Article

The predicting role of substance P in the neoplastic transformation of the hypoplastic bone marrow

Clinic of Pediatric Oncology Hematology and Transplantology, Poznan University of Medical Sciences, Posen, Greater Poland Voivodeship, Poland
Journal of Clinical Pathology (Impact Factor: 2.92). 10/2006; 59(9):935-41. DOI: 10.1136/jcp.2005.034629
Source: PubMed

ABSTRACT

To estimate the expression of substance P in the haematopoietic cells of hypoplastic bone marrow and define its relationship with the course of bone marrow hypoplasia.
Bone marrow specimens were obtained from 42 children with bone marrow hypoplasia who were hospitalised in the Department of Pediatric Oncology, Hematology and Transplantology, Poznan University of Medical Sciences, Poznan, Poland, between 1996 and 2003. Substance P and Ki-67 expression were evaluated using immunochemical and hybridocytochemical assays.
The expression of substance P (as evidenced by both immunocytochemical and hybridisation techniques) was confirmed in the cytoplasm of B lymphocytes in 8 of 11 children who developed acute leukaemia in 45 (SD 12) days. The percentage of substance P-positive cells ranged from 67.6 to 95.8 (mean of 81.5% cells with immunocytochemistry and 84.3% with in situ hybridisation). The risk of development of leukaemia secondary to bone marrow hypoplasia was found to be significant (p<0.001) in those children who expressed substance P in normal-looking lymphocytes at the initial bone marrow evaluation.
The presence of substance P in B lymphocytes of hypoplastic bone marrow may predict its neoplastic transformation. A marked correlation between substance P-positive bone marrow pattern and the expansion of tumour cells may prove the potential value of this oligopeptide in the pathogenesis of leukaemia.

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Available from: Bogdan Miskowiak, Oct 26, 2014
ORIGINAL ARTICLE
The predicting role of substance P in the neoplastic
transformation of the hypoplastic bone marrow
M Nowicki, D Ostalska-Nowicka, A Konwerska, B Miskowiak
...............................................................................................................................
See end of article for
authors’ affiliations
.......................
Correspondence to:
M Nowicki, Department of
Histology and Embryology,
Poznan University of
Medical Sciences, ul.
Swiecickiego 6, 60-781
Poznan
´
, Poland;
mnowicki@amp.edu.pl
Accepted for publication
30 December 2005
.......................
J Clin Pathol 2006;59:935–941. doi: 10.1136/jcp.2005.034629
Aims: To estimate the expression of substance P in the haematopoietic cells of hypoplastic bone marrow
and define its relationship with the course of bone marrow hypoplasia.
Methods: Bone marrow specimens were obtained from 42 children with bone marrow hypoplasia who
were hospitalised in the Department of Pediatric Oncology, Hematology and Transplantology, Poznan
University of Medical Sciences, Poznan, Poland, between 1996 and 2003. Substance P and Ki-67
expression were evaluated using immunochemical and hybridocytochemical assays.
Results: The expression of substance P (as evidenced by both immunocytochemical and hybridisation
techniques) was confirmed in the cytoplasm of B lymphocytes in 8 of 11 children who developed acute
leukaemia in 45 (SD 12) days. The percentage of substance P-positive cells ranged from 67.6 to 95.8
(mean of 81.5% cells with immunocytochemistry and 84.3% with in situ hybridisation). The risk of
development of leukaemia secondary to bone marrow hypoplasia was found to be significant (p,0.001)
in those children who expressed substance P in normal-looking lymphocytes at the initial bone marrow
evaluation.
Conclusions: The presence of substance P in B lymphocytes of hypoplastic bone marrow may predict its
neoplastic transformation. A marked correlation between substance P-positive bone marrow pattern and
the expansion of tumour cells may prove the potential value of this oligopeptide in the pathogenesis of
leukaemia.
B
one marrow hypoplasia seems to be a morphological
equivalent of clinically diagnosed haematopoietic insuf-
ficiency.
1
It is defined by the following laboratory values:
haemoglobin , 8.5 g/dl, mean corpuscular volume ,88 fl, a
white cell count ,2 g/l with a neutrophil count ,1 g/l, a
platelet count ,50 g/l and reticulocytes ,0.1%.
12
Bone
marrow aspirate contains hypocellularity. Transient bone
marrow hypoplasia is usually caused by infections (both viral
and bacterial) or by different chemical (including iatrogenic)
or physical factors.
