistent with that of type V, an emergent, antigenically diverse and increasingly prevalent GBS serotype.

A Phase I Clinical, Pharmacokinetic, and Pharmacodynamic Study of Weekly or Every Three Week Ixabepilone and Daily Sunitinib in Patients with Advanced Solid Tumors.

PURPOSE: To evaluate the safety, MTD, pharmacokinetics/pharmacodynamics, and early clinical activity of ixabepilone given either weekly or every 3 weeks in combination with daily sunitinib in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Eligible patients received either weekly (schedule A) or every 3 weeks (schedule B) ixabepilone at escalating doses (schedule A: 7.5, 15, or 20 mg/m(2); schedule B: 20, 30, or 40 mg/m(2)), and oral sunitinib (37.5 mg daily), starting on day 8 of cycle 1. Dose-limiting toxicities (DLT) were assessed during cycle 1. RESULTS: The ixabepilone and sunitinib combination was fairly well tolerated. DLTs were observed in 3 subjects (1 in schedule 3A and 2 in schedule 3B). The most common grade 3-4 hematologic and nonhematologic adverse events were leukopenia and fatigue, respectively. Four patients (3 in schedule A) achieved a partial response, while 13 patients had stable disease. Nine of 17 heavily pretreated colorectal cancer patients had clinical benefit. Coadministration of sunitinib with ixabepilone on a weekly (but not every 3 week) schedule was associated with a significant increase in the half-life and a significant decrease in the clearance of ixabepilone. Correlative studies demonstrated a significant association between higher baseline plasma angiogenic activity (PAA) and clinical benefit in schedule A patients. Weekly, but not every 3 weeks, ixabepilone led to a significant decrease in PAA postbaseline. CONCLUSIONS: Coadministration of ixabepilone with sunitinib has acceptable toxicity and encouraging clinical activity in heavily pretreated patients, particularly in patients with metastatic colorectal cancer. Clin Cancer Res; 22(13); 3209-17. ©2016 AACR.

Using unconjugated antibodies as an immunotherapeutic approach to treatment of B-cell neoplasms.

B-cell neoplasms have the sixth highest mortality incidence and one of the largest rates of increase of all malignancies. Frequent relapse and the development of refractory disease in this patient group have stimulated research to find alternative treatments. The use of unconjugated antibodies offers a new approach to the treatment of non-Hodgkin's lymphoma and chronic lymphocytic leukemia. This article will explore the pharmacology, dosage, and administration guidelines as well as side effect management of the available drugs in this classification. Expectations for future approaches to the treatment of B-cell neoplasms will be discussed.

Chemoimmunotherapy using pegylated liposomal Doxorubicin and interleukin-18 in recurrent ovarian cancer: a phase I dose-escalation study.

Recombinant interleukin (IL)-18 (SB-485232) is an immunostimulatory cytokine, with shown antitumor activity in combination with pegylated liposomal doxorubicin (PLD) in preclinical models. This phase I study evaluated the safety, tolerability, and biologic activity of SB-485232 administered in combination with PLD in subjects with recurrent ovarian cancer. The protocol comprised four cycles of PLD (40 mg/m(2)) on day 1 every 28 days, in combination with SB-485232 at increasing doses (1, 3, 10, 30, and 100 µg/kg) on days 2 and 9 of each cycle, to be administered over five subject cohorts, followed by discretionary PLD monotherapy. Sixteen subjects were enrolled. One subject withdrew due to PLD hypersensitivity. Most subjects (82%) were platinum-resistant or refractory, and had received a median of three or more prior chemotherapy regimens. SB-485232 up to 100 µg/kg with PLD had an acceptable safety profile. Common drug-related adverse events were grade 1 or 2 (no grade 4 or 5 adverse events). Concomitant PLD administration did not attenuate the biologic activity of IL-18, with maximal SB-485232 biologic activity already observed at 3 µg/kg. Ten of 16 enrolled subjects (63%) completed treatment, whereas five (31%) subjects progressed on treatment. A 6% partial objective response rate and a 38% stable disease rate were observed. We provide pilot data suggesting that SB-485232 at the 3 µg/kg dose level in combination with PLD is safe and biologically active. This combination warrants further study in a phase II trial.

