RU2612090C1 - Method for treatment of locally spread unresectable esophageal cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to oncology, and can be applied for treatment of locally spread unresectable esophageal cancer. For this purpose in conditions of procedure room after processing chest skin with antiseptic solution patients receive intradermally by 0.3 ml of autologous dendritic-cell vaccine, two injections from each side of chest approximately between III and IV, IV and V ribs. Dose constitutes 5,000,000 dendritic cells. 2 weeks after introduction of first vaccine second dendritic-cell vaccine is introduced in the same dose. 2 weeks after introduction of second vaccine third dendritic-cell vaccine is introduced in total dose 10,000,000 dendritic cells. 2 weeks after third introduction of vaccine fourth dendritic-cell vaccine is introduced in total dose 10,000,000 dendritic cells. During the entire course of vaccine therapy patients receive 30,000,000 cells.

EFFECT: invention makes it possible to increase duration and to improve quality of life in patients with locally spread unresectable esophageal cancer.

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Description

The invention relates to medicine, namely to Oncology, and can be used to treat locally advanced unresectable cancer of the esophagus.

In Russia, esophageal cancer is in 14th place in the structure of incidence of malignant neoplasms and in 7th place in the structure of mortality. In 2013, in our country esophageal cancer was registered in 7,403 patients, 6,532 patients died, the ratio of morbidity and mortality was 0.88. Five-year survival is about 10%, regardless of the histological type and degree of tumor differentiation. One of the reasons for the low five-year survival of patients with this nosology is the late diagnosis of esophageal cancer. In most cases (68%), esophageal cancer is diagnosed in stages III-IV (see Davydov M.I., Axel E.M. Statistics of malignant neoplasms in Russia and CIS countries in 2013, M., 2015).

The choice of tactics for treating patients with esophageal cancer depends on the location and level of the location of the tumor in the esophagus, the histological form of the tumor, the stage of the malignant process (see Davydov M.I., Stilidi I.S. Esophageal cancer. M .: Practical medicine, 2007). Currently, surgical, radiation, chemotherapeutic and combined treatment methods are used to treat esophageal cancer.

Surgical treatment is the main one, it is carried out with local and local-regional distribution, as well as for palliative purposes. In an isolated version, the surgical aid is ineffective, since by the time of diagnosis, more than half of patients with esophageal cancer have an unresectable or metastatic process (see Skvortsov MB Esophageal cancer: surgical treatment // Siberian Oncological Journal. - 2011. - No. 4. - C . 21-30).

Radiation therapy and chemotherapy alone are palliative in nature (see Davydov M.I., Stilidi I.S., Ter-Ovanesov M.D., Polotsky B.E. Esophageal cancer: modern approaches to diagnosis and treatment // Russian medical journal. - 2006. - No. 14. - S. 1006-1015). Chemotherapeutic treatment of patients with esophageal cancer is also used to increase the effectiveness of radiation therapy in neoadjuvant and adjuvant modes, however, esophageal tumors have moderate sensitivity to cytostatics. In addition, chemotherapy is often limited by the serious condition of patients (see Moiseenko V.M. Possibilities of modern drug treatment for patients with esophageal cancer // Practical Medicine. - 2003. - T. 4 - No. 2. - P. 83-90).

Regarding targeted therapy for esophageal cancer, a modest effect was achieved with the use of bevacuzumab in combination with capecitabine (see Garin AM, Bazin I.S. Drug treatment of tumors of the gastrointestinal tract // Effective Pharmacotherapy. - 2015. - No. 2. - P. 16-23). The multimodal approach, including neoadjuvant chemoradiation with subsequent surgery, is by far the most effective in the treatment of cancer of the esophagus (see Dengina N.V. Modern therapeutic options for cancer of the esophagus // Practical Oncology. - 2012. - T. 13. - No. 4.S. - 276-288). When using it, an increase in radically operated patients is noted, a slight improvement in three-year survival compared to the surgical method alone (see Urschel JD, Vasan N. A meta-analisis of randomized controlled trials that compared neoadjuvant chemoradiation and surgery alone for resectable esophageal cancer // Am. J. Surg. - 2003. - Vol. 185. - No. 3. - P. 538-543 .; Van Hagen P. Hulshof MC, van Lanschot JJ et al. Preoperative chemoradiotherapy for esophageal or junctional cancer // N. Engl. J. Med. - 2012. - Vol. 366. - No. 22. - P. 2074-2084.).