34
Inherited aplastic anaemia, or bone
marrow hypoplasia before non-haematopoietic tissue in bone
marrow (ie, neuroblastoma metastases in bone marrow), is a
much rarer phenomenon.
A considerable number of cases of bone marrow hypoplasia
may be subjected to spontaneous remission and may not even
be recognised. The first hospitalisation of a child with bone
marrow hypoplasia is usually after two or more ineffective
courses of antibiotic treatment (for some undiagnosed
chronic infection), followed by symptoms of anaemia and
thrombocytopenia. In such cases, bone marrow hypoplasia
can be diagnosed as myelodysplastic syndrome, severe
aplastic anaemia or neoplasia. The correct diagnosis usually
takes several weeks, during which time the affected children
are not treated but usually receive continuous monitoring,
with laboratory evaluation of peripheral blood, bone marrow,
chest radiographs and so on being carried out at least once a
month.
The acute leukaemia of both lymphoblastic and non-
lymphoblastic types, which develops secondary to bone
marrow hypoplasia, usually has a bad prognosis.
1
This may,
at least in part, result from the late introduction of a
chemotherapy regimen. In cases of neoplastic transforma-
tion, the type of chemotherapy used will depend on a precise
recognition of the neoplasm concerned. Such treatment,
however, can probably be introduced much faster if, in the
mean time, the expression of certain markers of early
neoplastic transformation can be detected sooner. The
difficulty at present is that the full list of possible markers
is not universally accepted. On the other hand, some
authors
5–7
emphasised that several of the cytokines which
regulate physiological haematopoiesis may themselves be
possible risk factors of neoplastic transformation in bone
marrow hypoplasia.
One of these cytokines is substance P, a neuromediator and
oligopeptide consisting of 11 amino acids (H-Arg-Pro-Lys-
Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2), with a C-terminal
fragment typical of tachykinins.
78
It is widely distributed in
the central and peripheral nervous systems, being responsible
for contraction of skeletal and smooth muscle, salivation,
micturition, vasodilatation and sensory perception.
Substance P and neurokinin A are products of the common
preprotachykinin I gene and are also capable of modulating
the functions of the immune and haematopoietic systems.
8
Substance P stimulates proliferative activity of cells with
surface receptors for substance P. It is generally accepted that
substance P shows a preference for neurokinin-1 receptor
(NK-1R) and has less affinity for NK-2R and NK-3R.
910
The
expression of NK-1R has been reported in T and B
lymphocytes, macrophages, haematopoietic progenitor
(CD34) cells, endothelial cells and CD10 lymphoblasts. It is
of particular significance that neoplastic lymphoblasts in
acute lymphoblastic leukaemia (ALL) in children carry a
threefold to fourfold excess of NK-1R as compared with
normal B and T lymphocytes.
11
According to some authors,
12 13
peptidergic nerve terminals
in closest proximity to immunocompetent cells in the
organs most exposed to exogenous antigens represent a
Abbreviations: ALL, acute lymphoblastic leukaemia; ANLL, acute non-
lymphoblastic leukaemia; ICC, immunocytochemistry; IL1, interleukin 1;
ISH, in situ hybridisation; NK-1R, nerokinin-1 receptor
935
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Page 1
morphological exponent of functional inter-relations
between the nervous and immune systems.
However, substance P present in the bone marrow can be
also of non-neural origin. It is synthesised in the macro-
phages, eosinophils and neoplastically transformed lympho-
blasts. It has been shown that substance P induces
interleukin 1 (IL1) and stem cell factor in bone marrow
stromal cells, with levels of IL1b (a major isoform of this
cytokine) being twice those of IL1a. Both cytokines also
perform a potent autoregulatory role in NK-1R expression in
bone marrow stromal cells.
7
Therefore, our study aimed to estimate the expression of
substance P in haematopoietic cells of hypoplastic bone
marrow and define its relationship with the future course of
bone marrow hypoplasia.
PATIENTS AND METHODS
Patients
Patients were children with bone marrow hypoplasia,
hospitalised in the Department of Pediatric Oncology,
Hematology and Transplantology (Poznan University of
Medical Sciences, Poznan, Poland) between 1 January 1996
and 31 December 2003. In all, 485 children were referred to
our department during that time. Bone marrow biopsies were
performed on all of them and evidence of bone marrow
hypoplasia was found in 42. Twenty children, all of whom
presented with only one enlarged lymph node, served as the
control group. Histopathological examination of the enlarged
node indicated an inflammatory response only. Subsequent
observation of these children in the outpatient clinic for
hyperplastic diseases for around 12 months detected no
clinical traits of a neoplastic disease. This research protocol
was approved by the ethical commission of the university.
