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N. N. Petrov Oncology Scientific-research InstituteN. N. Petrov Oncology Scientific-research Institute
I.I. Mechnikov Northwest State Medical UniversityI.I. Mechnikov Northwest State Medical University
NEOADJUVANT PHOTODYNAMIC THERAPY IN THENEOADJUVANT PHOTODYNAMIC THERAPY IN THE
SURGICAL TREATMENT OF PATIENTS WITHSURGICAL TREATMENT OF PATIENTS WITH
CUTANEOUS MELANOMACUTANEOUS MELANOMA
  
Moscow 2013Moscow 2013
Head:
M.D., Professor G. I. Gafton
Rapporteur:
M.Y. Myasnyankin
BackgroundBackground
 The significant increase in the morbidity rate of theThe significant increase in the morbidity rate of the
population of melanoma of the skin, as well as thepopulation of melanoma of the skin, as well as the
delay in the diagnosis of primary tumor dictates thedelay in the diagnosis of primary tumor dictates the
need to develop new methods of systemneed to develop new methods of system
neoadjuvant the treatment of patients.neoadjuvant the treatment of patients.
 In this respect, a promising, it is the use ofIn this respect, a promising, it is the use of
neoadjuvant photodynamic therapy (PDT) inneoadjuvant photodynamic therapy (PDT) in
patients with melanoma of the skin.patients with melanoma of the skin.
AimAim
 To study of the impact of neoadjuvant PDT onTo study of the impact of neoadjuvant PDT on
the indices of T - and B-cellar immune answer inthe indices of T - and B-cellar immune answer in
the surgical treatment of patients with melanomathe surgical treatment of patients with melanoma
of the skin of the I-III stage.of the skin of the I-III stage.
Objectives of the studyObjectives of the study
 To study the induction of apoptosis melanomaTo study the induction of apoptosis melanoma
cells of human skin under the influence of PDTcells of human skin under the influence of PDT
in the experiment in vitroin the experiment in vitro
 Evaluate the impact of operations with orEvaluate the impact of operations with or
without PDT on the immunocompetent cells ofwithout PDT on the immunocompetent cells of
peripheral blood in patients with localizedperipheral blood in patients with localized
melanoma skinmelanoma skin
Scientific innovationScientific innovation
In this workIn this work ::
 developed an original technique of neoadjuvant PDT indeveloped an original technique of neoadjuvant PDT in
the treatment of localized cutaneous melanomathe treatment of localized cutaneous melanoma
(prepared by the application for an invention)(prepared by the application for an invention)
 explore various modes of FD-effects of usingexplore various modes of FD-effects of using
photoditazini in experiments in vitrophotoditazini in experiments in vitro
 studied immunological parameters in patients withstudied immunological parameters in patients with
localized form of melanoma of the skin, receivinglocalized form of melanoma of the skin, receiving
neoadjuvant photodynamic therapy and surgicalneoadjuvant photodynamic therapy and surgical
treatmenttreatment
Research designResearch design
In accordance with the objectives of the research,In accordance with the objectives of the research,
the work was divided into two parts:the work was divided into two parts:
experimentalexperimental andand clinicalclinical
Method of experimental studiesMethod of experimental studies
 For experimental in vitro studies using a cellularFor experimental in vitro studies using a cellular
line melanoma of a skin of the person (Mel 226).line melanoma of a skin of the person (Mel 226).
..
 The level of apoptosis was assessed by theThe level of apoptosis was assessed by the
apoptosis index (AI)apoptosis index (AI)::
the number of apoptotic cellsthe number of apoptotic cells
AI = ________________________ х 100%AI = ________________________ х 100%
total number of cellstotal number of cells
AI considered separately for all the analyzed cells:AI considered separately for all the analyzed cells:
  
cells with a green staining, which are at the stage of earlycells with a green staining, which are at the stage of early
apoptosis (Annexin+, PI -), (1a);apoptosis (Annexin+, PI -), (1a);
cells with double staining (Annexin+, PI+) - late stage ofcells with double staining (Annexin+, PI+) - late stage of
apoptosis (1b)apoptosis (1b)
Fig. 1. The cells at the stage of «early» apoptosis (1A) and «late» apoptosis (1B)Fig. 1. The cells at the stage of «early» apoptosis (1A) and «late» apoptosis (1B)а b
Methodology of clinical studiesMethodology of clinical studies
 In a clinical study included patients whoIn a clinical study included patients who
received treatment in «Oncology researchreceived treatment in «Oncology research
Institute. N.N. Petrov» of the Ministry of healthInstitute. N.N. Petrov» of the Ministry of health
of Russia from July 2012 and January 2013of Russia from July 2012 and January 2013
..