Nevertheless, in patients with esophageal cancer there is a consistently low long-term survival rate in both developed and developing countries — 17% (see Siegel R., Ward E., Brawley O., Jenal A. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths // SA Cancer J. Clin. - 2011. - Vol. 61. - No. 4. - P. 212-236). The lethal outcome is due to local relapses and metastasis: during the first year from the moment of diagnosis, up to 65% of patients die, which puts esophageal cancer in first place for one-year mortality (see Skvortsov MB Esophageal cancer: surgical treatment // Siberian Oncology Journal . - 2011. - No. 4. - S. 21-30). Thus, the aggressive course of tumors of the esophagus, unsatisfactory treatment results dictate the need to find new approaches to the treatment of this severe oncopathology.

From patent sources known "Method for the treatment of cancer of the esophagus" (see patent RU 2474420 C1, publ. 02/10/2013, Bull. No. 4), in which in order to increase the effectiveness of the treatment, reduce the incidence of complications and their severity, reduce general toxicity caused by systemic use of cytostatics, in particular 5-fluorouracil, maintaining the quality of life of patients, 5-fluorouracil is administered intravenously 2 times a week at 300 mg / m ² for one administration with an interval of 2-3 days and simultaneously Coletex-gel is administered orally -DNA-L "3 times a day for 10-15 ml. On the remaining days of the week, the hydrogel material Coletex-5-Ftur hydrogel material is orally administered in a volume of 5 ml once a day, moreover, 10-15 minutes prior to the administration of the gel material, an additional administration of Coletex-gel-DNA-L is prescribed the volume of 10-15 ml. The duration of treatment is 4 weeks.

Also known is a "Method for the treatment of locally advanced inoperable cancer of the esophagus" (see patent RU 2468836 C1, publ. 10.12.2012, Bull. No. 34), including preliminary argon plasma coagulation of the tumor, intraluminal brachytherapy of high power in the 1st, 8th and 15th days of the course with a single focal dose of 7 Gy synchronously with intravenous injections of taxane and carboplatin preparations, subsequent remote irradiation of the tumor, ROD 1.8 Gy, 5 fractions / wk, SOD 40-45 Gy, characterized in that the remote irradiation of the tumor is accompanied application of zvodnyh fluoropyrimidine depending on the degree of dysphagia in the following doses: the degree of dysphagia 0-1 - oral capecitabine 600 mg / m ² during the radiation treatment, with dysphagia 2-3 degree - intravenous infusion of 5-fluorouracil 1000 mg / m ² weekly. The method provides optimization of the cytoreductive interaction of the components of chemoradiotherapy and a more complete implementation of the tumor response while reducing severe general and local reactions.

However, not all patients are shown radiation and chemotherapy. Contraindications are, in addition to the prevalence of the process, the somatic state of patients and concomitant pathology.

Authors Yu.A. Grinevich, V.L. Ganul et al. In the article “Results of thymus polypeptides in the combined treatment of patients with cancer of the esophagus or cancer of the stomach with the transition to the esophagus” (see the journal “Oncology”, T. 3, No. 4, 2001, pp. 278-282) data on the study of the clinical efficacy of thymus polypeptides (PTs) in the combined treatment of patients with cancer of the esophagus or cancer of the stomach with the transition to the esophagus are presented. In particular, the inclusion of PT in the active nonspecific immunotherapy regimen improved 5-year survival rates. The authors also found that adaptive immunotherapy in the form of autologous bone marrow transplantation preincubated with PT in vitro does not affect the effectiveness of the combined treatment of patients with esophageal or gastric cancer with passage to the esophagus.

