Diagnostic value of intraoperative histopathological examination of the sentinel nodes in breast cancer and skin melanoma—Preliminary results of single centre retrospective study

Objective

Intraoperative histopatological examination of the sentinel nodes enables selection of patients who need dissection of the regional lymphatic system during the same operation. The aim of this study is to evaluate the diagnostic value of intraoperative histopathological examination of the sentinel nodes in breast cancer and skin melanoma. Intraoperative histopathology of the sentinel nodes as a diagnostic method is used in patients with melanoma and breast cancer. Recent studies have proved it to be an effective method for evaluating the nodes in the final histopathology. Intraoperative histopathological examination of the sentinel nodes is not performed routinely and there is no clear position on this issue. In this paper we try to prove that intraoperative test gives patients the simultaneous benefits of removal of regional lymph nodes metastases and earlier initiation of adjuvant therapy.

Methods

The study comprises 137 patients with breast cancer and 35 patients with malignant skin melanoma. Sentinel nodes were intraoperatively sectioned and examined by means of the imprint method and frozen section evaluation. The patients with positive sentinel nodes underwent immediate dissection of regional lymph nodes. Those with negative sentinel nodes diagnosed in the intraoperative examination, but positive in final pathologic results, underwent subsequent dissection of regional lymph nodes.

Results

60 sentinel lymph nodes were found in 35 patients with skin melanoma. In 3 patients, 3 sentinel lymph nodes were false negative in the intraoperative histopathological examination. No false positive sentinel lymph nodes were found. 249 sentinel lymph nodes were found in the intraoperative histopathological examination in 137 patients with breast cancer. There were no false positive sentinel nodes, but there were 7 false negative sentinel nodes. In this study, only 5 (3.6%) patients with breast cancer and 3 (8.5%) patients with skin melanoma required another regional operation.

Conclusion

The method of intraoperative histopathological evaluation of the sentinel nodes enables identification of metastases in these lymph nodes and gives a possibility to carry out a one-step regional lymphadenectomy and start the adjuvant therapy earlier.     

Lymph node metastases of papillary thyroid cancer in immunohistochemical and molecular examination--preliminary report

BACKGROUND: Total thyroidectomy with lymphadenectomy is the most typical operation in a case of papillary thyroid cancer. Range of lymph node resection still remains a matter of controversy. In some publications treatment of lymph node metastases doesn't affect survival, so only selective lymph node resection is the extended enough operation. The others remark that, local relapse- the worst prognostic factor, appears the most often in the lymph nodes, so they suggest more aggressive treatment. To solve that problem we try to find more sensitive methods to examine lymph nodes. AIM: To compare the results of detection lymph node metastases of papillary thyroid cancer by immunohistochemistry with the results of RT- PCR for thyroglobulin (Tg) mRNA. MATERIAL AND METHODS: Each of one hundred eighty four cervical lymph nodes obtained from 24 patients, operated in our Department was divided into 2 halves: one was used for conventional histopathology and immunohistochemistry, the other part was investigated by RT- PCR for Tg mRNA. Immunohistochemical staining for Tg was performed on formalin-fixed, paraffin-embedded sections with anti-Tg antibodies. RESULTS: According to routine, histopathological examination 8 (33.5%) patients had involved lymph nodes. One hundred correspondence of the results of immunohistochemistry and histopathology was observed. We obtained different results of examination of the lymph nodes in 6 (25%) patients. In four patients (16.7%) RT-PCR was more sensitive in detection of positive lymph nodes, in two patients (8.3%) it revealed less metastasized lymph nodes than immunohistochemistry. The remaining 18 patients didn't have any differences, fourteen (58.3%) of them had the negative lymph nodes and four (16.7%) had positive, the same lymph nodes in all examinations. Finally, according to RT-PCR 10 (41.7%) of the patients had metastasized lymph nodes. COCLUSION: Tg RT-PCR is a sensitive method of detection of papillary thyroid cancer cells and may help to detect the metastases of papillary thyroid cancer in regional lymph nodes.     

