Immunologic and tumor responses of pegilodecakin with 5-FU/LV and oxaliplatin (FOLFOX) in pancreatic ductal adenocarcinoma (PDAC).

Background Treatment options for pancreatic ductal adenocarcinoma (PDAC) are limited and checkpoint blockade inhibitors have been disappointing in this disease. Pegilodecakin has demonstrated single agent anti-tumor activity in immune-sensitive tumors. Phase 1 and preclinical data indicate synergy of pegilodecakin with 5-FU and platins. We assessed the safety and activity of pegilodecakin+FOLFOX in patients with PDAC. Methods IVY (NCT02009449) was an open-label phase 1b trial in the United States. Here we report on all enrolled patients from cohort C. Heavily pretreated patients were treated with pegilodecakin (self-administered subcutaneously daily at 2.5, 5, or 10 µg/kg) + 5-flurouracil/leucovorin/oxaliplatin (FOLFOX), dosed per manufacturers prescribing information, until tumor progression. Eligible patients had measurable disease per immune-related response criteria (irRC), were ≥ 18 years of age, and had ECOG performance status of 0 or 1. Patients were evaluated for primary(safety) and secondary (tumor response per irRC) endpoints. Results From 5 August 2014-12 July 2016, 39 patients enrolled in cohort C. All patients were evaluable for safety. In this advanced population, regimen had manageable toxicities with no immune-related adverse events (irAEs) greater than grade 1. The most common grade 3/4/5 TEAEs were thrombocytopenia (21[53.8%] of 39) and anemia (17[43.6%] of 39). In evaluable PDAC patients, the best overall response of pegilodecakin+FOLFOX was 3(14%) with CRs in 2(9%) patients. Conclusions Pegilodecakin+FOLFOX had an acceptable tolerability profile in PDAC, with no substantial irAEs seen, and promising efficacy with the combination yielding a 2-year OS of 24% (95% CI 10-42). These data led to the phase 3 study with pegilodecakin+FOLFOX as second-line therapy of PDAC (SEQUOIA).

Diagnostic pitfalls of drug-induced immune hemolytic anemia.

Immune hemolytic anemia (IHA) is a rare complication of drug administration. However, its true incidence remains obscure, as there are a number of factors that may lead to misdiagnosis. The clinical and serologic pictures are variable, and there is a great deal of unawareness that certain drugs can cause IHA. Furthermore, serologic results can be easily misinterpreted, resulting in a wrong diagnosis.

Effects of 5-fluorouracil chemotherapy on fatigue: role of MCP-1.

Chemotherapy has been known to cause severe side effects, including fatigue. While the mechanisms for chemotherapy induced fatigue (CIF) are likely to be multi-factorial in origin, it is thought that inflammation and anemia may play a role. The purpose of this study was to examine the effect of chemotherapy on fatigue in mice, and further, to begin to determine if inflammation and anemia may contribute to this response. For experiment 1, C57BL/6 mice were assigned to: vehicle (PBS), low (20 mg/kg), medium (40 mg/kg), or high (60 mg/kg) doses of 5-fluorouracil (5-FU). Voluntary physical activity (PA) was measured throughout the treatment period (day 1-5) as well as during the recovery period (day 6-14). In experiment 2, we examined the effects of 5-FU (60 mg/kg) on the inflammatory mediator MCP-1 and on markers of anemia (RBC, Hct and Hb). Finally, using MCP-1(-/-) mice we examined the role of MCP-1 on CIF (experiment 3). 5-FU reduced voluntary PA in a dose response manner (p<0.05). Plasma MCP-1 was increased following 5-FU treatment on both days 5 (p=0.10) and 14 (p<0.05). In addition, RBCs, Hct and Hb were reduced with 5-FU on days 5 and 14 (p<0.05). Both C57BL/6 and MCP-1(-/-) mice saw similar decrements in PA through the duration of the treatment period (days 1-5), however the MCP-1(-/-) mice recovered much earlier than wildtype mice. This study provides evidence of the dose response effect of a standard chemotherapy agent on fatigue and demonstrates a potential role of MCP-1 and presumably inflammation, and anemia.