From the literature it is known the use of an immunocorrector - Transfer factor ™ in the form of monotherapy and as part of a comprehensive treatment of cancer of the esophagus (see Pinaev S.K., Pinaeva O.G., Chizhov A.Ya. First experience in the treatment of cancer of the esophagus using immunocorrector Transfer Factor ™ // Technologies of living systems. - 2014. - No. 4, - S. 59-62.). The authors cite the first two observations of the use of TF drugs in cancer of the esophagus. In one case of a patient with cancer of the lower third of the esophagus T1-2N0M0 due to the refusal of special treatment due to the presence of severe concomitant pathology, TF monotherapy was performed. After two months of treatment, complete tumor regression was achieved, confirmed by fibroesophagogastroduodenoscopy (FEGDS). Subsequently, after 4 months, local tumor progression developed without distant metastasis. Life expectancy was 26 months from the date of diagnosis. In the second observation, a patient with a cancer of the lower third of the esophagus T 1N0M0 TF therapy was carried out against the background of remote radiation therapy. Two months later, complete regression of the tumor, confirmed by FEGDS, was also achieved. Subsequently, the patient, according to dynamic monitoring with regular monitoring, including FEGDS, maintained stable remission for two years (in August 2012, at the age of 76, the patient died from myocardial infarction). Thus, a pronounced therapeutic effect of TF in esophageal cancer was noted, comparable with chemoradiotherapy. At the same time, the combination of immunotherapy with TF drugs and radiation therapy has a synergy effect and allows achieving complete persistent regression of the esophagus tumor. A proposal has been made when regression of “incurable” cancer of the esophagus against the background of TF drugs is returned to the question of the possibility of special treatment. Suggestions were also made to expand such studies in order to obtain representative data on the place and importance of TF drugs in programs for the comprehensive treatment of esophageal cancer.

The technical result of the invention is the development of a method for the treatment of patients with locally advanced unresectable cancer of the esophagus, which allows to increase the duration and improve the quality of life.

The technical result is achieved in that in the conditions of the treatment room after treatment of the skin of the anterior chest wall with an antiseptic solution, 0.3 ml of autologous dendritic-cell vaccine is administered intradermally, two injections on each side of the sternum approximately between the III and IV, IV and V ribs in this way that in total reach 5,000,000 dendritic cells, 2 weeks after the first dendritic cell vaccine is administered, the second dendritic cell vaccine is administered in the same dosage of 5,000,000 cells, 2 weeks after the second vaccine is administered the third dendritic cell vaccine in a total dosage of 10,000,000 dendritic cells, 2 weeks after the third administration of the dendritic cell vaccine, the fourth dendritic cell vaccine in a total dosage of 10,000,000 dendritic cells is administered in such a way that for the entire course of vaccine therapy patients receive 30,000,000 cells.

The method is as follows.

Peripheral venous blood was collected in sterile 50 ml tubes containing an anticoagulant (K ₃ - EDTA) and diluted with an equal volume of RPMI-1640 growth medium (Paneko, Russia). Then 10 ml of the resulting suspension was layered on 3 ml of Ficoll-Paque Premium and centrifuged in 15 ml sterile tubes at 1500 rpm for 40 min at room temperature (see Boyum A. Separation of leukocytes from blood and bone marrow // Scand. J. Clin. Lab. Investig. - 1968. - Vol. 21 - Suppl. 97. p. 1-9).

After centrifugation, erythrocytes and granulocytes remained at the bottom of the tube, and mononuclear cells (MNCs) remained at the interface. The transparent layer of the medium located directly above the opalescent layer containing the MNCs was removed, and the MNCs were collected over the entire cross-sectional area of the tube, achieving purification from other cells.

A suspension of MNCs was introduced into sterile 15 ml centrifuge tubes, diluted with a 4-fold excess of incomplete RPMI-1640 nutrient medium and carefully resuspended. MNCs were washed in incomplete RPMI-1640 nutrient medium by double centrifugation at 1000 rpm for 10 min.

Then, counting and assessment of the viability of MNCs were performed using the Goryaev camera and 0.4% trypan blue, 3.5-7 × 10 ⁶ or more cells were obtained with a viability of at least 98%.

To obtain DC from peripheral blood MNCs, the latter were placed in incomplete nutrient medium RPMI-1640 (inoculative dose of 1.5 × 10 ⁶ cells / ml) and 2 flat-bottomed culture bottles of 75 cm ² with vented plugs for growing adhesive cultures. Incubated under conditions of controlled 5% CO ₂ and 98% humidity at 37 ° C for 2 hours.

Then, RPMI-1640 medium containing non-adherent cells (mainly lymphocytes) was removed and re-washed with 4 ml of RPMI-1640 medium remaining on the cell substrate.

15 ml of CellGroDC medium was added to the culture bottles, growth factors and differentiation factors: GM-CSF (72 ng / ml) and IL-4 (20 ng / ml).

Culture bottles were incubated in a CO ₂ incubator.