Increased Lymph Node Yield in Colorectal Cancer Is Not Necessarily Associated with a Greater Number of Lymph Node Positive Cancers

The presence of lymph node metastasis is a key prognostic factor in colorectal cancer and lymph node yield is an important parameter in assessing the quality of histopathology reporting of colorectal cancer excision specimens. This study assesses the trend in lymph node evaluation over time in a single institution and the relationship with the identification of lymph node positive tumours. It compares the lymph node yield of a contemporary dataset compiled from the histopathology reports of 2178 patients who underwent surgery for primary colorectal cancer between 2005 and 2012 with that of a historic dataset compiled from the histopathology reports of 1038 patients who underwent surgery for colorectal cancer at 5 yearly intervals from 1975 to 2000. The mean lymph node yield was 14.91 in 2005 rising to 21.38 in 2012. In 2012 92.9% of all cases had at least 12 lymph nodes examined. Comparison of the mean lymph node yield and proportion of Dukes C cases shows a significant increase (Pearson correlation = 0.927, p = 0.001) in lymph node yield while there is no corresponding significant trend in the proportion of Dukes C cases (Pearson correlation = −0.138, p = 0.745). This study shows that there is increasing yield of lymph nodes from colorectal cancer excision specimens. However, this is not necessarily associated with an increase number of lymph node positive cancers. Further risk stratifying of colorectal cancer requires consideration of other pathological parameters especially the presence of extramural venous invasion and relevant biomarkers.     

Dynamics of lymph node function in disordered innervation

A mechanotron 6MX1C was used to study lymphodynamic indices of the dog popliteal lymph nodes after cutting the ischiatic and femoral nerves in the left limb at 1 week, 3 and 5 months. The lymph nodes of the contralateral limb and the lymph nodes from intact dogs were used as controls. It has been demonstrated that denervation causes acceleration of the periods of the filling and emptying of the lymph nodes both in the denervated and contralateral limbs as compared with the initial period. The amplitude of fluctuations under maximal filling of the lymph nodes decreases significantly at the long-term postoperative periods. The lymphodynamic changes seem to be determined by pathomorphological alterations in lymph node tissues.     

Urokinase-type plasminogen activator is required for the generation of a type 1 immune response to pulmonary Cryptococcus neoformans infection.

Urokinase-type plasminogen activator (uPA)(-/-) mice cannot mount protective host defenses during infection with the opportunistic yeast Cryptococcus neoformans (52D). Because effective host defense against C. neoformans requires specific immune responses and the generation of type 1 (T1) cytokines, we determined how the absence of uPA impacts these processes. Wild-type (WT) and uPA(-/-) mice were inoculated with C. neoformans. Macrophage antifungal activity was assessed histologically, T lymphocyte responses in vivo and proliferation in vitro were quantified, and cytokine concentrations were determined by ELISA. uPA(-/-) macrophages have impaired antimicrobial activity. Regional lymph nodes of infected uPA(-/-) mice contained fewer cells than WT, suggesting impaired T cell proliferation in response to the pathogen in vivo. In vitro, uPA(-/-) T lymphocytes had impaired proliferative responses to C. neoformans rechallenge compared with WT. Infected WT mice generated T1 cytokines in the lung, characterized by high levels of IFN-gamma and IL-12. uPA(-/-) mice had decreased levels of IFN-gamma and IL-12, and increased IL-5, a type 2 cytokine. In the absence of uPA, the cytokine profile of regional lymph nodes shifted from a T1 pattern characterized by IFN-gamma and IL-2 to a weak, nonpolarized response. We conclude that in the absence of uPA, lymphocyte proliferative responses are diminished, and mice fail to generate protective T1 cytokines, resulting in impaired antimicrobial activity. This study provides novel evidence that uPA is a critical modulator of immune responses and of immune cell effector functions in vivo.     

Senile changes in human lymph nodes

BACKGROUND: The degenerative process of lymph nodes is poorly documented. METHODS: 161 lymph nodes of seven fresh and one embalmed human cadavers in the head and neck were studied. We used 6% hydrogen peroxide, lead oxide injectant, and radiographs to demonstrate lymphatic vessels, and found both solidified and transparent lymph nodes. They were removed, fixed in 10% formalin and sent for histopathology cross section. RESULTS: Thirty-eight solidified and 123 transparent lymph nodes were found. A series of histopathological sections show the degenerative process is variable and continuous. Senile involution affects all elements of the lymph node including the cortex, the medulla, and the architecture. CONCLUSION: This study provides actual anatomical and histopathological images of lymph nodes in different degenerative stages in the head and neck region. It may help explain some clinical conditions in the elderly, especially their diminished immunological response to infection and cancer metastasis.     