To participate in the investigation, children had to meet the
following eligibility criteria: age 4–18 years, laboratory
values: haemoglobin ,8.5 g/dl, mean corpuscular volume
,88 fl, white cell count ,2 g/l with a neutrophil count ,1g/l,
platelet count ,50 g/l and reticulocytes , 0.1%, hypocellular-
ity of bone marrow aspirate, with no neoplastic cells evaluated
by microscopy and flow cytometry test; normal chest radio-
graph; no enlarged lymph nodes, spleen or liver; and sterile
blood cultures (obtained at least three times) at the time of the
first hospitalisation. All the children underwent reverse
transcriptase-polymerase chain reaction evaluation for hepa-
titis C virus, Epstein–Barr virus and cytomegalovirus. Table 1
presents the relevant data.
Bone marrow smear preparation
The samples of bone marrow were taken from the posterior
superior iliac spine. Bone marrow smears prepared on Super
Frost were fixed in 96% ethanol (30 min at room tempera-
ture) within 24 h of sampling and kept at 280
˚
C until
immunophenotyping and in situ hybridisation (ISH) were
carried out.
Antibodies and immunocytochemistry
To show the presence of substance P in bone marrow cells, an
indirect immunocytochemical procedure was carried out
using rabbit polyclonal antibodies against human substance
P (Serotec, PEP A40, Oxford, UK) and the streptavidin–biotin
complex method with the use of horseradish peroxidase
modified by application of biotinylated tyramine (Dako
Catalysed Signal Amplification System, Peroxidase, K 1500,
Dako, Copenhagen, Denmark).
14
Heat-induced antigen
demasking pretreatment was also carried out (Target
retrieval solution, Dako S 1699).
15
The endogenous activity
of peroxidase was blocked by 10-min pre-incubation in 10%
hydrogen peroxide. The smears were then incubated with
anti-substance P antibodies diluted 1:2000 for 12 h at 4
˚
C.
Incubation with a secondary antibody (biotinylated goat
anti-rabbit, Dako E 0432, diluted 1:300) was carried out at
room temperature for 60 min followed by incubation with
diaminobenzidine (Dako S 3000).
Ki-67 antigen detection included an immunocytochemical
procedure with mouse monoclonal antibodies directed
against human Ki-67 (Dako, M 7187). The smears were
incubated with anti-Ki-67 antibodies, diluted 1:100 for 12 h
at 4
˚
C. Incubation with the secondary antibody (biotinylated
goat anti-mouse, Dako E 0433, diluted 1:300) was carried out
at room temperature for 60 min.
After incubation with antibodies against human leucocyte
antigen-DR, CD3, CD10, CD13, CD14, CD15, CD20, CD33,
CD34, CD41, CD61 and glycophorin A were used in white cell
(including blasts) immunophenotyping.
16
In situ hybridisation
For detection of mRNA encoding the amino acid sequence of
human substance P, a 59-biotinylated probe of the nucleotide
sequence 59-TCT GGG TTC GGA GTC GTC AAG AAA CCT AAT
TAC 39, was used.
17–19
The probe was synthesised by the DNA-
Gdansk (Gdansk, Poland) and was complementary to the
nucleotide sequence of human substance P (locus = 7q21–
q22, gene = ‘‘TAC1’’, product = ‘‘substance P’’, bp = 68–100;
http://www.ncbi.nlm.nih.gov/entrez/
viewer.fcgi?val = M68907). The smears were incubated with
the probe (concentration: 200 ng/1 ml) for 18 h at 37
˚
Cina
hybridisation chamber. This was followed by incubation with
a streptavidin–biotin complex (Dako K 1500, diluted 1:100)
at room temperature for 60 min and incubation with
diaminobenzidine for 5 min.
Controls and microscopical analysis
Immunocytochemistry (ICC) and hybridocytochemistry com-
plied with the principles of positive and negative controls.