On the 3rd and 5th day of cultivation, a fresh portion of GM-CSF (72 ng / ml) and IL-4 (20 ng / ml) were added.

On the 7th day of cultivation, growth factors and differentiation factors GM-CSF (72 ng / ml), IL-4 (20-45 ng / ml) and TNF-α (20 ng / ml), as well as cellular HeLa cell culture lysate at the rate of 3 HeLa culture cells per 1 dendritic cell.

To prepare a lysate containing HeLa culture antigens, cells were cultured in vials in complete DMEM / F12 nutrient medium with 10% calf fetal serum supplemented with glutamine and gentamicin (50 μg / ml) under conditions of controlled 5% CO ₂ and 98% humidity at 37 ° C.

After reaching the confluent monolayer, the cells were reseeded and further cultured for a week.

After receiving a sufficient amount of cell mass, culture medium was taken from the vials, HeLa culture cells were removed from the substrate using a trypsin-Versen solution and resuspended to obtain a homogeneous cell suspension.

Cells were counted in the Goryaev chamber and the percentage of viable cells was determined by staining with 0.4% trypan blue.

To obtain a cell lysate, six consecutive cycles of instant freezing of a suspension of cells in liquid nitrogen and thawing to room temperature in phosphate-buffered saline without cryoprotectant were performed (the quality of cell lysis was controlled using 0.4% trypan blue).

Subsequently, cell detritus was precipitated by centrifugation (10 min, 3000 rpm, 20 ° C) and the supernatant was filtered through a sterile filter (pore diameter 0.2 μm).

The tumor lysate obtained from the HeLa culture was stored at −20 ° C. until use.

48 hours after the addition of the HeLa culture lysate, the culture medium was taken, centrifuged, and the supernatant was removed. 3 ml of trypsin-Versen solution was added to each vial and incubated in a CO ₂ incubator for 5 min.

Then the cells were removed with a scraper, precipitated by centrifugation (10 min, 1000 rpm, 20 ° C), the supernatant was removed. Cells from two vials were combined with the obtained cells from the supernatant by resuspending in incomplete RPMI 1640 medium (final volume 10 ml).

An aliquot of the cell suspension equal to 0.1 ml was taken and the cells were counted in the Goryaev chamber and the percentage of viable cells was determined by staining with 0.4% trypan blue.

Then 200,000 cells were selected to determine the immunophenotype of mature DCs on a flow cytometer, which was performed by isolating MNC-DCs on the population graphs and purifying it from debris and lymphocytes using CD45, CD3, CD4, CD8, CD16 + 56, CD19 tags.

The maturation stage of the mononuclear cells was evaluated using antibodies to CD1a, CD11c, CD14, CD33, CD38, CD83, CD86, HLA-DR.

The required number of dendritic vaccine cells was precipitated by centrifugation, washed in 10 ml of a 0.9% sodium chloride solution containing 10% human albumin, and counting and viability were performed using an automatic cell counter “Countess” and 0.4% trypan blue.

Under the above conditions, cells were obtained with a viability of at least 98% and an immunophenotype of mature DCs (CD14-, CD1alow, CD83high, HLA-DRhigh, CD80high, CD86high, CCR7high).

To administer DC vaccine, 1/2 cells (5 to 10 × 10 ⁶ ) resuspended in 1.5 ml of 0.9% isotonic NaCl solution containing 10% human albumin were transferred to an ampoule and delivered to the clinical unit for administration to the patient.

Another 1/2 DC was cryopreserved and stored in liquid nitrogen until the next vaccination.

Treatment begins with the introduction of the first dendritic cell vaccine in the abdominal treatment room, after treatment of the skin of the anterior chest wall with an antiseptic solution, 0.3 ml of autologous dendritic cell vaccine is administered intradermally, two injections on each side of the sternum approximately between III and IV, IV and V ribs in such a way that a total of 5,000,000 dendritic cells are reached.

Visually, a reaction is observed in the form of papules, slight hyperemia at the injection site. Subfebrile body temperature is noted within two days after the introduction of the vaccine. Allergic reactions, as well as other undesirable side effects, have not been recorded.

2 weeks after the administration of the first dendritic cell vaccine, the second dendritic cell vaccine was administered according to the same scheme, at the same dosage of 5.000.000 cells.

In just two sessions of vaccine therapy, patients receive 10,000,000 cells.