Initial axillary staging of breast cancer using ultrasound‐guided fine needle aspiration: a liquid‐based cytology study

A. Schiettecatte, C. Bourgain, C. Breucq, N. Buls, V. De Wilde and J. de Mey
Initial axillary staging of breast cancer using ultrasound‐guided fine needle aspiration: a liquid‐based cytology study Objective: To evaluate the preoperative detection of axillary metastasis combining ultrasound (US)‐guided fine needle aspiration cytology (FNAC) and liquid‐based cytology (Surepath^®) to reduce sentinel node procedures. Methods: In total, 148 patients with clinically negative lymph nodes and no preoperative therapy were included. All patients underwent preoperative ultrasound of the axilla with FNAC if suspicious lymph nodes were found. Complete axillary lymph node dissection was performed at primary surgery when FNAC was positive. All other patients underwent a sentinel node procedure. Results: US‐guided FNAC of the axilla revealed metastasis in 34 (23.0%) of the 148 patients. These 34 patients were 53.1% of all patients (n = 64) with proven axillary lymph node involvement. In 66 patients (44.6%), both ultrasound and histopathology were negative. Overall sensitivity of US‐guided FNAC was 50.0%, specificity 100%, positive predictive value 100% and negative predictive value 70.2%. In T1 tumours, all patients referred for sentinel node procedure were node‐negative. The correlation between malignant FNAC and histopathology was 100%. US‐guided liquid‐based FNAC in patients with no clinically positive lymph nodes reduced the necessity for a sentinel node procedure by 23.0%. Conclusions: We advocate that US‐guided fine needle aspiration (FNA) combined with liquid‐based cytology of axillary lymph nodes should be included in the preoperative staging of breast cancer.     

Preliminary report of histopathology associated with infection with tongue worms in Australian dogs and cattle

Tongue worms utilise herbivorous mammals as intermediate hosts and reside in the nasopharynx of carnivores as their definitive hosts. A recent study in south eastern Australia showed an unexpectedly high infection (67%) of wild dogs with these parasites. The present study aimed at determining the pathogenicity of the parasite in both definitive (dog) and intermediate (cattle) hosts by histopathology. The definitive host showed multifocal haemorrhage of the interstitium of the nasal mucosa, multifocal mucosal erosion, congestion and haemorrhage, with haemosiderin laden macrophages present in those foci and distortion and destruction of the nasal mucosa. Histopathologic examination of lymph nodes from an infected cow showed diffuse eosinophilic granulomatous necrotising lymphadenitis and perinodal panniculitis with intralesional parasitic remnants and comparatively large numbers of eosinophils. A large, ~300–500?μm diameter, area of necrosis was also observed in one lymph node. This is the first time a study has been undertaken in Australia to determine the pathogenicity of tongue worms in both their definitive and intermediate hosts. This is a preliminary study and to properly estimate the health impact of infection with this pathogenic parasites on Australian production and companion animals more studies are necessary.     

Use of monoclonal antibodies to human lymphocytes to identify lymphocyte subsets in lymph nodes of the rhesus monkey and the dog

Using commercially available monoclonal antibodies that bind to human lymphocyte subsets, we examined lymph nodes from the rhesus monkey and the dog for their binding ability to these monoclonal antibodies. The avidin biotin-peroxidase immunostaining procedure was used, and the following antibodies were reactive in the rhesus monkey: Leu 4, Leu 3a, OKT4, Leu 2a, OKT8, T8, Leu 5b, T11, Leu 14, B1, and Leu 12. No immunoreactivity was observed in the dog lymph node except for moderate reactivity of OKT8. The following antibodies failed to react in both the rhesus monkey and the dog: OKT3, T1, T2, T1B, T4, T8A, Pan B, and T29/33. Kappa and lambda immunoglobulin light chains were positive in both the dog and monkey.     

Cytologic diagnosis of axillary lymph node metastasis in breast cancer

OBJECTIVE: To compare imprint cytology with histology as a method for rapid intraoperative diagnosis of axillary lymph node metastasis in breast cancer. STUDY DESIGN: We evaluated imprint cytology, comparing it with histopathology. A sample of 635 axillary lymph nodes was studied by imprint cytology using both Giemsa stain and hematoxylin-eosin. The results were compared with each other and with those of histopathologic examination. RESULTS: The Giemsa stain method, as compared to histopathology, had 94% accuracy, 97% sensitivity, 90% specificity and 94% positive prognostic value. The hematoxylin-eosin stain method was less accurate than the Giemsa stain method as compared to histopathology (accuracy 91%, sensitivity 96%, specificity 83% and positive prognostic value 92%). CONCLUSION: These data confirm the value of imprint cytology as a rapid, reliable method of intraoperative assessment of axillary lymph node metastasis in breast cancer. It results in better staging of the disease. It can be used intraoperatively, as an alternative to frozen section, if a pathology laboratory is not available, to exclude stage I patients from further treatment.     

