Angiosarcoma and atypical vascular lesions of the breast: diagnostic and prognostic role of MYC gene amplification and protein expression.

MYC amplification has been reported as a prominent feature of secondary angiosarcomas (SAS). The differential diagnosis between atypical vascular lesion (AVL) and low-grade angiosarcoma (AS) can be occasionally very difficult or even impossible, and MYC amplification status has been pointed as an important diagnostic tool to distinguish cutaneous vascular lesions of the breast. We assessed MYC amplification and protein expression status by fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC), respectively, in 49 patients diagnosed with breast AS, and 30 patients diagnosed with post-radiation AVL of the breast. Clinical and pathological features, and follow-up data were collected, and survival analyses were performed. Among 37 patients with SAS, twenty patients had tumors with high-level MYC amplification and protein overexpression (54 %). None of primary angiosarcomas (PAS) or AVL cases showed MYC amplification or protein expression. Concordance between MYC amplification (FISH) and protein expression (IHC) was 100 % in AVL, PAS, and SAS. Survival analysis of the SAS patients demonstrates that those with MYC amplification had a significantly worse overall survival compared to cases without MYC amplification (P = 0.035). There was a non-significant trend toward a poor disease-free survival between cases with and without MYC amplification (P = 0.155). Our findings show that MYC amplification is a highly specific but poorly sensitive marker for SAS and, therefore, a negative result does not exclude the diagnosis of angiosarcoma. MYC amplification was associated with adverse prognosis, suggesting a prognostic role of MYC amplification status on SAS of the breast.

Second Axillary Sentinel Lymph Node Biopsy for Breast Tumor Recurrence: Experience of the European Institute of Oncology.

PURPOSE: This retrospective study aimed to determine the feasibility, accuracy, and recurrence rates of lymphoscintigraphy and the new sentinel lymph node biopsy (SLNB) for patients with ipsilateral breast tumor recurrences who were treated previously with conservative surgery and had negative SLNB results. METHODS: The study was conducted at the European Institute of Oncology in Milan and included 212 patients with the diagnosis of operable local breast cancer recurrence. They had been treated previously with conservative surgery and showed negative SLNB results. They subsequently underwent additional breast surgery and a second SLNB between May 2001 and December 2011. RESULTS: Preoperative lymphoscintigraphy demonstrated at least one new axillary sentinel lymph node (SLN) in 207 patients (97.7 %), whereas no drainage was observed in five patients (2.3 %). One or more SLNs were surgically removed from 196 of the 207 patients. Isolation of SLNs from the remaining 11 patients could not be accomplished. The success rate for the SLNB was 92.5 %. Extra-axillary drainage pathways were visualized in 17 patients (8 %). The annual axillary recurrence rate after a median follow-up period of 48 months was 0.8 %, and the cumulative incidence of axillary recurrence at 5 years was 3.9 %. CONCLUSIONS: A second SLNB should be considered for patients with operable local breast tumor recurrence who underwent conservative surgery and had negative SLNB results. The procedure is technically feasible and accurate for selected patients.

Clinicopathological and immunohistochemical study of 30 cases of post-radiation atypical vascular lesion of the breast.

Atypical vascular lesions (AVL) that occur in the field of prior radiation therapy for breast carcinoma are placed within the differential diagnosis with low grade angiosarcoma and other benign vascular lesions. Although considered a benign entity, the exact biological behavior of AVLs is not fully established because of the small number of cases reported in the literature. We aim to further characterize these lesions clinically and histopathologically, and to study their behavior. We report a series of 30 patients with AVL of the breast occurring after radiation exposure, diagnosed and treated at the European Institute of Oncology, Italy. Immunohistochemical study was performed in all cases, using CD31, D2-40, CD105, and Ki-67 antibodies. Twenty-seven patients were treated with standard doses of conventional adjuvant radiation therapy for the prior breast carcinoma. Three patients were treated with intraoperative radiotherapy with electrons. The post-radiation latency interval from breast carcinoma to AVL was 48.5 months (ranged from 1 to 146 months). Most of the lesions were classified as lymphatic type (78.6 %) based on D2-40 positivity. No extension into subcutaneous tissue or significant atypia was noted in all cases. Despite the fact that the AVL of our series have shown benign behavior in 93.3 %, one patient developed local recurrence of AVL, and two cases progressed to angiosarcoma at the previous AVL site. Further studies should be conducted to better understand the clinical behavior and to propose additional histopathologic diagnostic criteria to distinguish AVL from low grade angiosarcoma and those AVL with increased risk for malignant progression. Concerning current treatments of AVL, we recommend complete excision with free surgical margins and close follow up.

