Sabato 27 ottobre 2012 - Pacini Editore

COmuNiCaziONi ORali
microarray analysis in bone metastases from breast cancer (BrCa)
patients who developed secondary lesions also in visceral organs
or with metastases restricted to bone. Patients with metastases
restricted to bone showed a longer overall survival compared to
patients who rapidly developed multiple metastases involving
also visceral organs. Our bioinformatic elaboration unveiled a
cluster of genes that segregated the two groups. Among them, a
set of genes was involved in oxygen binding and transport. We
focused on β hemoglobin (HBB) that was not, so far, associated
with the metastatic phenotype of BrCa. Based on this observation,
we hypothesized a role of HBB in tumor progression 1.
Therefore, the aim of the present study was to investigate HBB
expression in primary human breast carcinoma and lymph node
metastases, in order to investigate a potential correlation between
such expression and known prognostic factors.
Material and methods. Samples from 36 consecutive and
unselected patients undergone surgery for breast cancer were
included in this prospective study. A total of 38 primary lesions
and 4 lymph node metastases from the same patients were investigated.
3 cases of fibroadenoma and 1 case of papilloma were also
included in the study. Histological sections were deparaffinized,
incubated with 0.07M citrate buffer (pH 6), 15 min at 98° C, for
antigen retrieval, treated with 3% H 2O 2 and incubated overnight
at +4°C with the anti-HBB mouse monoclonal antibody (clone
sc-21757, Santa Cruz Biotechnology Inc., Heidelberg, Germany).
The staining signals were revealed using the Dako LSAB+ System-HRP.
Slides were counterstained with Mayer’s hematoxylin.
Red blood cells were used as internal positive control and negative
controls were performed in parallel, by omitting primary antibody
for each case. HBB expression in carcinoma was recorded
as percentage of positively stained cells and correlated to known
prognostic factors, namely histologic type, grading, estrogen
(ER) and progesterone (PR) receptor expression, Ki-67 proliferation
index and HER-2 score.
Results. Positive staining was noted in our internal positive
controls, red blood cells, and in endothelial cells. Multifocal
immunostaining of extracellular space was noted in 8 cases and
possibly related to hemoglobin diffusion in the interstitial compartment.
Strong cytoplasmic positivity was noted in primary
invasive carcinoma in 22 cases, mainly in scattered elements
rESPIrATOrIO
EML4-ALK rearrangements detection by FISH and
rT-PCr in Formalin Fixed and Paraffin Embebbed
tissues of non Small Cell Lung Cancer.
N. Borrelli1 , R. Giannini1 , G. Alì2 , S. Pelliccioni2 , C. Niccoli2 ,
G. Fontanini1,2 1 2 Department of Surgery, University of Pisa, Pisa; Unit of Pathologic
Anatomy, Azienda Ospedaliera Universitaria Pisana, Pisa
Introduction. A transforming fusion gene joining the echinoderm
microtubule-associated protein-like 4 gene (EML4) and the
anaplastic lymphoma kinase gene (ALK) has recently been found
in a subset of non-small cell lung cancer (NSCLC). ALK encodes
a receptor tyrosine kinase, which belongs to the insulin receptor
superfamily. This protein comprises an extracellular domain, an
hydrophobic stretch corresponding to a single pass transmem-
Sabato, 27 ottobre 2012
Sala Donatello – ore 8,30-10,30
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or groups of cells. Percentage of positive cells ranged from 1 to
45%. Cases with more than 10% positive cells were arbitrarily
considered positive, accounting for 12 primary tumors. Two of
four nodal metastases expressed HBB in a higher percentage
than the primary tumor and one had the same percentage of the
primary tumor. Primary tumors positive for HBB were 9 poorly
differentiated (G3) infiltrating ductal carcinomas, 1 moderately
differentiated (G2) ductal carcinoma and 2 infiltrating lobular
carcinoma. The average age of the patients was 65.1 (range: 38-
83). All but one expressed significant levels of hormone receptors,
with an average proliferation index of 20 (range: 10-35) and
all but two did not overexpress HER-2. The percentage of HBB
positive cells was higher in the invasive lesions when compared
with the coexisting in situ lesions in the 87% of the cases. Benign
lesions tested, such as fibroadenoma and papilloma, as well as
adenosis, papillomatosis and normal tissue observed outside the
main tumor nodule, were all negative.
Conclusions. Significant positivity of HBB was detected in primary
BrCa tissue but not in normal breast epithelial cells and benign lesions,
such as fibroadenoma, papilloma and adenosis, indicating that
HBB expression is associated with the malignant cell phenotype.
Infiltrating lesions showed higher percentage of HBB positive cells
than in situ counterparts, as well as greater values of positivity were
found in lymph node metastases, compared to the corresponding
primary tumors. The majority of the HBB positive tumors were high
grade ductal carcinoma with moderately high proliferation index,
significant levels of hormone receptors and without HER2 overexpression.
These findings suggest that HBB expression by neoplastic
cells may identify a subgroup of lesions with a more aggressive
potential. Even though follow-up data and further investigation are
needed to clarify the prognostic value and the statistical significance
of HBB expression in primary breast cancers, our results suggest a
possible positive correlation between HBB expression and tumor
cell aggressiveness, paving the way for a possible use of HBB as a
novel marker for breast cancer characterization.
references
1 Capulli M, Angelucci A, Driouch K, et al. Increased expression of a
set of genes enriched in oxygen binding function discloses a predisposition
of breast cancer bone metastases to generate metastasis spread
in multiple organs. J Bone Mineral Research 2012; in press.
brane region, and an intracellular kinase domain. It plays an
important role in the cell proliferation, survival migration, and
alteration in cytoskeletal rearrangement 1 . EML4 is a cytoplasmic
protein that is a human homolog of echinoderm microtubule
associated protein, the major microtubule binding protein in
dividing sea urchin eggs, and is essential for the formation of
microtubules 1 . It is composed of a N-terminal basic region, a
HELP domain and four WD repeats in the C-terminus. EML4
and ALK are situated on the short arm of chromosome 2 (2p21
and 2p23, respectively) where they are separated by a distance
of 12.2 megabases and are oriented in opposite directions 2 . The
small inversion within chromosome 2p leads to the EML4-ALK
fusion gene. Multiple EML4-ALK variants have been identified
since EML4 truncation does not always occur in the same location
(at exons 2, 6, 13, 14, 15, 18 or 20); the intracellular tyrosine
kinase domain of ALK (encoded by exon 20) is conserved among
the variants. The most common variants were E13;A20 (the
nomenclature refers to the exons in EML4 (E) that are fused to