Interference localized surface plasmon resonance nanosensor tailored for the detection of specific biomolecular interactions.

In this paper, we present an innovative sensing nanomaterial for an interference localized surface plasmon resonance (iLSPR) sensor. The iLSPR is based on plasmonic gold nanoparticles with photonic thin-film multilayers of porous aluminum oxide (Al(2)O(3)) and aluminum (Al) on a substrate. With a controllable transparent Al(2)O(3) layer and a highly reflective Al layer, our new nanomaterial was able to detect refractive index (RI) changes of the surrounding environment and the specific interaction of biomolecules including biotin and avidin, 5- fluorouracil (5-FU) and its antibody, anti-5-fluorouracil (anti 5-FU), when the iLSPR surfaces were biologically functionalized. Our model nanostructure will open the way to display the plasmonic properties of other noble metal nanoparticles and to develop other functionally similar nanosensors, which could then be expanded into multiarrays.

Fatal immune haemolysis due to antibodies to individual metabolites of 5-fluorouracil.

Confusion still exists in the diagnosis of drug-induced immune haemolysis (DIH). The aim of this study was to demonstrate antibodies specific to 5-fluorouracil (5-FU) in a patient with fatal immune haemolysis (IH). The case of a patient who died due to protracted IH is described. A 57-year-old female underwent treatment with oxaliplatin, 5-FU and folinic acid due to cholangiocarcinoma. Following drug administration, she was transfused because of a mild non-haemolytic anaemia and died following haemolysis. Serological testing including antibody screening, direct antiglobulin test and detection of drug-dependent antibodies was performed using standard techniques. The patient's serum was observed to be red in colour due to the presence of free haemoglobin prior to and following blood transfusion, and contained antibodies reactive with RBCs only in the presence of urine from several patients treated with 5-FU (ex vivo antigens). Drug-induced immune haemolysis (DIH) and metabolite-dependent antibodies should always be taken into consideration when a patient being administered any type of drug develops haemolysis.

High levels of tumor-infiltrating lymphocytes showed better clinical outcomes in FOLFOX-treated gastric cancer patients.

Background: Tumor-infiltrating lymphocytes (TILs) and postoperative chemotherapeutics interact in the tumor micro-environment. This interaction has not been well investigated in gastric cancer. Materials & methods: A total of 129 patients were divided into high or low TILs based on the median number of positive CD3+ and FoxP3+ T cells, which was assessed by immunocytochemistry. Results: Cox regression analysis showed that the stage III disease with shorter overall survival was significant. The analysis showed that high numbers of CD3+ or FoxP3+ T cells have better clinical outcomes in FOLFOX-treated patients. Conclusion: High CD3+ and FoxP3+ T-cell infiltration was associated with better clinical outcomes in patients with gastric cancer treated with FOLFOX, suggesting TILs incorporated into algorithms to improve the therapeutic efficacy of optimal chemotherapy.

Systemic immune effects of adjuvant chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide and/or radiotherapy in breast cancer: a longitudinal study.