The indocyanine green method is equivalent to the 99mTc-labeled radiotracer method for identifying the sentinel node in breast cancer: a concordance and validation study.

AIMS: The aim of this study was to assess concordance between the indocyanine green (ICG) method and (99m)Tc-radiotracer method to identify the sentinel node (SN) in breast cancer. Evidence supports the feasibility and efficacy of the ICG to identify the SN, however this method has not been prospectively compared with the gold-standard radiotracer method in terms of SN detection rate. METHODS: Between June 2011 and January 2013, 134 women with clinically node-negative early breast cancer received subdermal/peritumoral injection of (99m)Tc-labeled tracer for lymphoscintigraphy, followed by intraoperative injection of ICG for fluorescence detection of SNs using an exciting light source combined with a camera. In all patients, SNs were first identified by the fluorescence method (ICG-positive) and removed. A gamma ray-detecting probe was then used to determine whether ICG-positive SNs were hot ((99m)Tc-positive) and to identify and remove any (99m)Tc-positive (ICG-negative) SNs remaining in the axilla. The study was powered to perform an equivalence analysis. RESULTS: The 134 patients provided 246 SNs, detected by one or both methods. 1, 2 and 3 SNs, respectively, were detected, removed and examined in 70 (52.2%), 39 (29.1%) and 17 (12.7%) patients; 4-10 SNs were detected and examined in the remaining 8 patients. The two methods were concordant for 230/246 (93.5%) SNs and discordant for 16 (6.5%) SNs. The ICG method detected 99.6% of all SNs. CONCLUSIONS: Fluorescent lymphangiography with ICG allows easy identification of axillary SNs, at a frequency not inferior to that of radiotracer, and can be used alone to reliably identify SNs.

Mastectomy without radiotherapy: outcome analysis after 10 years of follow-up in a single institution.

The aim of this study was to identify the prognostic factors associated with the risk of loco-regional recurrence (LRR) of women undergoing mastectomy and complete axillary dissection without radiotherapy. We analyzed data from 650 women operated between 1997 and 2001 in a single institution. Median follow-up was 10 years. Overall survival was 89.8 % at 5 years and 76.6 % at 10 years. The 10-year cumulative incidence of LRRs was 10.0 % (5.0, 10.5, 15.8, and 18.5 % in patients with 0, 1-3, 4-9, and ≥10 positive lymph nodes (LNs), respectively). Sixty-two (9.5 %) LRRs were observed, 5 (0.8 %) of which occurred in the axillary LNs. Supraclavicular LNs recurrences (n = 16, 2.5 %) occurred more frequently in patients with four or more positive LNs, Ki-67 ≥ 20 % or extensive peritumoral vascular invasion (PVI). At multivariable analysis, nodal status was the only prognostic factor for local events, while nodal status, Ki-67 and PVI were significant prognostic factors for recurrences in the regional LNs. Moreover, within each category of positive LNs, high values of Ki-67 and extensive PVI were associated with the highest risk of LRR while low values of Ki-67 and absence of extensive PVI were associated with the lowest risk of LRR. Women with node-negative tumors have the lowest risk of LRR and represent the group of patients that might benefit the least from radiotherapy. PVI and Ki-67 might help tailoring PMRT indications among patients with positive LNs. Finally, the very low incidence of recurrences in the axillary LNs raises questions about the inclusion of the axilla in the radiation field.

Unavoidable mastectomy for ipsilateral breast tumour recurrence after conservative surgery: patient outcome.