20
Both analyses were carried out blind on coded samples. In
Table 1 Study group characteristics: the evolution of bone marrow hypoplasia during the 12-month observation period
Study group 12-month observation (n)
Subgroup
No of
patients Age (year)
RT-PCR viral infection markers (n)
Spontaneous
remission
Severe
aplastic
anaemia
Neoplastic
transformation
(diagnosis) No evolutionHCV EBV CMV
I 17 10 (6.2) 1 17
II 10 8 (2.5) 1 1 10
III 11 10 (1.8) 6 8 (ALL), 3 (ANLL)
IV 4 14 (2.7) 2 4
Age was expressed as the mean (SD).
ALL, acute lymphoblastic leukaemia; ANLL, acute non-lymphoblastic leukaemia; CMV, cytomegalovirus; EBV, Epstein–Barr virus; HCV, hepatitis C virus; RT-PCR,
reverse transcriptase-polymerase chain reaction. –, absence of the trait in all patients from a given group.
936 Nowicki, Ostalska-Nowicka, Konwerska, et al
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Page 2
control incubations, primary antibodies or oligoprobes were
replaced by rabbit or mouse serum (positive control) or
phosphate-buffered saline (negative control). The results of
the immunocytochemical reactions and of the ISH were
examined under a light microscope (Eclipse 600, Nikon), at
magnifications of 1:200–1:400. On the basis of the results of
the haematological staining, which were analysed through
the use of Microimage (Olympus) morphometric software,
the content of reaction-positive cells was determined by
comparing the number of cells with a positive reaction for
substance P, Ki-67 or mRNA substance P with the total
number of blasts. A percentage of substance P-positive cells
,5% was deemed to reflect technical errors and was
classified as a negative result.
Statistical analysis
As the group studied was relatively small, statistical analysis
was based on McNemar analysis and Fisher’s exact test. The
first was used to verify the difference in percentage of
substance P-positive cells noted by the immunocytochemical
and in ISH techniques. The relationship between substance
P-positive cases and an unfavourable course of bone marrow
hypoplasia was determined using the Fisher’s exact test.
Significance was defined at p,0.05.
RESULTS
12-month monitoring
Every child from the study group underwent a 12-month
period of observation. During that time, laboratory values of
peripheral blood were evaluated at least once a week; bone
marrow, depending on the clinical course, was aspirated
every 30–60 days; and chest radiographs and abdomen
ultrasonography were carried out every 3 months. Reverse
transcriptase-polymerase chain reaction markers of viral
infection (see Patients and methods) were estimated only
once. Of the patients, 17 children underwent a spontaneous
remission (subgroup I, median time of observation 93
(standard deviation (SD) 23) days) and 10 children devel-
oped severe aplastic anaemia (subgroup II, median time 212
(162) days). Eleven children were diagnosed with acute
leukaemia (subgroup III): eight had ALL with a median time
of 44 (12) days and three had acute non-lymphoblastic
leukaemia (ANLL) with a median time of 48 (13) days. In the
remaining 4 children (subgroup IV), bone marrow hypoplasia
did not develop during the 12-month period of observation,
and these children were excluded from further analysis
(table 1).
Outcome
The 17 children who underwent spontaneous remission are
still alive. The median time of observation is 37
(10.2) months. These patients are regularly evaluated for
clinical traits of aplastic anaemia or neoplastic disease.
Of the 10 patients who developed severe aplastic anaemia
(haemoglobin ,7.0 g/dl, mean corpuscular volume ,85 fl,
white cell count ,1.0 g/l with a neutrophil count ,0.5 g/l,
platelet count ,20 g/l), 6 underwent allogenic bone marrow
transplantation and they are alive at the first remission.
Three children in this group were given thymoglobulin (they
are also alive at the first remission). The one remaining child
died 50 days after first hospitalisation due to a cytomegalo-
virus infection. The median time of observation of the
children of subgroup II who survived is 32 (12.5) months.
The children of subgroup III had the worst outcome. Six of
the eight patients with ALL died, with no clinical and
haematological signs of remission (median time of observa-
tion after the development of ALL was 45 (12) days). In the
remaining two children ALL remission was diagnosed, but
they later relapsed (median time of observation 284
(68) days) and underwent allogenic bone marrow transplan-
tation. They are still alive in the second remission, but the
period of observation is not long (12 (4.5) months). Of the
three patients, two with ANLL died, with no clinical and
haematological signs of remission (median time of observa-
tion after ANLL was recognised as 34 (16) days). The
remaining child with ANLL is in remission and is still alive
(time of observation 24.5 months).