Immunotherapy is being increasingly utilized for adjuvant treatment for breast cancer (BC). We have previously described immune functions during primary therapy for BC. The present study describes immune recovery patterns during long-term, unmaintained follow-up after completion of adjuvant therapy.A group of patients with primary BC had been treated with adjuvant radio-chemotherapy (RT + CT) 5-fluorouracil, epirubicin and cyclophosphamide (FEC) (n = 21) and another group with radiotherapy (RT) (n = 20) alone. Immunological testing of NK and T-cell functions was performed initially at the end of adjuvant treatment and repeated after 2, 6 and 12 months. NK cell cytotoxicity was significantly higher (P < 0.05) at all time-points in patients than in age-matched controls and did not differ between the two treatments groups during one year observation. In contrast, lower numbers of CD4 T-cells and lower expression of CD28 on T-cells was observed particularly in RT + CT patients and did not normalize during the observation period. The numbers of T(reg) cells (CD4(+)CD25(high)) were low in the RT + CT group during follow-up, as well as expression of TCRxi, Zap70, p56(lck), P59(fyn) and PI3 k in CD4(+) cells. In contrast, expression of intracellular cytokines (IFN-gamma, IL-2, IL-4) in CD4 and CD8 T cells were significantly higher in RT + CT patients than in the RT group and the difference increased during follow-up. In conclusion, NK-cell cytotoxicity increased during unmaintained long-term follow-up whereas CD4 and regulatory T cells as well as signal transduction molecules remained low following adjuvant radio-chemotherapy.

Sensitization to low dose 5-fluorouracil. Subsequent enhancement of its systemic antitumor effect in the rat.

This report describes a novel method of immunochemotherapy; the active immunization to the drug 5-fluorouracil (5-FU) with enhanced antitumor activity resulting from its subsequent systemic administration. Two metastasizing carcinomas in the Fischer strain (F344) rat have been used: a chemically induced bladder carcinoma (FBCa) and a spontaneous mammary adenocarcinoma (MACa). Both tumors grow rapidly and result in 100% mortality within 10 wk of implantation. Neither tumor is sensitive to systemic 5-FU alone. Intradermal sensitization to 5-FU before FBCa tumor implantation, followed by 5-FU administered systemically, resulted in significant tumor regression and improvement in survival with eradication of all tumor and cure in 20% of animals. A similar antitumor effect was observed with the MACa. A comparable drug effect was observed when methotrexate sensitization was given before FBCa implantation followed by systemic MTX. Specificity to the sensitizing drug was demonstrated by the lack of effect of sensitization with either 5-FU or MTX unless followed by systemic therapy with the requisite sensitizing agent. Sensitization to 5-FU has also been assessed after FBCa implantation followed by resection of the local tumor. Resection was performed after distant tumor metastases had occurred, and was followed by systemic 5-FU therapy. Whereas tumor resection alone failed to cure any animal, sensitization to 5-FU increased cure rate fourfold over animals receiving systemic 5-FU alone. Antibody to 5-FU in the sera of sensitized animals has been suggested by an immunoenzymatic staining technique and its specificity confirmed in a radioimmunoassay. It is postulated that a combination of the systemic agent and the antibody elicited to it by sensitization produces the significant antitumor effect observed. The antitumor effect observed with this new approach to immunochemotherapy warrants further experimental and clinical study.

Delayed hypersensitivity to 5-fluorouracil following topical chemotherapy of cutaneous cancers.

Skin tests to various common antigens, dinitrochlorobenzene, and 5-fluorouracil (5-FU) were performed on patients being treated for cutaneous neoplasms with topical 5-FU cream. Eleven of 15 patients tested both before and after therapy converted from skin test negative to positive with respect to 5-FU. This conversion correlated with positive dinitrochlorobenzene skin tests and therapeutic cure. The relation between the induction of delayed hypersensitivity reactions to 5-FU following treatment with topical 5-FU and the cure rate for cutaneous neoplasms showed a trend toward correlation.

[Preparation and characterization of polyclonal antibody against 5-fluorouracil].

OBJECTIVE: To prepare 5-fluorouracil (5-FU) immunogen and develop polyclonal antibodies against 5-FU. METHODS: The derivant of 5-FU (5-fluorouracil-1-yl-aceto amino acid, 5-FUAA) was synthesized, and then conjugated with bovine serum albumin (BSA) or ovalbumin (OVA) by carbodiimide (CDI) method. 5-FUAA conjugating BSA (5-FUAA-BSA) was used to immunize BALB/c mice to produce antiserum, and 5-FUAA conjugating OVA (5-FUAA-OVA) was used as coating antigen to detect the titer of the antiserum by indirect ELISA. Furthermore, specificity of the polyclonal antiserum was identified by ELISA and Western blotting. RESULTS: 5-FU derivant 5-FUAA was successfully synthesized and conjugated with BSA or OVA. Indirect ELISA showed that the titer of the antiserum from BALB/c mice immunized with 5-FUAA-BAS reached 1:1 280 000. Moreover, ELISA and Western blotting proved that the anti-serum could combine 5-FU specifically. CONCLUSION: The experiment has prepared high-specific and high-titer polyclonal antibody against 5-FU.