Phase II study of gemcitabine, oxaliplatin, and cetuximab in advanced pancreatic cancer.

OBJECTIVE: Little progress has been made in the treatment of pancreatic cancer (PC). This study evaluated the clinical activity of gemcitabine, oxaliplatin, and cetuximab (GOC) in patients with locally advanced or metastatic PC. METHODS: The study primary endpoint was progression-free survival (PFS). Eligible, chemotherapy-naive PC patients were treated with gemcitabine (1000 mg/m(2) over 100 min) on day 1, oxaliplatin (100 mg/m(2)) on day 2, every 2 weeks, and weekly cetuximab, (loading dose of 400 mg/m(2) on cycle 1 day 1 and 250 mg/m(2) thereafter). It was expected that GOC treatment would extend the median PFS from 5.8 to 7.54 months, a relative increase of 30%, compared with gemcitabine and oxaliplatin (historical control). RESULTS: A total of 41 evaluable patients were enrolled. The overall response rate was 24%. Median PFS time was 6.9 months and median overall survival (OS) was 11.3 months. Patients with locally advanced disease had longer median PFS (12.4 vs. 4.7 mo) and OS (15.7 vs. 6.4 mo) compared with patients with metastatic disease. The most common grade 3 to 4 toxicities included neutropenia (32%), infection (with normal or grade 1 to 2 neutropenia, in 24%), neuropathy (17%), fatigue (15%), and rash (7%). Five patients (12%) discontinued study treatment without evidence of progression. Rash was not a significant prognostic factor affecting PFS or OS. CONCLUSIONS: GOC is a feasible combination with an acceptable toxicity profile. However, GOC did not significantly extend PFS in the overall patient population to consider it for further development.

Development of a Pulsed-Field Gel Electrophoresis (PFGE) method for molecular typing of clinical isolates of Arcanobacterium haemolyticum.

We developed a two-block PFGE method to study molecular variation among clinical isolates of Arcanobacterium haemolyticum, an often overlooked human pathogen. Three main macrorestriction profiles were defined among 15 isolates. PFGE was an objective method for characterizing A. haemolyticum and may be useful in molecular epidemiological studies of this organism.

Molecular characterization of nontypeable group B streptococcus.

Traditionally, the capsular polysaccharide (CPS) antigen has been used to distinguish between the nine known serotypes of group B streptococcus (GBS) by classical antibody-antigen reactions. In this study, we used PCR for all CPSs and selected protein antigens, multilocus sequencing typing (MLST), and pulsed-field gel electrophoresis (PFGE) to molecularly characterize 92 clinical isolates identified as nontypeable (NT) by CPS-specific antibody-antigen reactivity. The PCR and MLST were performed on blinded, randomly numbered isolates. All isolates contained the cfb gene coding for CAMP factor. While most (56.5%) contained a single CPS-specific gene, 40 isolates contained either two or three CPS-specific genes. Type V CPS-specific gene was present in 66% of the isolates, and all serotypes except types IV, VII, and VIII were represented. Most (44.5%) of the isolates contained a single protein antigen gene (bca, bac, rib, alp1, or alp3), and the remaining isolates had multiple protein antigen genes. Of the 61 isolates that had the V CPS-specific gene, 48 (78.6%) had the alp3 gene. PFGE analysis classified the isolates into 21 profile groups, while MLST analysis divided the isolates into 16 sequence types. Forty-two (69%) of 61 isolates with the V CPS-specific gene were in PFGE profile group 4; 41 of these 42 were sequence type 1 by MLST. These data shed new light on the antigenic complexity of NT GBS isolates, information that can be valuable in the formulation of an effective GBS vaccine.

DNA macrorestriction analysis of nontypeable group B streptococcal isolates: clonal evolution of nontypeable and type V isolates.