 The scheme of the study:The scheme of the study:
1)1) Evaluation of IP → Operation → 7 days:Evaluation of IP → Operation → 7 days:
evaluation of IPevaluation of IP
2)2) Evaluation of IP → PDT → Operation → 7 days:Evaluation of IP → PDT → Operation → 7 days:
evaluation of IPevaluation of IP
3)3) IP - immune statusIP - immune status
Characteristics of patientsCharacteristics of patients
Characteristics
Pathiens Total
1 2
abs. % (ДИ) abs. % (ДИ) abs. %
number of patients 9 16 25 100
Age
average
interval
54
39-87
53
34-85
56
34-87
men
women
4
5
44
(12-77)
56
(23-88)
3
13
19
(4-41)
81
(59-96)
7
18
28
(10-46)
72
(54-90)
Stagies
I
II
III
5
2
2
56 (23-88)
22 (3-53)
22 (3-53)
10
6
0
63 (39-86)
37 (14-61)
0 (0-6)
15
8
2
60 (41-79)
32 (14-50)
8 (1-22)
..
Methods of statistical processingMethods of statistical processing
of the dataof the data
 For statistical processing of the data usedFor statistical processing of the data used
methods of descriptive statistics: the calculationmethods of descriptive statistics: the calculation
of the 95% confidence interval (CI) for AI andof the 95% confidence interval (CI) for AI and
the criterion of characters Уилкоксона forthe criterion of characters Уилкоксона for
dependent observationsdependent observations
 Calculations have been carried out using theCalculations have been carried out using the
SPSS v.17. The study was accepted significanceSPSS v.17. The study was accepted significance
level p≤0,05level p≤0,05
The resultsThe results (1)(1)
Fig.2. AI in 1 hour after PD-exposure exposure 6 min. (a) and 10 min. (b):Fig.2. AI in 1 hour after PD-exposure exposure 6 min. (a) and 10 min. (b):
the early and the late apoptosisthe early and the late apoptosis
а б
The resultsThe results (2)(2)
Fig.3. AI 4 hours after the PD-exposure exposure 6 min. (a) and 10 min.Fig.3. AI 4 hours after the PD-exposure exposure 6 min. (a) and 10 min.
(b): the early and the late apoptosis(b): the early and the late apoptosis
а б
Evaluation of the undesirableEvaluation of the undesirable
phenomena PDTphenomena PDT
 Of the complications and adverse events inOf the complications and adverse events in
patients receiving neoadjuvant PDT, there was apatients receiving neoadjuvant PDT, there was a
local hyperemia in the «zone illumination»local hyperemia in the «zone illumination»
The content of the main subpopulations of lymphocytes in the blood ofThe content of the main subpopulations of lymphocytes in the blood of
patients with localized melanoma of the skin prior to treatment, and the 7-patients with localized melanoma of the skin prior to treatment, and the 7-
th day after operationth day after operation
Lymphocytic gate Group Before the
operation
M±m
After the operation
M±m
р
CD3+
CD19-
PDT(-) 1.32±0.16 1.50±0.6 0.036
PDT(+) 1.2±0.09 1.3±0.4 0.18
CD3+
CD4+
PDT(-) 0.83±0.1 0.95±0.4 0.092
PDT(+) 0.72±0.05 0.91±0.3 0.02
CD3+
CD8+
PDT(-) 0.46±0.09 0.48±0.24 0.263
PDT(+) 0.47±0.06 0.47±0.2 0.19
CD3+
CD4+
CD8+
PDT(-) 0.02±0.01 0.02±0.02 0.063
PDT(+) 0.02±0.01 0.03±0.04 0.75
CD3-
CD19+
PDT(-) 0.18±0.04 0.23±0.1 0.31
PDT(+) 0.16±0.01 0.19±0.08 0.02
CD3+
CD4+
HLADR+
PDT(-) - - -
PDT(+) 0.03±0.004 0.05±0.009 0.05
ConclusionsConclusions (1)(1)
 The increase of concentration of photoditaziniThe increase of concentration of photoditazini
does not lead to an increase in the number ofdoes not lead to an increase in the number of
cells at early stage of apoptosis (p>0.05).cells at early stage of apoptosis (p>0.05).
