Comparison of three validated PD-L1 immunohistochemical assays in urothelial carcinoma of the bladder: interchangeability and issues related to patient selection.

Different programmed cell death-ligand 1 (PD-L1) assays and scoring algorithms are being used in the evaluation of PD-L1 expression for the selection of patients for immunotherapy in specific settings of advanced urothelial carcinoma (UC). In this paper, we sought to investigate three approved assays (Ventana SP142 and SP263, and Dako 22C3) in UC with emphasis on implications for patient selection for atezolizumab/pembrolizumab as the first line of treatment. Tumors from 124 patients with invasive UC of the bladder were analyzed using tissue microarrays (TMA). Serial sections were stained with SP263 and SP142 on Ventana Benchmark Ultra and with 22C3 on Dako Autostainer Link 48. Stains were evaluated independently by two observers and scored using the combined positive score (CPS) and tumor infiltrating immune cells (IC) algorithms. Differences in proportions (DP), overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen κ were calculated for all comparable cases. Good overall concordance in analytic performance was observed for 22C3 and SP263 with both scoring algorithms; specifically, the highest OPA was observed between 22C3 and SP263 (89.6%) when using CPS. On the other hand, SP142 consistently showed lower positivity rates with high differences in proportions (DP) compared with 22C3 and SP263 with both CPS and IC, and with a low PPA, especially when using the CPS algorithm. In conclusion, 22C3 and SP263 assays show comparable analytical performance while SP142 shows divergent staining results, with important implications for the selection of patients for both pembrolizumab and atezolizumab.

TSC loss is a clonal event in eosinophilic solid and cystic renal cell carcinoma: a multiregional tumor sampling study.

Eosinophilic, solid and cystic (ESC) renal cell carcinoma (RCC) is characterized by a solid and cystic architecture with cells showing abundant eosinophilic cytoplasm with hobnail arrangement and a cytokeratin 7-negative/cytokeratin 20-positive immunophenotype. Recent studies have suggested that bi-allelic events affecting TSC genes might play an important role for such tumors. However, only indirect evidence of the clonal origin of TSC mutation has been gathered so far. Therefore, in this paper we aimed to perform multi-regional tumor sampling molecular analysis in four ESC RCC cases that had been completely embedded, three sporadic and one occurring in a patient with tuberous sclerosis complex (TSC). Histologically, the 4 cases showed cystic and solid architecture and cells with abundant eosinophilic cytoplasm with cytoplasmic stippling and round to oval nuclei. Immunohistochemistry showed at least focal expression of cytokeratin 20 in all tissue samples and negative cytokeratin 7, as well as diffuse positivity for S100A1 and at least focal expression of cathepsin K in three out of four cases. The sporadic cases showed the same somatic TSC1 mutations in all tissue samples analyzed, while the TSC-associated case showed the same TSC1 alteration in both normal tissue and all tumor samples analyzed, proving the germline nature of the alteration. In conclusion, our data demonstrate that clonal TSC loss is a key event in ESC RCC and support considering ESC RCC as an entity given its distinct morphologic, immunophenotypical and molecular characteristics.

Impact of PD-L1 and PD-1 Expression on the Prognostic Significance of CD8+ Tumor-Infiltrating Lymphocytes in Non-Small Cell Lung Cancer.

The immune infiltrate within tumors has proved to be very powerful in the prognostic stratification of patients and much attention is also being paid towards its predictive value. In this work we therefore aimed at clarifying the significance and impact of PD-L1 and PD-1 expression on the prognostic value of CD8+ tumor infiltrating lymphocytes (TILs) in a cohort of consecutive patients with primary resected non-small cell lung cancer (NSCLC). Tissue microarrays (TMA) were built using one representative formalin fixed paraffin embedded block for every case, with 5 cores for each block. TMA sections were stained with PD-L1 (clone SP263), PD-1 (clone NAT105) and CD8 (clone SP57). Number of CD8+ cells per mm2 were automatically counted; median, 25th and 75th percentiles of CD8+ cells were used as threshold for statistical clinical outcome analysis and evaluated in patients subgroups defined by expression of PD-L1 and PD-1 within tumors. We found an overall strong prognostic value of CD8+ cells in our cohort of 314 resected NSCLC, especially in PD-L1 negative tumors lacking PD-1+ TILs, and demonstrated that in PD-L1 positive tumors a higher density of CD8+ lymphocytes is necessary to improve the prognosis. Our data strengthen the concept of the importance of the assessment and quantification of the immune contexture in cancer and, similarly to what has been carried on in colorectal cancer, promote the efforts for the establishment of an Immunoscore for NSCLC for prognostic and possibly predictive purposes.

