Application of tissue microarrays for receptor immunohistochemistry in breast carcinoma

Abstract: The current treatment of breast cancer, the most frequent malignancy found in females, requires the study of biomarkers. The standard set of these includes at least an estrogen receptor, a progesterone receptor and a HER2 receptor, although many other factors have been shown to contribute to the prognosis. Tissue microarrays have been introduced to decrease costs and workload of immunohistochemistry applied to large collections of samples. The aim of the study was to test the performance of this technology on three basic biomarkers of breast carcinoma in 106 cases of invasive breast carcinoma. Tissue microarrays composed of 3 cores sized 0.6 mm per case were constructed and stained by standard immunohistochemistry. The results were assessed on virtual slides created with an Aperio scanner. A sensitivity and specificity of 0.83 and 0.88 was obtained for the estrogen receptor, 0.76 and 0.88 for the progesterone receptor, 0.69 and 0.96 for HER2. In conclusion, TMA technology may give results comparable to the diagnosis based on whole sections, and the clinicopathologic correlations for the immunohistochemistry performed by both methods are fairy similar

CD207+/langerin positive dendritic cells in invasive and in situ cutaneous malignant melanoma

Introduction: Dendritic cells are crucial for cutaneous immune response. Their role in melanoma progression is however a matter of controversy. Material and methods: The number of dendritic cells within epidermis and in peri- and intratumoral location was analyzed using CD207 immunostain in 17 cases of in situ and 25 case of invasive melanoma. Results: Average peritumoral CD207+ cells count was 22.88 for all cases, 17.94 for in situ lesions and 26.24 for invasive cases. Average epidermal CD207+ cells count was 164.47 for all cases, 183.00 for in situ lesions and 150.78 - for invasive cases. In case of invasive melanomas, peritumoral CD207+ cells count was positively correlated with Breslow stage (R = 0.59) mitotic activity within the tumor (R = 0.62). Invasive cases with regression showed higher intratumoral and epidermal CD207+ cells count than the ones without (275.00 vs. 95.32 and 173.20 vs. 148.35) but lower peritumoral CD207+ cells count (17.60 vs. 27.26). Invasive cases with ulceration showed higher intratumoral and peritumoral CD207+ cells count than the ones without ulceration (220.08 vs. 55.67 and 44.17 vs. 9.69). Conclusions: CD207+ cells play a role in both progression and regression of melanoma but their exact role needs further studies

The composition of T cell infiltrates varies in primary invasive breast cancer of different molecular subtypes as well as according to tumor size and nodal status

T lymphocytes are the most numerous immune cells in tumor-associated infiltrates and include several subpopulations of either anticancer or pro-tumorigenic functions. However, the associations between levels of different T cell subsets and breast cancer molecular subtypes as well as other prognostic factors have not been fully established yet. We performed immunohistochemistry for CD8 (cytotoxic T cells (CTL)), FOXP3 (regulatory T cells (Tregs)), and GATA3 (Th2 cells) in 106 formalin-fixed paraffinembedded invasive breast cancer tissue samples and analyzed both the numbers and percentages of investigated cells in tumorassociated infiltrates. We observed that triple-negative breast cancer (TNBC) and HER2+ non-luminal breast tumors were associated with more numerous CTLs and Tregs and a higher Treg/Th2 cell ratio as compared with luminal A subtype. A higher Treg percentage was related to a decreased hormone receptor expression, an increase in the Ki67 level, a greater tumor size of luminal tumors, and the presence of lymph node metastases. Moreover, differences in the composition of T cell infiltrates were associated with HER2 status and histologic grade and type, and a distinct immune pattern was observed in tumors of different phenotypes regarding pT stage and nodal status. The results of our work show the diversity of T cell infiltrates in primary invasive breast cancers of different phenotypes and suggest that progression of luminal or non-luminal tumors is related to distinct tumorassociated T cell composition

Melanomas and dysplastic nevi differ in epidermal CD1c+ dendritic cell count

Background. Dendritic cells could be involved in immune surveillance of highly immunogenic tumors such as melanoma. Their role in the progression melanocytic nevi to melanoma is however a matter of controversy. Methods. The number of dendritic cells within epidermis, in peritumoral zone, and within the lesion was counted on slides immunohistochemically stained for CD1a, CD1c, DC-LAMP, and DC-SIGN in 21 of dysplastic nevi, 27 in situ melanomas, and 21 invasive melanomas. Results. We found a significant difference in the density of intraepidermal CD1c+ cells between the examined lesions; the mean CD1c cell count was 7.00/mm2 for invasive melanomas, 2.94 for in situ melanomas, and 13.35 for dysplastic nevi. The differences between dysplastic nevi and melanoma in situ as well as between dysplastic nevi and invasive melanoma were significant. There was no correlation in number of positively stained cells between epidermis and dermis. We did not observe any intraepidermal DC-LAMP+ cells neither in melanoma in situ nor in invasive melanoma as well as any intraepidermal DC-SIGN+ cells in dysplastic nevi. Conclusion. It was shown that the number of dendritic cells differs between dysplastic nevi, in situ melanomas, and invasive melanomas. This could eventually suggest their participation in the development of melanoma