Prolongation of pancreatic allograft survival by 5-fluorouracil.

Recipient survival in canine pancreatic allotransplantation was significantly prolonged when animals were treated with 5-fluorouracil in combination with low-dose azathioprine. Untreated dogs and those receiving only azathioprine survived for only short periods of time, and pancreatitis was frequently encountered. In contrast, animals treated with 5-fluorouracil were protected from pancreatitis and survived an average of almost three weeks.

The combination of a low-dose chemotherapeutic agent, 5-fluorouracil, and an adenoviral tumor vaccine has a synergistic benefit on survival in a tumor model system.

Standard cancer therapies, particularly those involving chemotherapy, are in need of modifications that both reduce short-term and long-term side effects as well as improve the overall survival of cancer patients. Here we show that combining low-dose chemotherapy with a therapeutic vaccination using an adenovirus encoding a model tumor-associated antigen, ovalbumin (Ad5-OVA), had a synergistic impact on survival in tumor-challenged mice. Mice that received the combinatorial treatment of Ad5-OVA plus low-dose 5-fluorouracil (5-FU) had a 95% survival rate compared to 7% and 30% survival rates for Ad5-OVA alone and 5-FU alone respectively. The presence of 5-FU enhanced the levels of OVA-specific CD8(+) T lymphocytes in the spleens and draining lymph nodes of Ad5-OVA-treated mice, a phenomenon that was dependent on the mice having been tumor-challenged. Thus 5-FU may have enhanced survival of Ad5-OVA-treated mice by enhancing the tumor-specific immune response combined with eliminating tumor bulk. We also investigated the possibility that the observed therapeutic benefit may have been derived from the capacity of 5-FU to deplete MDSC populations. The findings presented here promote the concept of combining adenoviral cancer vaccines with low-dose chemotherapy.

A thymidylate synthase ternary complex-specific antibody, FTS, permits functional monitoring of fluoropyrimidines dosing.

5-Fluorouracil (5FU) and similar fluoropyrimidines induce covalent modification of thymidylate synthase (TS) and inhibit its activity. They are often used to treat solid cancers, but drug resistance and toxicity are drawbacks. Therefore, there is an unmet need for a functional assay to quantify fluorouracil activity in tissues, so as to individually tailor dosing. It is cumbersome to separately quantify unmodified and 5FU-modified TS using currently available commercial anti-TS antibodies because they recognize both forms. We report here the first monoclonal antibody (FTS) specific to 5FU-modified TS. By immunoblot assay, the FTS antibody specifically recognizes modified TS in a dose-dependent manner in 5FU-treated cells, in cancer xenograft tissues of 5FU-treated mice, and in the murine tissues. In the same assay, the antibody is nonreactive with unmodified TS in untreated or treated cells and tissues. Speculatively, a high-throughput assay could be enabled by pairing anti-TS antibodies of two specificities, one recognizing only modified TS and another recognizing both forms, to structurally quantify the TS-inhibiting effect of fluorouracil at a cellular or tissue level without requiring prior protein separation. Such a development might aid preclinical analytic studies or make practical the individual tailoring of dosing.

Infections of Blastocystis hominis and microsporidia in cancer patients: are they opportunistic?