ConclusionsConclusions (2)(2)
 The prolonging exposure time leads to increaseThe prolonging exposure time leads to increase
in the late forms of apoptosis (p<0.05)in the late forms of apoptosis (p<0.05)
Conclusions (3)Conclusions (3)
 The use of photoditazini in the dose of 50 mgThe use of photoditazini in the dose of 50 mg
followed by irradiation (662 nm, 400 j) 2 days priorfollowed by irradiation (662 nm, 400 j) 2 days prior
to surgical intervention contributes to theto surgical intervention contributes to the
activation of T - and b-cellular link of the immuneactivation of T - and b-cellular link of the immune
system (p<0.05)system (p<0.05)
The main task of any medical practiceThe main task of any medical practice
is the memory of what medicineis the memory of what medicine
should serve the benefit of the sickshould serve the benefit of the sick
people.people.
«Вопросы хирургической«Вопросы хирургической
деонтологии»деонтологии»
Н.Н.Петров, 1956 г.Н.Н.Петров, 1956 г.
M.D., Professor G. I. GaftonM.D., Professor G. I. Gafton
Senior laboratory assistant of the Department ofSenior laboratory assistant of the Department of
OncologyOncology
1976-19811976-1981
1 University named after academician I. P.1 University named after academician I. P.
Pavlova, Leningrad, St. L. TolstoyPavlova, Leningrad, St. L. Tolstoy
Junior researcherJunior researcher
1981-19911981-1991
1 University named after academician I. P.1 University named after academician I. P.
Pavlova, Leningrad, St. L. TolstoyPavlova, Leningrad, St. L. Tolstoy
Senior researchSenior research
officerofficer
1991-20031991-2003
1 University named after academician I. P.1 University named after academician I. P.
Pavlova, Leningrad, St. L. TolstoyPavlova, Leningrad, St. L. Tolstoy
Department headDepartment head
a total of Oncology and urologya total of Oncology and urology
from 2003 - «scientific research Institute offrom 2003 - «scientific research Institute of
Oncology named N.N. Petrov» of the Ministry ofOncology named N.N. Petrov» of the Ministry of
health of Russia, St. Petersburg, str. Leningrad,health of Russia, St. Petersburg, str. Leningrad,
etc. 68etc. 68

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NEOADJUVANT PHOTODYNAMIC THERAPY IN THE SURGICAL TREATMENT OF PATIENTS WITH CUTANEOUS MELANOMA

  • 1. N. N. Petrov Oncology Scientific-research InstituteN. N. Petrov Oncology Scientific-research Institute I.I. Mechnikov Northwest State Medical UniversityI.I. Mechnikov Northwest State Medical University NEOADJUVANT PHOTODYNAMIC THERAPY IN THENEOADJUVANT PHOTODYNAMIC THERAPY IN THE SURGICAL TREATMENT OF PATIENTS WITHSURGICAL TREATMENT OF PATIENTS WITH CUTANEOUS MELANOMACUTANEOUS MELANOMA    Moscow 2013Moscow 2013 Head: M.D., Professor G. I. Gafton Rapporteur: M.Y. Myasnyankin
  • 2. BackgroundBackground  The significant increase in the morbidity rate of theThe significant increase in the morbidity rate of the population of melanoma of the skin, as well as thepopulation of melanoma of the skin, as well as the delay in the diagnosis of primary tumor dictates thedelay in the diagnosis of primary tumor dictates the need to develop new methods of systemneed to develop new methods of system neoadjuvant the treatment of patients.neoadjuvant the treatment of patients.  In this respect, a promising, it is the use ofIn this respect, a promising, it is the use of neoadjuvant photodynamic therapy (PDT) inneoadjuvant photodynamic therapy (PDT) in patients with melanoma of the skin.patients with melanoma of the skin.
  • 3. AimAim  To study of the impact of neoadjuvant PDT onTo study of the impact of neoadjuvant PDT on the indices of T - and B-cellar immune answer inthe indices of T - and B-cellar immune answer in the surgical treatment of patients with melanomathe surgical treatment of patients with melanoma of the skin of the I-III stage.of the skin of the I-III stage.