The relationship between breast cancer molecular subtypes and mast cell populations in tumor microenvironment

Mast cells (MCs) are a part of the innate immune system. The MC functions toward cancer are partially based on the release of chymase and tryptase. However, the MC effect on breast cancer is controversial. The aim of our study was to investigate the presence of MCs in breast cancer tumors of different molecular subtypes and their relationships with other pathological prognostic factors. Tryptase- and chymase-positive mast cell densities were evaluated by immunohistochemistry in 108 primary invasive breast cancer tissue samples. Positive cells were counted within the tumor bed and at the invasive margin. For all analyzed MC subpopulations, we observed statistically significant differences between individual molecular subtypes of breast cancer. The significantly higher numbers of intratumoral chymase- and tryptase-positive mast cells were observed in luminal A and luminal B tumors compared to triple-negative and HER2+ non-luminal lesions. A denser MC infiltration was associated with lower tumor grade, higher ER and PR expression, lower proliferation rate as well as the lack of HER2 overexpression. The results obtained in our study indicate a possible association of chymase- and tryptase-positive MCs with more favorable cancer immunophenotype and with beneficial prognostic indicators in breast cancer

Prostate cancer with different ERG status may show different FOXP3-positive cell numbers

Prostatic carcinoma is the most frequent cancer in males in the Western world. A significant proportion of these cancers have a recurrent translocation involving ETS family genes, which leads to the overexpression of ERG transcription factor. Prostate cancers, which bear this mutation, differ in a number of features, including tumor microenvironment. One of the components of the tumor microenvironment is FOXP3 positive lymphocytes, which may participate in breaking immunosurveillance and promoting tumor growth. The aim of the study was to analyze the relationships between ERG expression, number of FOXP3 positive cells and other features of the tumor. The study group consisted of 65 cases. Tissue microarrays composed of 2 mm tissue cores were used for immunohistological evaluation. Immunohistochemistry for ERG and FOXP3 was performed according to the routinely applied protocol. The FOXP3 positive cells were counted and the results were expressed as the number of cells per mm2. The average number of FOXP3 positive cells was 33.30/mm2 for all cases, 21.43/mm2 for the ERG negative and 42.28/mm2 for the ERG positive group (p < 0.02). There were no significant relationships between FOXP3 positive cell count and any other parameters studied. Our results suggest that the immune response may differ between ERG negative and ERG positive prostatic carcinomas

Application of tissue microarrays to the assessment of estrogen, progesterone and HER2 receptors in breast carcinoma