2 weeks after the introduction of the second vaccine, the third dendritic cell vaccine is administered according to the same scheme in a total dosage of 10,000,000 dendritic cells. Visually, a reaction is observed in the form of papules, slight hyperemia at the injection site. Subfebrile body temperature is noted within two days after the introduction of the vaccine.

Allergic reactions, as well as other undesirable side effects, have not been recorded.

2 weeks after the third administration of the dendritic cell vaccine, the fourth dendritic cell vaccine is administered in a total dosage of 10,000,000 dendritic cells. In just 3 and 4 sessions of vaccine therapy, patients receive 20,000,000 cells.

For the entire course of vaccine therapy, patients received 30,000,000 cells.

For a better understanding of the essence of the claimed invention, as well as to confirm its compliance with the condition "industrial applicability", we give an example of a specific implementation of the method.

Example No. 1. Patient P., born in 1937

July 15, 2015, she turned to the polyclinic of the Research Institute of Nuclear Research, where during the examination she was diagnosed with Cancer with / 3 of the esophagus St IV (T3N1M1) (LYM), cl. column 2. Dysphagia III Art. ". Histological analysis (GA) - squamous keratinizing cancer. Concomitant pathology: IHD, hypertension 2 tbsp., The risk is high.

SKRT OGK (chest organs), BP (abdominal cavity) and OMT (pelvic organs) from 07.15.2015: infiltrative cancer of the middle and lower thoracic esophagus for 10 cm narrows its lumen. L / a posterior mediastinum up to 2.5 cm.

FES dated 07.15.2015. The mouth of the esophagus 17 cm from the incisors. At 29 cm, a circular fine-tuberous infiltration of the mucosa is determined. At 39 cm, the lumen of the esophagus is significantly narrowed, it is difficult to pass for the endoscope due to ulceration with raised edges. Cardia 41 cm, elastic. Gatekeeper, KDP without features.

07/16/2015, endoscopic stenting of the area of tumor stenosis was performed. Moved satisfactorily.

On July 17, 2015, after treatment of the skin of the anterior chest wall with an antiseptic solution, 0.3 ml of autologous dendritic-cell vaccine was injected intradermally between approximately the III and IV, IV and V ribs, two injections on each side of the sternum in a manner that totals 5.000. 000 dendritic cells.

07/31/2015, the second dendritic cell vaccine is administered in the same dosage of 5.000.000 cells.

On August 14, 2015, the third dendritic cell vaccine was administered in a total dosage of 10,000,000 dendritic cells.

On August 28, 2015, the fourth dendritic cell vaccine was administered in a total dosage of 10,000,000 dendritic cells.

Thus, for the entire course of vaccine therapy, the patient received 30,000,000 cells.

The patient tolerated the introduction of vaccines satisfactorily. No local reactions were noted. After 2-3 days from the introduction, an increase in body temperature to 37.1-37.2 ° C was noted. The temperature stopped on their own.

SKRT OGK, BP and OMT from 08/31/2015: tumor with / 3 of the esophagus over 7.5 cm, the condition after stenting. Thoracic lymph nodes are not enlarged.

Today (after 6 months), the patient feels satisfactory, eats on her own, notes the normal passage of solid food through the esophagus.

This method was treated 5 patients. The method allows to increase the duration and improve the quality of life.

The invention is industrially applicable, can be repeatedly reproduced in medical institutions of oncological profile, providing high-tech assistance.

Claims (1)

A method of treating locally unresectable esophageal cancer, which consists in the fact that in the treatment room after treatment of the skin of the anterior chest wall with an antiseptic solution, 0.3 ml of autologous dendritic-cell vaccine is administered intradermally, two injections on each side of the sternum approximately between III and IV, IV and V ribs in such a way that they reach 5.000.000 dendritic cells in total, 2 weeks after the first dendritic cell vaccine is administered, the second dendritic cell vaccine is administered in the same dosage of 5.000.000 glue ok, 2 weeks after the second vaccine is administered, the third dendritic cell vaccine is introduced in a total dosage of 10,000,000 dendritic cells, 2 weeks after the third administration of the dendritic cell vaccine the fourth dendritic cell vaccine in a total dosage of 10,000,000 is administered dendritic cells in such a way that for the entire course of vaccine therapy, patients receive 30,000,000 cells.