Overall, 9 of the 42 patients (21.4%) investigated in this
study died between 1 January 1996 and 31 December 2003,
27 (64.3%) are alive at the first remission and 2 (4.8%) at the
second remission. Four patients (9.5%) were excluded from
the analysis. The relevant data are presented in fig 1.
White cell immunophenotyping
In all the children of the study group the population of
nucleated cells in the hypoplastic bone marrow was mainly
composed of lymphocytes (mean of 96.4%). The percentage
of B lymphocytes (CD20) was .95% (mean of 95.1%),
whereas T lymphocytes (CD3) constituted ,4% of all
lymphocytes. Other individual nucleated cells were first
represented by neutrophils (fig 2A). After neoplastic trans-
formation B lymphocytes constituted ,5% of all nucleated
cells (fig 2B), which consisted mostly CD10-positive blasts in
cases of ALL, or CD13/CD14-positive cells in cases of ANLL
(data not shown).
ICC and ISH
Before and at the time of neoplastic transformation the
expression of substance P (in both the immunocytochemical
and hybridisation techniques) was confirmed exclusively in
the cytoplasm of B lymphocytes in 8 of the 11 children
assigned to subgroup III (fig 2C,E). The number of substance
P-positive cells ranged from 67.6% to 95.8% (mean of 81.5%
cells for ICC and 84.3% with ISH). This positive expression
was also found in the subsequent bone marrow examination
and the range of positive cells did not differ markedly from
that in the initial findings. During the 12-month observation
period, seven substance P-positive patients developed ALL
and one developed ANLL. The mean percentages of substance
P-positive cells at this stage of the diseases were 82.7 (ICC,
fig 2D) and 88.9 (ISH, fig 2F), respectively. No differences
were observed among the densities from substance P staining
in individual patients.
The expression of Ki-67 antigen in the bone marrow
haematopoietic cells was not evaluated at the first hospita-
lisation. However, it was detected at the time of (fig 2G), and
after (fig 2H), neoplastic transformation, when the percen-
tage of Ki-67-positive cells (blasts) ranged from 39.6% to
49.8% (mean of 42.6% blast cells) of all nucleated cells.
Substance P was absent from the cytoplasm and Ki-67
from the nucleus of the normal haematopoietic cells of the 20
children in the control group (data not shown). Table 2
presents the information in detail.
Statistical analysis
McNemar analysis showed no significant difference between
the results obtained by the ICC and ISH techniques (p values
ranged from 0.25 to 1.00).
When all the patients were considered (regardless of
leukaemia type), the risk of developing leukaemia secondary
to bone marrow hypoplasia was found to be significantly
greater (p,0.001, Fisher’s exact test) in those children who
showed expression of substance P in normal-looking
lymphocytes at the initial bone marrow evaluation—that is,
before neoplastic transformation. However, in subgroup III
the risk of death resulting from progression of the leukaemia
was not significantly greater in the children who were
substance P-positive at the initial bone marrow examination
Predicting role of substance P 937
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Page 3
(ALL-related death: p = 0.248; ANLL-related death: p = 1.0;
death regardless of leukaemia type: p = 0.153). On the other
hand, statistical analysis of those patients in whom no
treatment failure was noted during the observation period
showed that the probability of remission of bone marrow
hypoplasia was significantly greater in those children who
were initially substance P-negative (p,0.001).
No marked relationship was found between initially
detected viral infection and the outcome of bone marrow
hypoplasia.
DISCUSSION
The search for causes and aids to the diagnosis of bone
marrow hypoplasia includes taking a thorough history, a
detailed physical examination and several laboratory tests,
including microscopic analysis of bone marrow, sampled by
needle biopsy or during surgery, determination of infectious
diseases and, occasionally, genetic studies of patients and
their families. The application of ICC techniques in the
diagnosis of bone marrow hypoplasia has been tested
already.
21
However, attempts to use this technique have been
restricted to the differentiation of myelodysplastic syndromes
from various grades of bone marrow aplasia.
21–24
Among other
antigens, Ki-67 antigen and proliferating cell nuclear antigen
are useful in such a procedure.
24
In patients with bone
marrow aplasia the markers could not be seen in nucleated
cells, whereas in patients with myelodysplastic syndromes
the percentage of immunopositive cells ranged from 20% to
60%.