Wilms' Tumor Primary Cells Display Potent Immunoregulatory Properties on NK Cells and Macrophages.

The immune response plays a crucial defensive role in cancer growth and metastasis and is a promising target in different tumors. The role of the immune system in Wilm's Tumor (WT), a common pediatric renal malignancy, is still to be explored. The characterization of the immune environment in WT could allow the identification of new therapeutic strategies for targeting possible inhibitory mechanisms and/or lowering toxicity of the current treatments. In this study, we stabilized four WT primary cultures expressing either a blastematous (CD56+/CD133-) or an epithelial (CD56-/CD133+) phenotype and investigated their interactions with innate immune cells, namely NK cells and monocytes. We show that cytokine-activated NK cells efficiently kill WT cells. However, after co-culture with WT primary cells, NK cells displayed an impaired cytotoxic activity, decreased production of IFNγ and expression of CD107a, DNAM-1 and NKp30. Analysis of the effects of the interaction between WT cells and monocytes revealed their polarization towards alternatively activated macrophages (M2) that, in turn, further impaired NK cell functions. In conclusion, we show that both WT blastematous and epithelial components may contribute directly and indirectly to a tumor immunosuppressive microenvironment that is likely to play a role in tumor progression.

PD-L1 expression in non-small cell lung cancer: evaluation of the diagnostic accuracy of a laboratory-developed test using clone E1L3N in comparison with 22C3 and SP263 assays.

Different studies have evaluated the comparability of various immunohistochemical assays for PD-L1 expression evaluation, with contrasting results. Besides the important issues related to analytic performance and comparability of validated assays, not all platforms are available in all laboratories; moreover, standardized assays are very expensive, and funding for PD-L1 testing is hard to obtain, especially in the research setting. One of the most widely used and inexpensive PD-L1 clones is E1L3N (Cell Signaling Technology, Danvers, MA), which is labeled for research use only. In this work, we wanted to further study and validate in a larger cohort the analytical performance of E1L3N clone on Ventana platform (Ventana Medical Systems, Tucson, AZ) and its comparability with assays SP263 and 22C3 run onto their dedicated platforms. Serial sections of tissue microarrays built from 165 cases of resected lung cancer were stained for E1L3N onto Ventana platform following a previously reported protocol and for 22C3 and SP263 assays onto their respective platforms following manufacturer's instructions. Overall, we found very high concordance when comparing E1L3N with SP263 at both 1% and 50% cutoffs. Lower concordance was found between E1L3N and 22C3 at both cutoffs; however, 100% sensitivity was found for E1L3N compared with both SP263 and 22C3 at 50% cutoff. Given the 100% sensitivity at 50% cutoff demonstrated by E1L3N in comparison with both SP263 and 22C3 and therefore the lack of false-negative cases, we propose an algorithm for PD-L1 testing in NSCLC when considering pembrolizumab as first-line therapy.

Expression of programmed cell death ligand 1 in non-small cell lung cancer: Comparison between cytologic smears, core biopsies, and whole sections using the SP263 assay.

BACKGROUND: Evaluation of programmed cell death ligand 1 (PD-L1) expression can be made on both resection specimens and diagnostic biopsies; however, more than 30% of patients with advanced non-small cell lung cancer (NSCLC) do not have adequate histologic material to perform PD-L1 assays and require additional biopsies. In addition, in our practice, more than 16% of cases have cytological smears as the only available material. Our aim was to validate the PD-L1 immunocytochemistry assay on cytological smears and compare its accuracy with the results obtained from tissue cores and whole tumor sections using the clinically relevant cutoff of 50%. METHOD: We compared the PD-L1 staining results of cytological smears to those from tissue cores or whole sections in 50 and 53 NSCLC cases, respectively, using the SP263 assay after scanning hematoxylin and eosin slides. RESULTS: We found an overall agreement of 90.6% between cytological smears and whole sections; specifically, we found absolute concordance between smears with PD-L1 expressed in <10% and ≥50% of cells and whole sections with PD-L1 expressed in <50% and ≥50% of cells, respectively. In addition, slightly lower diagnostic accuracy was found for the cytological smears in comparison with the tissue cores, but the difference was not statistically significant. We found excellent intraobserver and good interobserver agreement in the evaluation of PD-L1 on smears. CONCLUSION: Immunocytochemistry on cytological smears is a reliable method for determination of PD-L1 at the 50% cutoff when positive cells are <10% or ≥50%; for cases showing PD-L1 expression in 10% to 49% of cells, additional tissue sampling may be necessary.