Chemotherapy can cause immunosuppression, which may trigger latent intestinal parasitic infections in stools to emerge. This study investigated whether intestinal parasites can emerge as opportunistic infections in breast and colorectal cancer patients (n=46 and n=15, respectively) undergoing chemotherapy treatment. Breast cancer patients were receiving a 5-fluorouracil/epirubicin/cyclophosphamide (FEC) regimen (6 chemotherapy cycles), and colorectal cancer patients were receiving either an oxaliplatin/5-fluorouracil/folinic acid (FOLFOX) regimen (12 cycles) or a 5-fluorouracil/folinic acid (Mayo) regimen (6 cycles). Patients had Blastocystis hominis and microsporidia infections that were only present during the intermediate chemotherapy cycles. Thus, cancer patients undergoing chemotherapy should be screened repeatedly for intestinal parasites, namely B. hominis and microsporidia, as they may reduce the efficacy of chemotherapy treatments.

[Study of the immunological mechanism of anti-tumor effects of 5-FU by establishing EL4 tumor-bearing mouse models].

AIM: To investigate the immunological mechanism of anti-tumor effect of 5-FU by establishing lymphoma EL4 tumor-bearing mouse models in wild type C57BL/6 mice and nude C57BL/6 mice, respectively. METHODS: The mouse lymphoma EL4 cells were inoculated subcutaneously into wild type C57BL/6 mice (immune-competent mice). Twelve days later, 5-FU of different doses was administered intraperitoneally to treat these wild type C57BL/6 tumor-bearing mice. The size of tumors in the wild type C57BL/6 mice was observed and recorded to explore the minimal dose of 5-FU that could cure the tumor-bearing mice. Then the same amount of EL4 tumor cells was inoculated subcutaneously into wild type C57BL/6 mice and nude C57BL/6 mice (T cell-deficient mice) simultaneously, which had the same genetic background of C57BL/6. Twelve days later, 5-FU of the minimal dose was given intraperitoneally to treat both the wild type and nude C57BL/6 tumor-bearing mice. The size of tumors in the two different types of mice was observed and recorded. RESULTS: A single dose of 5-FU (75 mg/kg) cured both the EL4 tumor-bearing wild type C57BL/6 mice and the EL4 tumor-bearing nude C57BL/6 mice in the first week. Two weeks after 5-FU treatment, all of the nude mice died of tumor relapse while most of the wild type C57BL/6 mice were fully recovered. CONCLUSION: A single dose of 5-FU has marked anti-tumor effects on lymphoma EL4 tumor-bearing C57BL/6 mice with or without T lymphocytes. The relapse of tumors after 5-FU treatment might be related to the function of T lymphocytes.

Efecto inmunomodulador de la solución CM-95 tratada magnéticamente en ratones Balb/c inoculados con 5-fluorouracilo / Immunomodulator effect of the CM-95 Solution treated magnetically in Balb/c mouse with 5- fluorouracil inoculated

El 5-fluorouracilo es un antineoplásico usado en la terapia del cáncer y posee acción inmunosupresora al inhibir la proliferación de células del tejido hematopoyético. En este trabajo se evaluó el efecto inmunomodulador de la solución CM-95 tratada magnéticamente en ratones Balb/c inoculados con 5-fluorouracilo a través de su acción protectora rehabilitadora sobre parámetros celulares y tisulares del tejido hematopoyético. A los ratones, entre 20-22 g de peso y 6 semanas de nacidos, se les administró esta solución CM-95 tratada magnéticamente por vía intraperitoneal en un esquema de dos inoculaciones; luego se administró por la misma vía el 5_fluorouracilo a una dosis de 150 mg/m2 de superficie corporal de cada ratón. Se evaluó el conteo total y diferencial de leucocitos antes de inocular el 5-fluorouracilo, y a los tres y siete días de aplicado este. La celularidad de médula ósea y bazo y la observación microscópica del corte histológico de hígado y bazo por la técnica de inclusión en parafina y tinción con eosina hematoxilina al 10 por ciento se evaluaron a los siete días de haber aplicado el 5- fluorouracilo. La solución CM-95, tratada magnéticamente, logró modular los efectos del 5-fluorouracilo con una actividad protectora rehabiltadora, para los parámetros evaluados en médula ósea, sangre periférica, bazo e hígado. Estos resultados abren nuevas perspectivas para la aplicación del sistema acuoso tratado magnéticamente como inmunomodulador(AU)