24
These studies, however, have not attempted to
determine the future trend of such bone marrow hypoplastic
lesions.
Our selection of substance P as an indicator of the potential
evolving trend of bone marrow hypoplasia was prompted by
detection of its role and the role of other neuropeptides in the
physiological control of haematopoiesis. Owing to the
peptidergic nerve endings in bone marrow, substance P
Figure 1 Diagrammatic representation of sample outcome. ALL, acute lymphoblastic leukaemia; ANLL, acute non-lymphoblastic leukaemia; BM, bone
marrow; BMT, bone marrow transplantation; SAA, severe aplastic anaemia.
938 Nowicki, Ostalska-Nowicka, Konwerska, et al
www.jclinpath.com
Page 4
easily comes into contact with haematopoietic and stromal
cells of the bone marrow.
25
Previous investigations have
proved that substance P is released in bone marrow by
macrophages,
26
eosinophils
27 28
and vascular endothelial
cells.
29–31
Most bone marrow cells—that is, haematopoietic
cells,
32
the cells forming bone marrow stroma,
33
and T and B
lymphocytes present in bone marrow
34
—are equipped with
the substance P-specific NK-1R receptor. Substance P, tested
in short-term human bone marrow cultures in methylcellu-
lose, is capable of supporting haematopoiesis in vitro.
32
This
indicates that at a concentration of 10
28
–10
211
mol/l it can
substitute for IL3 and for granulocyte–monocyte colony
stimulating factor, the presence of which is indispensable
for growth of the colonies. However, substance P cannot
Figure 2 (A) Immunohistochemical expression of CD20 in the hypoplastic bone marrow of a 7-year-old child assigned to subgroup III. Individual cells
present at the time of neoplastic transformation are mostly B lymphocytes. (B) A bone marrow specimen of a 7-year-old child who developed acute
lymphoblastic leukaemia. Mature CD20-positive B lymphocytes form a minority of all blasts. (C) Substance P (SP) immunoreactivity in normal-looking B
lymphocytes before neoplastic transformation of a 5-year-old child. Its expression is present in the cytoplasm of most nucleated cells. (D) The same5-
year-old child (as in C), who developed acute non-lymphoblastic leukaemia. Weak substance P expression in all blasts. (E) A hypoplastic bone marrow
of a 9-year-old child assigned to subgroup III. Expression of substance P mRNA in individual groups of nucleated cells. (F) Expression of SP mRNA in
the same 9-year-old child (as in E) after neoplastic transformation (common acute lymphoblastic leukaemia). SP transcript immunoreactivity in all
nucleated cells. (G) Expression of Ki-67 antigen at the moment of neoplastic transformation. Strong nuclear immunocytochemical reaction in about half
of the normal-looking B-lymphocytes (7-year-old child assigned to subgroup III). (H) Immunocytochemical expression of Ki-67 in the bone marrow
specimen of a 7-year-old child who developed acute leukaemia. Ki-67-positive cells constitute one third of all nucleated cells. Scale bar = 50 mm. ICC,
immunocytochemistry; ISH, in situ hybridisation.
Predicting role of substance P 939
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Page 5
substitute for erythropoietin, although, given in parallel, it
stimulates its effects. The specificity of such a stimulatory
function of substance P was confirmed when it was given
with blockers of the known subtypes of substance P receptor.
Substance P given with the blocker for the NK-1R subtype of
the receptor yielded results similar to those in negative
controls. On the other hand, substance P, when given with a
blocker of the NK-2R receptor, exerted no effect on the
activity of substance P.
732
Substance P was also found to act
indirectly on haematopoietic cells through the stromal cells,
stimulating them to produce cytokines. In substance P-
supplemented cultures, IL1-specific, IL3-specific, IL6-specific
and granulocyte–monocyte colony stimulating factor-specific
antibodies induced only partial growth inhibition, indicating
that substance P acts by inducing synthesis of the cyto-
kines.
73235
Substance P can also induce synthesis of IL1 and
of stem cell factor in cells of the bone marrow sublayer.
73235
Some investigators have suggested that substance P
released from nerve endings not only augments IL1 synthesis
but also increases the density of IL1 receptors.
7
In turn, IL1,
released by the sublayer cells, augments the density of NK-1R
receptors on the surface of haematopoietic and sublayer cells.
We have previously reported that substance P is also
secreted by blast cells in ALL.