Distinct roles for IL-4 and IL-10 in regulating T2 immunity during allergic bronchopulmonary mycosis

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis that has also been used to test a number of immunomodulatory agents. Our objective was to determine the role of IL-4 and IL-10 in the development/manifestation of the T2 response to C. neoformans in the lungs and lung-associated lymph nodes. In contrast to wild-type (WT) mice, which develop a chronic infection, pulmonary clearance was significantly greater in IL-4 knockout (KO) and IL-10 KO mice but was not due to an up-regulation of a non-T cell effector mechanism. Pulmonary eosinophilia was absent in both IL-4 KO and IL-10 KO mice compared with WT mice. The production of IL-4, IL-5, and IL-13 by lung leukocytes from IL-4 KO and IL-10 KO mice was lower but IFN-gamma levels remained the same. TNF-alpha and IL-12 production by lung leukocytes was up-regulated in IL-10 KO but not IL-4 KO mice. Overall, IL-4 KO mice did not develop the systemic (lung-associated lymph nodes and serum) or local (lungs) T2 responses characteristic of the allergic bronchopulmonary C. neoformans infection. In contrast, the systemic T2 elements of the response remained unaltered in IL-10 KO mice whereas the T2 response in the lungs failed to develop indicating that the action of IL-10 in T cell regulation was distinct from that of IL-4. Thus, although IL-10 has been reported to down-regulate pulmonary T2 responses to isolated fungal Ags, IL-10 can augment pulmonary T2 responses if they occur in the context of fungal infection.     

Visceral leishmaniasis with cardiac involvement in a dog: a case report

A dog presented with cutaneous nodules, enlarged lymph nodes and oedema in limbs, face and abdomen. The diagnosis of visceral leishmaniasis was established by identification of Leishmania amastigotes within macrophages from skin and popliteal lymph node biopsies. At necropsy, lesions were found in different organs, but it was particularly striking to observe large areas of pallor in the myocardium. Histological examination revealed an intense chronic inflammatory reaction in many organs, and numerous macrophages were found to contain amastigote forms of Leishmania. The inflammatory reaction was especially severe in the heart, where large areas of the myocardium appeared infiltrated with huge numbers of mononuclear immune cells, causing cardiac muscle atrophy and degeneration. Despite the severe inflammation, the number of parasitized macrophages was low in the myocardium, as revealed by immunohistochemical staining of Leishmania amastigotes. Because cardiac involvement is not usually described in this condition, this dog represents a very rare case of canine visceral leishmaniasis with affection of the myocardium.     

Suppression of systemic lupus erythematosus in NZBWF1 mice infected with Hymenolepis microstoma

Intestinal helminths induce immune suppressive responses thought to regulate inflammatory diseases including allergies and autoimmune diseases. This study was designed to evaluate whether helminthic infections suppress the natural development of systemic lupus erythematosus (SLE) in NZBWF1 mice. Infection of NZBWF1 SLE-prone mice with two nematodes failed to establish long-lasting settlement. However, the Hymenolepis microstoma (Hm) rodent tapeworm successfully established long-term parasitization of NZBWF1 mice and was used to evaluate the suppressive effects of helminth infection. Ten-month-old NZBWF1 mice developed symptoms including autoantibody generation, proteinuria, glomerular histopathology, and splenomegaly, but mice infected with Hm at 2 months of age did not show any clinical signs. Furthermore, infection with Hm reduced lymphocyte activation and increased regulatory T cells in the spleen and mesenteric lymph nodes. These results indicate that infection with Hm protects NZBWF1 mice from naturally developing SLE and suggest that pathological immunity is attenuated, presumably because of the induction of regulatory T cells.     