5-Fluorouracil is an antineoplastic drug used in cancer chemotherapy. It has immunosuppressant effects by inhibiting cell proliferation from the haematopoietic tissue. In this paper, the immunomodulating effect of the magnetically treated CM-95 solution in Balb/c mice inoculated with 5- fluorouracil by its rehabilitating protecting action on cell parameters of the haematopoietic tissue. Six-week old mice weighing 20-22 g were inoculated twice with magnetically treated CM-95 solution by intraperitoneal route. Then, they received 5-fluoracile in a dose of 150 mg/m2 of body surface by the same route. Haematopoietic parameters such as total and differential leukocyte count were evaluated before applying the antineoplastic drug and at the third and seventh days after the application. Bone marrow and spleen cellularity as well as the microscopic observation of the histological cut of the liver and of spleen, by the technique of inclusion in paraffin dyes with 10 percent eosin haematoxylin, were evaluated at seven days after the application of 5 fluorouracil. The magnetically treated CM-95 solution could modulate the effects of 5-fluorouracil with protecting and rehabilitating activity for the parameters evaluated in the bone marrow, peripheral blood, spleen and liver. These results open new perspectives for the application of the magnetically treated aqueous system as immunomodulator(AU)

The technical problems shoot for the production of anti low molecular weight hapten antibody.

Four kinds of small molecular weight haptens, Desmosine, Adriamycin, 4'-carboxycotinine and 5'-Fluorouracil were used as the immunizing antigens to obtain rabbits polyclonal antisera. All of these haptenes were conjugated to Bovine Serum Albumin (BSA) using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDCl) reagent. The functional amino or/and carboxyl group in hapten molecules was utilized for conjugation with BSA by EDCl reagent. The hapten conjugated with BSA was mixed with Freund's Complete or Incomplete Adjuvant, and immunized to rabbits abdominal subcutaneous regions. More than 3 times immunized rabbit's antisera were evaluated the titer, and the specificity was investigated by ELISA method. The specific antisera against desmosine and adriamycin were produced. Anti-desmosine antibodies had no cross-reaction to molecular structure resemble pyridinoline. Anti-adriamycin antibodies reacted well to adriamycin and it could distinguish the differences of -epi type of adriamycin. The ELISA assay systems used here had such sensitivity. However the specificity possessing antisera against 4'-carboxycotinine and 5-Fluorouracil could not produce even though the antisera titers were elevated. The reasons why and what kinds of problems were there are discussed in this paper. For the production of the specific affinity possessing antisera, the length and distance between the carrier protein and the hapten and the shape of the functional groups were the important factors for production of the specific antisera which recognize free form hapten molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

The antitumor activity and immunosuppressive effects of 5-fluorouracil suppositories in rectal cancer patients.

The antitumor activity and immunological effects of the local administration of 5FU were investigated by determining the tissue concentration of 5FU, histological appearance of the primary tumor, and lymphocyte subsets of the regional lymph nodes in 23 rectal cancer patients. Twelve patients were treated with 5FU suppositories preoperatively, being the 5FU group, while 11 patients were given no preoperative treatment, being the control group. The 5FU concentrations in the primary tumors were higher than those in the regional lymph nodes and appeared to remain high for an extended period. No histological changes peculiar to the 5FU group were observed in the primary tumors. An analysis of the lymphocyte subsets in the pararectal nodes revealed that Leu2a+15- cells, or cytotoxic T lymphocytes, were significantly decreased in numbers in the 5FU group compared to the control group. These results suggest that the local use of 5FU may not only exert an antitumor effect against rectal cancer, but can also cause the suppression of antitumor immunity in the regional lymph nodes.