18 19
A possible connection
between the substance P-positive blast pattern and leukae-
mia relapse may prove that substance P is associated with the
proliferation of the tumour cells.
Our studies of the bone marrow of healthy people (who
formed the control group) showed that the immunocyto-
chemical expression of peripherally located substance P
(most probably coupled to NK-1R) on nucleated cells of the
bone marrow was (5%. These results prompted us to carry
out analogous determinations in the material sampled from
patients with clinical symptoms of bone marrow hypoplasia.
In some of them the proportion of substance P-positive cells
among all nucleated cells was 67.6–95.8% (mean of 81.5%
cells for ICC and 84.3% with ISH), with an absence of
neoplastic cells. Subsequent observation of these patients
showed that they developed a proliferative disease of the
bone marrow. Throughout the entire time preceding the
neoplastic transformation, an increased number of substance
P-positive cells was noted even if the proportion of Ki-67-
positive cells was similar to control values. Neoplastic
transformation was manifested by the presence of poorly
differentiated cells with a positive reaction for Ki-67 (39.6–
49.8%) in bone marrow. In line with the above, we suggest
that the presence of substance P in normal-looking B
lymphocytes of hypoplastic bone marrow becomes the second
(after nerve endings) source of this peptide, which may
stimulate other neoplastically transformed cells for uncon-
trolled proliferation. Substance P possibly accelerates the
already initiated development of leukaemia. The origin of
leukaemia, however, seems to be independent of substance P
expression.
The expression of substance P in normal-looking bone
marrow lymphocytes before neoplastic transformation could
not predict the death of the patient, which is certainly related
to the small number of patients in subgroup III and the
different chemotherapy protocols used in the treatment of
ALL and ANNL.
Bone marrow hypoplasia, followed by neoplastic transfor-
mation, often remains asymptomatic. Only a few patients
receive specialist care before the symptoms of bone marrow
proliferative disease become evident. In these few patients,
the diagnosis of bone marrow hypoplasia leads to subsequent
check-ups, during the course of which immunocytochemical
analysis of cellular inducers of differentiation on the surface
of bone marrow cells may provide a simple screening test
pointing to the potential trend of evolution of the lesion.
CONCLUSIONS
1. Substance P expressed in the B lymphocytes of hypo-
plastic bone marrow may predict its leukaemic transfor-
mation.
2. The significant correlation between a substance P-
positive bone marrow pattern and expansion of tumour
cells in the bone marrow may prove the potential role of
this oligopeptide in the pathogenesis of leukaemia.
3. The results reported here, although promising, require
confirmation by the investigation of a larger group of
patients.
ACKNOWLEDGEMENTS
We thank Professor Geoffrey Shaw for his invaluable help in English
text editing.
Authors’ affiliations
.....................
M Nowicki, Department of Pediatric Oncology, Hematology and
Transplantology, Poznan University of Medical Sciences, Poznan,
Poland
M Nowicki, A Konwerska, B Miskowiak, Department of Histology and
Embryology, Poznan University of Medical Sciences
D Ostalska-Nowicka, Department of Pediatric Cardiology and
Nephrology, Poznan University of Medical Sciences
B Miskowiak, Department of Optometry and Biology of the Visual
System, Poznan University of Medical Sciences
Competing interests: None.
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Table 2 Immunocytochemical and hybridocytochemical expression of substance P and Ki-67 antigen in the children of the
study group during a 12-month observation period (shown as the mean percentage of immunopositive cells)
Subgroup
Initial BM evaluation BM evaluation after the neoplastic transformation
ICC
ISH
ICC
ISHSP (%) Ki-67 (%) SP (%) Ki-67 (%)
I ,5 ,5 ,5———
II ,5 ,5 ,5———
III 81.5 ,5 84.3 82.7 42.6 88.9
IV ,5 ,5 ,5———
BM, bone marrow; ICC, immunocytochemistry; ISH, in situ hybridisation; SP, substance P. –, absence of the trait in all patients from a given group.