  • 4. Objectives of the studyObjectives of the study  To study the induction of apoptosis melanomaTo study the induction of apoptosis melanoma cells of human skin under the influence of PDTcells of human skin under the influence of PDT in the experiment in vitroin the experiment in vitro  Evaluate the impact of operations with orEvaluate the impact of operations with or without PDT on the immunocompetent cells ofwithout PDT on the immunocompetent cells of peripheral blood in patients with localizedperipheral blood in patients with localized melanoma skinmelanoma skin
  • 5. Scientific innovationScientific innovation In this workIn this work ::  developed an original technique of neoadjuvant PDT indeveloped an original technique of neoadjuvant PDT in the treatment of localized cutaneous melanomathe treatment of localized cutaneous melanoma (prepared by the application for an invention)(prepared by the application for an invention)  explore various modes of FD-effects of usingexplore various modes of FD-effects of using photoditazini in experiments in vitrophotoditazini in experiments in vitro  studied immunological parameters in patients withstudied immunological parameters in patients with localized form of melanoma of the skin, receivinglocalized form of melanoma of the skin, receiving neoadjuvant photodynamic therapy and surgicalneoadjuvant photodynamic therapy and surgical treatmenttreatment
  • 6. Research designResearch design In accordance with the objectives of the research,In accordance with the objectives of the research, the work was divided into two parts:the work was divided into two parts: experimentalexperimental andand clinicalclinical
  • 7. Method of experimental studiesMethod of experimental studies  For experimental in vitro studies using a cellularFor experimental in vitro studies using a cellular line melanoma of a skin of the person (Mel 226).line melanoma of a skin of the person (Mel 226).
  • 8. ..  The level of apoptosis was assessed by theThe level of apoptosis was assessed by the apoptosis index (AI)apoptosis index (AI):: the number of apoptotic cellsthe number of apoptotic cells AI = ________________________ х 100%AI = ________________________ х 100% total number of cellstotal number of cells
  • 9. AI considered separately for all the analyzed cells:AI considered separately for all the analyzed cells:    cells with a green staining, which are at the stage of earlycells with a green staining, which are at the stage of early apoptosis (Annexin+, PI -), (1a);apoptosis (Annexin+, PI -), (1a); cells with double staining (Annexin+, PI+) - late stage ofcells with double staining (Annexin+, PI+) - late stage of apoptosis (1b)apoptosis (1b) Fig. 1. The cells at the stage of «early» apoptosis (1A) and «late» apoptosis (1B)Fig. 1. The cells at the stage of «early» apoptosis (1A) and «late» apoptosis (1B)а b
  • 10. Methodology of clinical studiesMethodology of clinical studies  In a clinical study included patients whoIn a clinical study included patients who received treatment in «Oncology researchreceived treatment in «Oncology research Institute. N.N. Petrov» of the Ministry of healthInstitute. N.N. Petrov» of the Ministry of health of Russia from July 2012 and January 2013of Russia from July 2012 and January 2013
  • 11. ..  The scheme of the study:The scheme of the study: 1)1) Evaluation of IP → Operation → 7 days:Evaluation of IP → Operation → 7 days: evaluation of IPevaluation of IP 2)2) Evaluation of IP → PDT → Operation → 7 days:Evaluation of IP → PDT → Operation → 7 days: evaluation of IPevaluation of IP 3)3) IP - immune statusIP - immune status
  • 12. Characteristics of patientsCharacteristics of patients Characteristics Pathiens Total 1 2 abs. % (ДИ) abs. % (ДИ) abs. % number of patients 9 16 25 100 Age average interval 54 39-87 53 34-85 56 34-87 men women 4 5 44 (12-77) 56 (23-88) 3 13 19 (4-41) 81 (59-96) 7 18 28 (10-46) 72 (54-90) Stagies I II III 5 2 2 56 (23-88) 22 (3-53) 22 (3-53) 10 6 0 63 (39-86) 37 (14-61) 0 (0-6) 15 8 2 60 (41-79) 32 (14-50) 8 (1-22) ..