Rak piersi w Polsce od lat zajmuje pierwsze lub drugie miejsce wśród nowotworów, zarówno pod względem liczby zachorowań jak i zgonów wśród kobiet. Histologiczny podział wyróżnia: raka przedinwazyjnego (in situ) przewodowego i zrazikowego oraz raka inwazyjnego (naciekającego) przewodowego i zrazikowego. Rak inwazyjny przewodowy stanowi ok. 85 – 90% wszystkich raków inwazyjnych sutka; w tej grupie wyszczególnia się także inne, rzadziej spotykane podtypy tego nowotworu, np.: rak rdzeniasty, rak koloidowy i rak cewkowaty.W diagnostyce i ocenie zaawansowania tej choroby kluczową rolę odgrywa badanie histopatologiczne. Obecnie coraz większe znaczenie w tym zakresie ma analiza markerów biologicznych, ze szczególnym uwzględnieniem białkowych markerów komórkowych. Analiza steroidowych receptorów hormonalnych (ER - estrogenowych i PR - progesteronowych) oraz błonowych receptorów HER2 ma dla pacjentów z rakiem sutka wartość zarówno prognostyczną jak i predykcyjną. Stwierdzona ekspresja ER i PR uzasadnia zastosowanie w terapii choroby tamoksyfenu, fulvestrantu lub inhibitorów aromatazy, natomiast nadekspresja HER2 jest wskazaniem do wdrożenia leczenia trastuzumabem (Herceptin ®). Zastosowanie techniki mikromacierzy tkankowych (TMA) w połączeniu z różnymi metodami badawczymi może znacznie ułatwić badania naukowe nad markerami biologicznymi (przeprowadzane na szeroką skalę) oraz przyspieszyć czas analizy i zmniejszyć jej koszty w przypadku rutynowej diagnostyki raka piersi. W niniejszej pracy przeanalizowano skuteczność wykorzystania TMA w połączeniu z badaniem immunohistochemicznym do oceny ekspresji receptorów ER, PR i HER2 w raku piersi. Do badań wykorzystano materiał tkankowy pobrany od 106 osób ze zdiagnozowanym inwazyjnym rakiem przewodowym. Przygotowując multibloczki z każdego preparatu pobrano po 3 cylindry tkankowe. Wybarwione preparaty zeskanowano (ScanScope, Aperio Technologies, USA), a ekspresję poszczególnych receptorów w cylindrach oceniono na preparatach wirtualnych (ImageScope 10, Aperio Technologies, USA). Analizę statystyczną przeprowadzono przy pomocy programu Statistica 10 (StatSoft Inc., USA), wykorzystując test χ2, test dokładny Fishera oraz współczynniki korelacji: Pearsona i gamma. Dodatkowo w programie GraphPad (http://graphpad.com/quickcalcs/) obliczono wartość statystyki Kappa. Poziom istotności przyjęto na poziomie 0,05. Wyniki uzyskane przy użyciu TMA porównano z odczytami uzyskanymi w analizie pełnego skrawka tkankowego.Dla wszystkich trzech analizowanych receptorów (ER, PR i HER2) wyniki, zarówno w ocenie poszczególnych cylindrów jak i całych przypadków, wykazywały zgodność w analizie obiema technikami. Stopień zgodności wyników między tymi technikami dla ER i PR, przy ocenie stanu receptora jako dodatni/ujemny, były określane jako „duże”. W przypadku analizy receptora HER2 duży stopień zgodności uzyskano, gdy jego ekspresję oceniano w skali od 0 do 3+, zaś umiarkowany przy ocenie w kategoriach dodatni/ujemny. Ponadto zanotowano dość dużą ilość strat materiału w obrębie TMA (od 25,8% dla PR do 38,05% dla HER2). Otrzymane wyniki potwierdzają skuteczność zastosowania TMA do oceny wyżej wymienionych receptorów w diagnostyce raka piersi.Breast carcinoma is the most frequent female cancer worldwide. Histologically it can be classified as in situ (ductal or lobular) or invasive (ductal or lobular) carcinoma. Invasive ductal carcinoma constitutes about 85 – 90% of all invasive breast cancer cases; in this group special subtypes can be distinguished, e.g.: medullary, colloid and tubular carcinoma.In diagnosis and staging of cancer histopathology plays the crucial role. Currently, analysis of biomarkers, especially cellular proteins, is becoming of prime importance. Assessing steroid hormonal receptors (ER – estrogen and PR – progesterone receptor) and membrane HER2 receptor has a predictive and a prognostic value breast cancer patients. Positive ER and PR status allows treatment with tamoxifen, fulvestrant and aromatase inhibitors, while overexpression of HER2 receptor allows treatment with trastuzumab (Herceptin ®).Tissue microarray (TMA) coupled with immunohistochemistry or FISH shortens time and lowers costs of biomarker analysis. In this study TMAs were used for assessing ER, PR and HER2 expression in breast cancer samples and effectiveness of such approach was evaluated. Paraffin-embedded tissue from 106 patients with diagnosed invasive ductal breast carcinoma was used for the study. The TMA was constructed with Tissue Microarrayer semi-automatic device, with 3 cores for each case. Immunohistochemistry was performed using standard protocol. The slides were scanned into virtual slides with ScanScope device (Aperio Technologies, USA), and virtual slides were used for assessing the expression of ER, PR and HER2 receptors, otherwise by standard methodology. Statistical analysis was done with Statistica 10 (StatSoft Inc., USA), using the χ2 test, Fisher’s test and Pearson’s and gamma correlation coefficient. GraphPad (http://graphpad.com/quickcalcs/) was used for calculating kappa statistics. The level of significance was set to 0,05. The results obtained with TMA were compared with previously done routine receptor assessment. For each of 3 analyzed receptors (ER, PR and HER2) similar results were obtained both with TMA and standard method, analyzing both individual tissue cores or entire cases. The concordance rate between those techniques was particularly high for ER and PR if positive or negative result of the test was analyzed. For HER2 high concordance rate was obtained when its expression was estimated in 0 to 3+ scale, but concordance was estimated as moderate if positive versus negative status was assessed. Relatively high number of cores was lost from the TMA (from 25,8% for PR to 38,05% for HER2). These results confirm that TMA might be a valuable choice for of analysis of PR, ER and HER2 in breast cancer