BACKGROUND: In the case of ipsilateral breast tumour recurrence (IBTR) after breast-conserving surgery (BCS), a second conservative surgical approach maybe considered in some motivated patients whereas in others mastectomy is unavoidable. PATIENTS AND METHODS: From 1997 to 2004, 282 patients presented at the European Institute of Oncology with an operable invasive IBTR after BCS. One hundred and sixty-one (57%) underwent a second conservative surgery, whereas 121 patients (43%) were given a mastectomy and represent the study population. We investigated the prognosis and determined predictive factors of outcome. RESULTS: Median time from primary breast cancer to IBTR was 41 months (range 5-213). Recurrences were T2-T4 and/or multifocal in 83 cases (68.6%). With a median follow-up of 5 years after mastectomy, 5-year overall survival (OS) and disease-free survival (DFS) were 73.3% [95% confidence interval (CI) 65.0% to 81.6%] and 50.4% (95% CI 40.9% to 59.8%), respectively. At the multivariate analysis, early onset of IBTR, presence of vascular invasion and Ki67 >or=20 of the recurrent tumour were found to significantly affect both DFS and OS. CONCLUSIONS: In women who need mastectomy for IBTR, early onset of the relapse, high proliferation index and presence of vascular invasion represent the worst prognostic factors.

Expression of NGF, Trka and p75 in human cartilage.

Nerve growth factor (NGF) exerts its action through two types of receptor: high-affinity tyrosine kinase A receptor (trkA) and low-affinity p75 receptor. NGF has a neurotrophic role in central and peripheral nervous system development, but there is also clear evidence of its involvement in the developing skeleton. The aim of the present immunohistochemical study was to investigate the expression and distribution of NGF, trkA, and p75 in normal cartilaginous tissues from adult subjects: articular and meniscal cartilage of the knee, cartilage from the epiglottis, and intervertebral disc tissue. Detection of NGF mRNA was also performed by in situ hybridization. Immunoreaction for NGF and the two receptors in articular chondrocytes, chondrocyte-like cells of meniscus and annulus fibrosus, and chondrocytes of the epiglottis demonstrated that they are all expressed in hyaline, fibrous and elastic cartilaginous tissues, suggesting that they could be involved in cartilage physio-pathology.

Altered balance of proteinase inhibitors in atrophic muscle after denervation.

Skeletal muscle denervation leads to an increase of proteolytic activity, which is also favoured by reduced levels of alpha1 antichymotrypsin and nexin II, two serine-proteinase inhibitors normally acting at the neuromuscular junction. In the present experiments we extended our investigation to other muscular proteinase inhibitors after denervation. In all muscles examined (soleus, plantaris, extensor digitorum longus) specific immunoreactivity for alpha2macroglobulin (alpha2M) and alpha1proteinase inhibitor (alpha-1-antitrypsin, ATI) was distributed in peri-endomysial structures as well as in small patches inside the fibres. By contrast, inter-alpha-trypsin inhibitor (ITI) was mainly localized in the extracellular matrix. These localization patterns did not change substantially in 15-days denervated muscles. Dot-blot analysis revealed a small decrease (about 15%) of alpha2M in 15-days denervated muscles, while ATI and ITI specific activities were substantially unchanged. RT-PCR allowed us to detect the above protease inhibitor mRNAs in normal muscle homogenates. Denervation atrophy induced by section of the sciatic nerve resulted in a remarkable reduction of (2macroglobulin mRNA (60%) and ITI (30%), but not ATI, as measured by computer-assisted semiquantitative densitometry of electrophoresed RT-PCR bands. The marked decrease of alpha2M we have detected in denervated muscle may be responsible, at least in part, for the proteolytic increase which is known to occur in skeletal muscle during denervation atrophy.

Characterization of human bone cells in culture.

Osteoblast-like cells isolated from human bone bioptic specimens were established in culture. Their osteoblast-like phenotype was studied by biochemical, histochemical and immunohistochemical methods and by electron microscopy examination. Third-passage cell cultures exhibited high level of alkaline phosphatase activity and the exposure to human parathyroid hormone produced an increase of intracellular cAMP. Cultured cells were immunoreactive for type I and type III collagen, osteonectin, and fibronectin; when ascorbic acid and beta-glycerophosphate were added, they synthesized a rich extracellular matrix. This characterization ensures the reliability of osteoblast-like cultures when they are used as experimental models.

Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis.