  • 13. Methods of statistical processingMethods of statistical processing of the dataof the data  For statistical processing of the data usedFor statistical processing of the data used methods of descriptive statistics: the calculationmethods of descriptive statistics: the calculation of the 95% confidence interval (CI) for AI andof the 95% confidence interval (CI) for AI and the criterion of characters Уилкоксона forthe criterion of characters Уилкоксона for dependent observationsdependent observations  Calculations have been carried out using theCalculations have been carried out using the SPSS v.17. The study was accepted significanceSPSS v.17. The study was accepted significance level p≤0,05level p≤0,05
  • 14. The resultsThe results (1)(1) Fig.2. AI in 1 hour after PD-exposure exposure 6 min. (a) and 10 min. (b):Fig.2. AI in 1 hour after PD-exposure exposure 6 min. (a) and 10 min. (b): the early and the late apoptosisthe early and the late apoptosis а б
  • 15. The resultsThe results (2)(2) Fig.3. AI 4 hours after the PD-exposure exposure 6 min. (a) and 10 min.Fig.3. AI 4 hours after the PD-exposure exposure 6 min. (a) and 10 min. (b): the early and the late apoptosis(b): the early and the late apoptosis а б
  • 16. Evaluation of the undesirableEvaluation of the undesirable phenomena PDTphenomena PDT  Of the complications and adverse events inOf the complications and adverse events in patients receiving neoadjuvant PDT, there was apatients receiving neoadjuvant PDT, there was a local hyperemia in the «zone illumination»local hyperemia in the «zone illumination»
  • 17. The content of the main subpopulations of lymphocytes in the blood ofThe content of the main subpopulations of lymphocytes in the blood of patients with localized melanoma of the skin prior to treatment, and the 7-patients with localized melanoma of the skin prior to treatment, and the 7- th day after operationth day after operation Lymphocytic gate Group Before the operation M±m After the operation M±m р CD3+ CD19- PDT(-) 1.32±0.16 1.50±0.6 0.036 PDT(+) 1.2±0.09 1.3±0.4 0.18 CD3+ CD4+ PDT(-) 0.83±0.1 0.95±0.4 0.092 PDT(+) 0.72±0.05 0.91±0.3 0.02 CD3+ CD8+ PDT(-) 0.46±0.09 0.48±0.24 0.263 PDT(+) 0.47±0.06 0.47±0.2 0.19 CD3+ CD4+ CD8+ PDT(-) 0.02±0.01 0.02±0.02 0.063 PDT(+) 0.02±0.01 0.03±0.04 0.75 CD3- CD19+ PDT(-) 0.18±0.04 0.23±0.1 0.31 PDT(+) 0.16±0.01 0.19±0.08 0.02 CD3+ CD4+ HLADR+ PDT(-) - - - PDT(+) 0.03±0.004 0.05±0.009 0.05
  • 18. ConclusionsConclusions (1)(1)  The increase of concentration of photoditaziniThe increase of concentration of photoditazini does not lead to an increase in the number ofdoes not lead to an increase in the number of cells at early stage of apoptosis (p>0.05).cells at early stage of apoptosis (p>0.05).
  • 19. ConclusionsConclusions (2)(2)  The prolonging exposure time leads to increaseThe prolonging exposure time leads to increase in the late forms of apoptosis (p<0.05)in the late forms of apoptosis (p<0.05)
  • 20. Conclusions (3)Conclusions (3)  The use of photoditazini in the dose of 50 mgThe use of photoditazini in the dose of 50 mg followed by irradiation (662 nm, 400 j) 2 days priorfollowed by irradiation (662 nm, 400 j) 2 days prior to surgical intervention contributes to theto surgical intervention contributes to the activation of T - and b-cellular link of the immuneactivation of T - and b-cellular link of the immune system (p<0.05)system (p<0.05)
  • 21. The main task of any medical practiceThe main task of any medical practice is the memory of what medicineis the memory of what medicine should serve the benefit of the sickshould serve the benefit of the sick people.people. «Вопросы хирургической«Вопросы хирургической деонтологии»деонтологии» Н.Н.Петров, 1956 г.Н.Н.Петров, 1956 г.
  • 22. M.D., Professor G. I. GaftonM.D., Professor G. I. Gafton Senior laboratory assistant of the Department ofSenior laboratory assistant of the Department of OncologyOncology 1976-19811976-1981 1 University named after academician I. P.1 University named after academician I. P. Pavlova, Leningrad, St. L. TolstoyPavlova, Leningrad, St. L. Tolstoy Junior researcherJunior researcher 1981-19911981-1991 1 University named after academician I. P.1 University named after academician I. P. Pavlova, Leningrad, St. L. TolstoyPavlova, Leningrad, St. L. Tolstoy Senior researchSenior research officerofficer 1991-20031991-2003 1 University named after academician I. P.1 University named after academician I. P. Pavlova, Leningrad, St. L. TolstoyPavlova, Leningrad, St. L. Tolstoy Department headDepartment head a total of Oncology and urologya total of Oncology and urology from 2003 - «scientific research Institute offrom 2003 - «scientific research Institute of Oncology named N.N. Petrov» of the Ministry ofOncology named N.N. Petrov» of the Ministry of health of Russia, St. Petersburg, str. Leningrad,health of Russia, St. Petersburg, str. Leningrad, etc. 68etc. 68