PD-L1 expression comparison between primary and relapsed non-small cell lung carcinoma using whole sections and clone SP263.

We assessed the concordance, in terms of PD-L1 expression, between primary and metastatic non-small cell lung carcinoma (NSCLC) of different histotypes using validated SP263 clone. A few samples of local recurrences have also been analyzed. Whole sections of consecutive cases of primary NSCLC and paired relapses undergone surgical resection have been stained with PD-L1 clone SP263; for scoring purposes, a three-tiered system was applied using the following thresholds: <1%, 1-49% and ≥50%. Eighty-four cases of paired primary and relapsed tumors from 83 patients were analyzed, including 75 metastases and 9 local recurrences. Regarding metastases, when considering a cutoff of 1%, discrepancy in PD-L1 expression occurred in 9/75 (12%) paired samples (kappa value = 0.75); at 50% cutoff, discrepancy in PD-L1 expression was detected in 7/75 (9.3%) of paired samples (kappa value = 0.61). Regarding recurrences, at 1% cutoff, the discrepancy in PD-L1 expression was seen in 3/9 (33%) paired samples and in all cases there was a gained PD-L1 expression; at 50% cutoff, 1/9 (11%) paired samples showed gained PD-L1 expression. Our data provide important information regarding the concordance between primary and relapsed NSCLC and the degree of reliability of metastatic sites in terms of PD-L1 expression evaluation.

PD-L1 Assays 22C3 and SP263 are Not Interchangeable in Non-Small Cell Lung Cancer When Considering Clinically Relevant Cutoffs: An Interclone Evaluation by Differently Trained Pathologists.

Pembrolizumab is the only programmed cell death 1/programmed death-ligand 1 inhibitor for treatment of patients with non-small cell lung cancer, with a companion diagnostic assay, the 22C3 PharmDx. Although in many studies 22C3 and Ventana's SP263 appear to yield overlapping results, they show discrepancies at clinically relevant cutoffs (1% and 50%). We provide a solid comparison between 22C3 and SP263 assays in a large cohort of non-small cell lung cancer cases taking into account interobserver variability between trained pathologists who are used to either clone in their clinical practice. Serial sections of tissue microarrays, built from 198 cases of resected lung cancer, were stained for 22C3 on the Dako Link-48 platform and for SP263 on the Ventana Benchmark Ultra, following manufacturer's instructions. A protocol was also developed to run the 22C3 antibody on the Ventana platform. The pathologist used to 22C3 scored consistently higher than the pathologist used to SP263 at both 1% and 50% cutoff for all assays. For 22C3 and SP263 on respective platforms, we found statistically significant differences in terms of proportion of positive cases at both cutoffs; at 50% cutoff, around half of the cases positive with SP263 would have been defined negative with 22C3 by both pathologists. Important differences were also observed, when comparing clone 22C3 and SP263, both run on the Ventana platform. The lowest differences were seen with 22C3 run on both platforms. Assays 22C3 and SP263 show important discrepancies in identifying programmed death-ligand 1-positive cases at clinically relevant cutoffs, with possible underestimation of patients suitable for pembrolizumab therapy.

NK-cell Editing Mediates Epithelial-to-Mesenchymal Transition via Phenotypic and Proteomic Changes in Melanoma Cell Lines.