To investigate the pathogenesis of the degenerative changes of the ligamentum flavum occurring in lumbar spine stenosis, yellow ligament cells from patients with lumbar spine stenosis were cultured for the first time and subjected to biochemical, histochemical and immunohistochemical study. Stenotic ligamentum flavum (SLF) cells were seen to express high levels of alkaline phosphatase (ALP) activity and to produce a matrix rich in type I and III collagen, fibronectin and osteonectin. The matrix mineralized only following beta-glycerophosphate (betaGP) and ascorbic acid supplementation. Stimulation with human parathyroid hormone (PTH) increased intracellular cAMP concentration. These findings indicate that there was significant evidence of osteoblast-like activity in these cells. SLF cells also stained for S100 protein, type II and type X collagen, and co-localized type II collagen and ALP labelling, reflecting the presence of hypertrophic chondrocyte-like cells. Cultures from control patients showed neither osteoblastic nor chondrocytic features: they expressed type I and type III collagen and fibronectin, but did not stain for osteonectin, nor were bone-like calcifications observed in presence or absence of betaGP. Normal ligamentum flavum (NLF) cells did not synthesized S100 protein or type II or type X collagen, and showed a weaker response to PTH stimulation. Our data demonstrated the presence of hypertrophic chondrocytes with an osteoblast-like activity in the ligamentum flavum of patients with spinal stenosis suggesting that they could have a role in the pathophysiology of the heterotopic ossification of ligamentum flavum (OLF) in lumbar spine stenosis.

Evidence of type II estrogen receptor in human osteoblast-like cells.

Osteoblast-like cells isolated from human bone bioptic specimens were characterized and analysed for the presence of type II estrogen receptor (type II EBS). The amount of type II EBS was measured by a whole-cell assay at 4 degrees C for 2.5 h using [(3)H]-estradiol as tracer. Saturation analysis, used to investigate the binding characteristic of type II EBS, resulted in a sigmoid curve. Scatchard analysis showed the binding affinity of the estrogen receptor, yielding a concave plot. The dissociation constant (K(d)), determined from the [(3)H]-estradiol concentration required for half saturation was about 12+/-2 nM (SD). The number of type II EBS, estimated at maximum binding, was 197,000+/-8800 sites per cell. If the regulation of the receptor by flavonoids would be confirmed, the evidence of type II EBS in osteoblast-like cells could suggest a direct action of ipriflavone and others flavonoids on bone density in postmenopausal osteoporosis.

Effect of skeletonization of the internal thoracic artery on vessel wall integrity.

BACKGROUND: This study was conceived to evaluate the effect of internal thoracic artery (ITA) skeletonization on vessel wall integrity. METHODS: Forty consecutive patients undergoing coronary artery bypass were randomized to receive a skeletonized (n = 22) or a pedicled (n = 18) ITA graft. ITA harvesting was performed by 2 experienced surgeons using the same instrumentation and technique. Specimens were examined by light and electron microscope in order to assess vascular wall integrity. A specific immunohistochemical staining and a computerized method were used to quantify the degree of endothelial integrity after surgical preparation. RESULTS: Morphologic analysis revealed 2 cases of limited subadventitial hemorrhage (one for each group) and no case of major arterial damage. Immunohistochemical staining demonstrated an extremely high degree of maintenance of the endothelial integrity in both groups (97.2% +/- 1.9% in the skeletonized and 96.8% +/- 2.1% in the pedicled one; p = 0.53). CONCLUSIONS: Skeletonization does not affect ITA wall integrity in humans submitted to coronary artery bypass procedures.

Central and peripheral myelin in the rat cochlear and vestibular nerves.

In the bipolar neurons of vertebrate cochlear and vestibular nerves, the myelin envelopes without interruption the axon, the perikaryon and the dendrite. The perikaryal myelin is thin and partially loose, whereas axon and dendrite are enveloped by compacted myelin. The expression of protein 0 and myelin basic protein, constituents of peripheral and central myelin respectively, has been investigated in the rat by immunohistochemical study at the light microscopic level. Our data indicate that both in the cochlear and vestibular nerves the myelin of the perikaryon and dendrite is composed by specific peripheral myelin proteins. The axon segment between the perikaryon and the transitional zone expresses peripheral myelin proteins in the cochlear nerve, while both types of myelin proteins are present in the vestibular nerve. Between the transitional zone and the brainstem the myelin of the axon is exclusively of the central type. The peripheral-central myelin transitional zone is in close proximity to the axonal pole in the vestibular ganglion cells, while in the cochlear nerve it is near the spiral foramina, at variable distance from the axonal pole of ganglion cells.