Group B streptococci (GBS) are serotyped according to capsular polysaccharide (CPS) type (Ia to VIII); an isolate is classified as nontypeable (NT) if no detectable CPS is found. Surface-localized protein antigens (alpha, beta, R1, and R4) serve as additional markers to classify GBS isolates, which is particularly useful since NT isolates often express one or more of these proteins. To compare genetic resemblance among isolates with similar protein profiles, we studied 58 NT isolates digested with the SmaI macrorestriction enzyme prior to pulsed-field gel electrophoresis (PFGE). Of these 58, 15.5% expressed alpha only, 20.7% expressed alpha+beta, 15.5% expressed R4, and 25.8% expressed R1,R4, while 22.4% of the isolates expressed no detectable proteins. The largest PFGE profile group, with 48% of the isolates, was group 4, composed primarily of isolates that expressed R1,R4 or no proteins. The second most common profiles were 3 and 32, each with 13.8% of the isolates. Since NT isolates in profile group 4 were highly related to type V isolates, as demonstrated by PFGE profiles, we investigated 45 type V isolates. Two-thirds of the type V isolates within profile group 4 were classified into subgroup 4a, compared to 28.2% of 39 NT isolates. Only 11% of the V/R1,R4 isolates were identical to the prototype group 4 profile, in contrast to 75% of the NT/R1,R4 isolates. A shift of type V isolates into profile 4 subgroups may be indicative of a genetic change over time. PFGE is a valuable approach for comparison of GBS isolate relatedness and for monitoring of NT and typeable GBS isolates for potential clonal divergence.

Improved methods for typing nontypeable isolates of group B streptococci.

Group B streptococci (GBS) are classified by capsular polysaccharide (CPS) type and by cell surface-expressed proteins (c and R). Isolates lacking detectable CPS are considered nontypeable (NT) although they frequently express surface proteins. Immunological and genetic methods were used to study 91 NT GBS isolates collected during surveillance studies for invasive disease or colonization in pregnant or non-pregnant women and neonates less than seven days of age. CPS production was upregulated by the addition of glucose and sodium phosphate to Todd-Hewitt broth (THB) and cells were extracted using hot HCl or mutanolysin. Extracts were tested with antisera for specific CPS types Ia, Ib, and II - VIII by double immunodiffusion (DD) in agarose. By mutanolysin extraction, 12 (13.2%) of the 91 isolates were typeable. In contrast, only four of these 12 newly typeable isolates tested positive for CPS with the HCl extracts of cells grown in modified THB. DNA was analyzed by pulsed-field gel electrophoresis (PFGE) using SmaI restriction with NT isolates grouped by protein profile to facilitate analysis. PFGE results of the NT isolates were compared to DNA profiles of typeable isolates and were correlated with the DD results. The DNA profiles of the newly typeable isolates were similar to profiles of isolates with corresponding defined CPS type. Of the remaining 78 NT isolates digested by SmaI, 63 (80.8%) had DNA profiles that resembled those of specific types of GBS. These approaches will be useful for classification of NT isolates in continued epidemiological surveillance associated with GBS vaccine trials.

Molecular analysis of group B protective surface protein, a new cell surface protective antigen of group B streptococci.

Group B streptococci (GBS) express various surface antigens designated c, R, and X antigens. A new R-like surface protein from Streptococcus agalactiae strain Compton R has been identified by using a polyclonal antiserum raised against the R protein fraction of this strain to screen a lambda Zap library. DNA sequence analysis of positive clones allowed the prediction of the primary structure of a 105-kDa protein designated BPS protein (group B protective surface protein) that exhibited typical features of streptococcal surface proteins such as a signal sequence and a membrane anchor region but did not show significant similarity with other known sequences. Immunogold electron microscopy using a BPS-specific antiserum confirmed the surface location of BPS protein on S. agalactiae strain Compton R. Anti-BPS antibodies did not cross-react with R1 and R4 proteins expressed by two variant type III GBS strains but reacted with the parental streptococcal strain in Western blot and immunoprecipitation analyses. Separate R3 and BPS immunoprecipitation bands were observed when a cell extract of strain Compton R was tested with an antiserum against Compton R previously cross-absorbed to remove R4 antibodies. Immunization of mice with recombinant BPS protein by the subcutaneous route produced an efficient antigen-specific response, and immunized animals survived challenge with a lethal dose of a virulent strain. Therefore, BPS protein represents a new R-like protective antigen of GBS.