Paracoccidioidomycosis in a Dog: Case Report of Generalized Lymphadenomegaly

Paracoccidioidomycosis (PCM) is a severe systemic mycosis, endemic in Latin America and highly prevalent in Brazil, where it ranks eighth as a mortality cause among infectious and parasitic diseases in humans. The disease in animals has been little explored. It is observed that armadillos can harbor the fungus at high frequencies, although the active disease has not been well documented in this wild mammal. Dogs are susceptible to experimental infection, and the naturally acquired PCM-disease was reported only recently in a dog from Brazil. The present work reports the second case of naturally acquired PCM in a 6-year-old female dog that presented emaciation, lymphadenomegaly, and hepatosplenomegaly. Biochemical and pulmonary radiographic evaluation did not reveal any abnormalities. PCM was diagnosed by clinical findings, culturing, immunohistochemistry, and histopathology of popliteal lymph node. The fungus was recovered from popliteal lymph node, and the molecular analysis showed respective sequencing similarities of 99 and 100% for 803 nucleotides of the Gp43 gene and 592 nucleotides from the ITS-5.8S region of Paracoccidioides brasiliensis. Immunohistochemistry revealed severe lymphadenitis and presented numerous yeasts, which reacted against the gp43 antibody. Histopathology revealed a severe granulomatous lymphadenitis associated with numerous single or multiple budding yeasts. After diagnosis, the dog was successfully treated with itraconazol for 2 years. Veterinarians should be aware of the importance of considering PCM for differential diagnosis, especially in dogs from PCM-endemic areas, whose monophagocytic system involvement is evident.     

Imaging Lymphatic System in Breast Cancer Patients with Magnetic Resonance Lymphangiography

Objective

To investigate the feasibility of gadolinium (Gd) contrast-enhanced magnetic resonance lymphangiography (MRL) in breast cancer patients within a typical clinical setting, and to establish a Gd-MRL protocol and identify potential MRL biomarkers for differentiating metastatic from non-metastatic lymph nodes.

Materials and Methods

32 patients with unilateral breast cancer were enrolled and divided into 4 groups of 8 patients. Groups I, II, and III received 1.0, 0.5, and 0.3 ml of intradermal contrast; group IV received two 0.5 ml doses of intradermal contrast. MRL images were acquired on a 3.0 T system and evaluated independently by two radiologists for the number and size of enhancing lymph nodes, lymph node contrast uptake kinetics, lymph vessel size, and contrast enhancement patterns within lymph nodes.

Results

Group III patients had a statistically significant decrease in the total number of enhancing axillary lymph nodes and lymphatic vessels compared to all other groups. While group IV patients had a statistically significant faster time to reach the maximum peak enhancement over group I and II (by 3 minutes), there was no other statistically significant difference between imaging results between groups I, II, and IV. 27 out of 128 lymphatic vessels (21%) showed dilatation, and all patients with dilated lymphatic vessels were pathologically proven to have metastases. Using the pattern of enhancement defects as the sole criterion for identifying metastatic lymph nodes during Gd-MRL interpretation, and using histopathology as the gold standard, the sensitivity and specificity were estimated to be 86% and 95%, respectively.

Conclusion

Gd-MRL can adequately depict the lymphatic system, can define sentinel lymph nodes, and has the potential to differentiate between metastatic and non-metastatic lymph nodes in breast cancer patients.     

Mycosis Due to Tropicoporus tropicalis (= Inonotus tropicalis) in a Domestic Dog

Mycopathologia - Mycelial basidiomycetes rarely produce mycoses in animals including humans. We report a case of a 9-year-old female mongrel dog with lesions in the prescapular lymph nodes. The...     

Murine superficial lymph node surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x10(6) cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.     

Chronic alcohol consumption decreases the percentage and number of NK cells in the peripheral lymph nodes and exacerbates B16BL6 melanoma metastasis into the draining lymph nodes

NK cells in the lymph nodes play important roles in inhibiting tumor metastasis into draining lymph nodes. Previously, we reported that chronic alcohol consumption interferes with NK cell trafficking from the bone marrow to the spleen. Herein, we found that alcohol consumption decreases the numbers of NK cells in lymph nodes. Adoptive transfer experiments indicated that continued exposure of donor splenocytes to alcohol inhibits NK but not T cell trafficking to lymph nodes. Alcohol did not negatively affect CCR7⁺ and CXCR3⁺ NK cells, but decreased the percentage and number of CD62L⁺ NK cells in the spleen, which are an important source of NK cell trafficking into the lymph nodes. These data suggest that modulation of the microenvironment associated with alcohol consumption impairs the trafficking of NK cells to lymph nodes. The decreased number of NK cells in the lymph nodes was associated with increased melanoma metastasis into the draining lymph nodes.