Protease inhibitors in mouse skeletal muscle: tissue-associated components of serum inhibitors and calpastatin.

The proteinase inhibitor set in skeletal muscle is poorly characterized at present. This study was aimed to investigate in mouse skeletal muscle 1) the tissue-associated counterpart, if any, of serum protease inhibitors (which may also play antiproteolytic functions in tissues) and 2) calpastatin, a tissue inhibitor of calcium-activated neutral proteases (calpains). Triton-extracts were prepared from muscle homogenates of mice, which had been perfused extensively with phosphate buffered saline (PBS) (under deep anesthesia) to remove blood inhibitors. Among various inhibitors tested, the following muscle-associated inhibitors were identified by western-blotting: alpha-2-macroglobulin (185, 165, 35 kDa), alpha-1-antitrypsin (52 kDa), inter-alpha-trypsin inhibitor (220, 180 kDa) and calpastatin (70 kDa). Combined light microscope and confocal immunohistochemical experiments revealed that, in all muscles examined (soleus, plantaris, extensor digitorum longus) the above specific immunoreactivities were localized outside the muscle fibers (in periendomysium, blood vessel wall) as well as within them. Inter-alpha-trypsin inhibitor, however, completely lacked the intracellular localization. This wide distribution of proteinase inhibitors suggests that numerous muscular structures may be normally protected from unwanted proteolysis, thus providing an essential background for further studies on pathological models with altered proteolysis (m. dystrophy, denervation atrophy, etc.).

Immunohistochemical study of the corpora cavernosa of the human clitoris.

The microarchitecture of the corpora cavernosa of the human clitoris was investigated by immunohistochemistry. The distribution pattern of the nerve network was demonstrated by S-100 and neuron specific enolase immunoreactivity. Vascular and nonvascular muscle cells were identified by desmin and/or vimentin expression, and fibroblasts and endothelial cells by vimentin immunoreactivity. The findings show that tissue organisation in the corpora cavernosa of the clitoris is essentially similar to that of the penis except for the absence of the subalbugineal layer interposed between the tunica albuginea and erectile tissue. This has functional implications, suggesting that the clitoral erection cycle differs from that of the penis.

Evidence for tissue-associated alpha(2) macroglobulin in mouse skeletal muscle.

Alpha(2)-Macroglobulin (alpha(2)M), a major serum protease inhibitor, was localized in mouse skeletal muscle by immunoperoxidase histochemistry. In all muscles examined (mm. soleus, plantaris, and extensor digitorum longus) specific immunoreactivity occurred diffusely in extracellular structures (periendomysium, blood vessel wall) as well as inside about a half of the muscle fibers. This localization pattern did not change substantially by extensively perfusing deeply anesthetized mice with phosphate buffered saline (PBS) to remove serum alpha(2)M. In release experiments on fresh (nonfixed) cryostat sections, specific immunoreactivity persisted after an extensive prewash with PBS (up to 5-6 h), but a new specific staining appeared inside those fibers that were originally negative. Western blotting experiments were negative on the soluble fraction of muscle homogenate, thus confirming that the perfusion procedure was effective in removing serum alpha(2)M. By contrast, three specific bands (185, 165, and 35 kDa) appeared in detergent-solubilized extracts (0.3% Triton X-100), indicating the occurrence of tissue-associated alpha(2)M. Confocal immunofluorescence microscopy revealed that the intracellular specific staining was associated to a longitudinal network, probably corresponding to the sarcoplasmic reticulum. A multifunctional role of alpha(2)M in skeletal muscle was hypothesized.

Immunohistochemical detection of three serum protease inhibitors in mouse skeletal muscle by confocal laser scanning microscopy.