940 Nowicki, Ostalska-Nowicka, Konwerska, et al
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Predicting role of substance P 941
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    • "These findings suggest an inhibitory role for HK-1 on developing B cells, and that it is part of the bone marrow microenvironment that supports and regulates the proliferation and differentiation of hematopoietic cells [96]. In another study, the presence of SP in B lymphocytes of hypoplastic bone marrow predicted neoplastic transfor- mation [97]. Also, HK-1 enhances B-cell proliferation and antibody production [99], promotes survival of dendritic cells [100], enhances the proliferation of T-cell precursors, and increases the number of thymocytes [101] as well as decreases blood pressure [102]. "
    [Show abstract] [Hide abstract] ABSTRACT: The recent years have witnessed an exponential increase in cancer research, leading to a considerable investment in the field. However, with few exceptions, this effort has not yet translated into a better overall prognosis for patients with cancer, and the search for new drug targets continues. After binding to the specific neurokinin-1 (NK-1) receptor, the peptide substance P (SP), which is widely distributed in both the central and peripheral nervous systems, triggers a wide variety of functions. Antagonists against the NK-1 receptor are safe clinical drugs that are known to have anti-inflammatory, analgesic, anxiolytic, antidepressant, and antiemetic effects. Recently, it has become apparent that SP can induce tumor cell proliferation, angiogenesis, and migration via the NK-1 receptor, and that the SP/NK-1 receptor complex is an integral part of the microenvironment of inflammation and cancer. Therefore, the use of NK-1 receptor antagonists as a novel and promising approach for treating patients with cancer is currently under intense investigation. In this paper, we evaluate the recent scientific developments regarding this receptor system, its role in the microenvironment of inflammation and cancer, and its potentials and pitfalls for the usage as part of modern anticancer strategies.
    Full-text · Article · Apr 2012 · The Scientific World Journal
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    • "Our data do not rule out the possibility of NGF or BDNF acting on gene expression in BMSC through one, or several, intermediate factors. Increased expression or release of Substance-P following neurotrophic stimulation of cells has been described in diverse settings documenting its important role in the hematopoieticneuro-immune axis in inflammation, as well as normal and malignant hematopoiesis575859606162 . Relevant to our model, Substance-P has been shown to participate in upregulation of inflammatory cytokines in fibroblastic cells[63,64] and more recently in a model of increased IL-6, IL-1b, and TNF-a in a model of burn-induced lung injury[65]. "
    [Show abstract] [Hide abstract] ABSTRACT: The host's response to infection is characterized by altered levels of neurotrophins and an influx of inflammatory cells to sites of injured tissue. Progenitor cells that give rise to the differentiated cellular mediators of inflammation are derived from bone marrow progenitor cells where their development is regulated, in part, by cues from bone marrow stromal cells (BMSC). As such, alteration of BMSC function in response to elevated systemic mediators has the potential to alter their function in biologically relevant ways, including downstream alteration of cytokine production that influences hematopoietic development. In the current study we investigated BMSC neurotrophin receptor expression by flow cytometric analysis to determine differences in expression as well as potential to respond to NGF or BDNF. Intracellular signaling subsequent to neurotrophin stimulation of BMSC was analyzed by western blot, microarray analysis, confocal microscopy and real-time PCR. Analysis of BMSC Interleukin-6 (IL-6) expression was completed using ELISA and real-time PCR. BMSC established from different individuals had distinct expression profiles of the neurotrophin receptors, TrkA, TrkB, TrkC, and p75(NTR). These receptors were functional, demonstrated by an increase in Akt-phosphorylation following BMSC exposure to recombinant NGF or BDNF. Neurotrophin stimulation of BMSC resulted in increased IL-6 gene and protein expression which required activation of ERK and p38 MAPK signaling, but was not mediated by the NFkappaB pathway. BMSC response to neurotrophins, including the up-regulation of IL-6, may alter their support of hematopoiesis and regulate the availability of inflammatory cells for migration to sites of injury or infection. As such, these studies are relevant to the growing appreciation of the interplay between neurotropic mediators and the regulation of hematopoiesis.
    Full-text · Article · Mar 2010 · PLoS ONE
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    [Show abstract] [Hide abstract] ABSTRACT: The role of substance P (SP) in physiological haematopoiesis is well established. However, it also seems to be important in the neoplastic transformation of bone marrow, leading to the development of acute leukaemia in children, and also metastases to bone marrow of solid tumours (particularly neuroblastoma and breast cancer) in early stages of these diseases. This review summarises the available data on SP involvement in both processes. In the future, SP antagonists may be used as anti-neoplastic drugs, for example by direct or indirect blocking of tumour cell proliferation through inhibition of growth factor production and interleukin-1b synthesis.
    Full-text · Article · Aug 2007 · Journal of Clinical Pathology
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