The tissue-associated counterpart of some plasmatic protease inhibitors has been studied in mouse skeletal muscle by combining immunoperoxidase confocal microscopy and Western blot analysis. To remove serum contamination all experiments were performed on C57 BL/10 adult mice perfused extensively with physiological solution under deep anesthesia. The following serum inhibitors were investigated in skeletal muscle by immunoperoxidase staining: alpha-2-macroglobulin (alpha2M), antithrombin III (ATIII) and inter-alpha-trypsin inhibitor (ITI). The resulting localization patterns were analysed by laser transmittance scanning at 488 nm using a confocal microscope. Images obtained from a series of optical sections were then digitally intensified by a computerized program, allowing detection of even negligible amounts of immunoreaction product. In all muscles examined (soleus and extensor digitorum longus mm.) an extracellular (endomysial) localization was apparent for all inhibitors. By contrast remarkable differences were observed for the intracellular component: in fact alpha2M was present in about a half of the muscle fibers; ATIII was present inside all fibers; intracellular ITI was completely absent. Western blotting analysis of muscle homogenate was performed to biochemically characterize the above immunoreactivities. In preliminary experiments alpha2M-related immunoreactivity could not be found in the soluble fraction of perfused muscle, confirming an absence of serum contamination after in vivo perfusion. By contrast experiments on detergent-solubilized extracts (0.3% Triton X-100) revealed that tissue-bound alpha2M consisted of two main bands (168-166 KDa) and a minor component (35 KDa); ATIII of a single band (50 KDA); ITI of four bands (180, 50, 45, 40 KDa). These results confirmed that the specific immunoreactivities visualized by morphological techniques corresponded to muscle-associated plasmatic inhibitors. The present data suggest that in mouse skeletal muscle i) numerous tissue-associated plasmatic inhibitors may protect the extracellular matrix from an excess of proteolysis; ii) a more restricted set of inhibitors may be also involved in the down-regulation of intracellular proteolytic processes.

Distribution and developmental expression of lamin-like immunoreactivity in the central nervous system.

Lamins are the major proteic constituents of the nuclear lamina, the innermost layer of the nuclear membrane. The immunolocalization of lamins in the rat central nervous system was studied using polyclonal antibodies. Besides an ubiquitarious localization in the nuclear membranes of neurons and glial cells, an intense lamin-like immunoreactivity was found in the soma and dendrites of cerebellar Purkinje cells. The same specific reaction was also observed in the human cerebellum. Experiments performed in newborn animals demonstrated that the cytoplasmic expression of lamins in Purkinje cells begins during postnatal development.

The ultrastructure of peripheral neurofibroma: the role of mast cells and their interaction with perineurial cells.

The authors analyze the ultrastructure of mast cells and perineurial cells when both are present in neurofibroma of the nerve sheath. Samples of pathologic tissue taken from three patients with neurofibroma of a peripheral nerve sheath were analyzed by light and transmission electron microscopy. The observations document the characteristics of the tumor cells (Schwann cells and perineurial cells) as well as the presence of numerous mast cells, typically in close contact with the perineurial cells and never with the Schwann cells. Many electron-dense vesicles were found between the cells; these vesicles are created when the cell membrane of the mast cell buds, and then they come into contact with the adjacent perineurial cell. Endocytosis vesicles are often present in the cytoplasm of perineurial cells. Analysis of these observations led the authors to assume the existence of a metabolic interaction between the two cell type in contact with each other and an active role of the mast cells in the evolution of the tumor. The following two theories are plausible: either the mast cells actively stimulate tumor growth, or they alter the phenotype of the tumor cell. These findings could have interesting clinical applications. The use of treatment protocols which inhibit mast cell activity could, in theory, stop either the proliferation of the neurofibroma or its malignant transformation.

Ultrastructural analysis of the adaptation of articular cartilage to mechanical stimulation.

Ultrastructural analysis was conducted on samples of articular cartilage taken from both load-bearing and non-load-bearing areas with the aim of evaluating the morphologic adaptation of the articular cartilage to mechanical stimulation and identifying the mechanisms of interaction of the chondrocyte and the matrix. Through this analysis we were able to better define the adaptation process of the cartilage as well as the modalities of mechanical stress transmission. We believe that the complex formed by the chondrocyte, the pericellular matrix, and the pericellular capsule constitutes the biomechanical unit of the articular cartilage which serves as the sensor and transducer of mechanical stress. The arrangement of the collagen fibers and the proteoglycans which make up the pericellular capsule and membrane around the chondrocyte can be compared, from a mechanical standpoint, to a dynamic structure constructed in order to absorb the load stresses and protect the internal environment. From a biological standpoint, these are comparable to an extracellular-scaffold constructed with the aim of mediating the interaction between the chondrocyte and the territorial and inter-territorial compartments.