Tumor cell plasticity is a major obstacle for the cure of malignancies as it makes tumor cells highly adaptable to microenvironmental changes, enables their phenotype switching among different forms, and favors the generation of prometastatic tumor cell subsets. Phenotype switching toward more aggressive forms involves different functional, phenotypic, and morphologic changes, which are often related to the process known as epithelial-mesenchymal transition (EMT). In this study, we report natural killer (NK) cells may increase the malignancy of melanoma cells by inducing changes relevant to EMT and, more broadly, to phenotype switching from proliferative to invasive forms. In coculture, NK cells induced effects on tumor cells similar to those induced by EMT-promoting cytokines, including upregulation of stemness and EMT markers, morphologic transition, inhibition of proliferation, and increased capacity for Matrigel invasion. Most changes were dependent on the engagement of NKp30 or NKG2D and the release of cytokines including IFNγ and TNFα. Moreover, EMT induction also favored escape from NK-cell attack. Melanoma cells undergoing EMT either increased NK-protective HLA-I expression on their surface or downregulated several tumor-recognizing activating receptors on NK cells. Mass spectrometry-based proteomic analysis revealed in two different melanoma cell lines a partial overlap between proteomic profiles induced by NK cells or by EMT cytokines, indicating that various processes or pathways related to tumor progression are induced by exposure to NK cells.Significance: NK cells can induce prometastatic properties on melanoma cells that escape from killing, providing important clues to improve the efficacy of NK cells in innovative antitumor therapies. Cancer Res; 78(14); 3913-25. ©2018 AACR.

PD-L1 Expression Heterogeneity in Non-Small Cell Lung Cancer: Defining Criteria for Harmonization between Biopsy Specimens and Whole Sections.

INTRODUCTION: Determination of programmed death ligand 1 (PD-L1) expression defines eligibility for treatment with pembrolizumab in patients with advanced NSCLC. This study was designed to better define which value across core biopsy specimens from the same case more closely reflects the PD-L1 expression status on whole sections and how many core biopsy specimens are needed for confident classification of tumors in terms of PD-L1 expression. METHODS: We built tissue microarrays as surrogates of biopsies collecting five cores per case from 268 cases and compared PD-L1 staining results obtained by using the validated clone SP263 with the results obtained by using whole tumor sections. RESULTS: We found an overall positivity in 39% of cases at a cutoff of 1% and in 10% of cases at a cutoff of 50%. The maximum value across cores was associated with high concordance between cores and whole sections and the lowest number of false-negative cases overall. To reach high concordance with whole sections, four and three cores are necessary at cutoffs of 1% and 50%, respectively. Importantly, with 20% as the cutoff for core biopsy specimens, fewer than three cores showed high sensitivity and specificity in identifying cases with 50% or more of tumor cells positive for PD-L1 on whole sections. Specifically, for PD-L1 expression values of 20% to 49% on cores, the probabilities of a tumor specimen expressing PD-L1 in at least 50% of cells on a whole section were 46% and 24% with one and two biopsy specimens, respectively. CONCLUSIONS: An accurate definition of the criteria to determine the PD-L1 status of a given tumor may greatly help in selecting those patients who could benefit from anti-programmed cell death 1/PD-L1 treatment.

Correction: PD-L1 expression heterogeneity in non-small cell lung cancer: evaluation of small biopsies reliability.

[This corrects the article DOI: 10.18632/oncotarget.21485.].

PD-L1 expression heterogeneity in non-small cell lung cancer: evaluation of small biopsies reliability.

Immunotherapy with checkpoint inhibitors, allowing recovery of effector cells function, has demonstrated to be highly effective in many tumor types and represents a true revolution in oncology. Recently, the anti-PD1 agent pembrolizumab was granted FDA approval for the first line treatment of patients with advanced non-small cell lung cancer (NSCLC) whose tumors show PD-L1 expression in ≥ 50% of neoplastic cells and as a second line treatment for patients with NSCLC expressing PD-L1 in ≥1% of neoplastic cells, evaluated with a validated assay. For the large majority of patients such evaluation is made on small biopsies. However, small tissue samples such as core biopsies might not be representative of tumors and may show divergent results given the possible heterogeneous immunoexpression of the biomarker. We therefore sought to evaluate PD-L1 expression concordance in a cohort of 239 patients using tissue microarrays (TMA) as surrogates of biopsies stained with a validated PD-L1 immunohistochemical assay (SP263) and report the degree of discordance among tissue cores in order to understand how such heterogeneity could affect decisions regarding therapy. We observed a discordance rate of 20% and 7.9% and a Cohen's κ value of 0.53 (moderate) and 0,48 (moderate) for ≥ 1% and ≥ 50% cutoffs, respectively. Our results suggest that caution must be taken when evaluating single biopsies from patients with advanced NSCLC eligible for immunotherapy; moreover, at least 4 biopsies are necessary in order to minimize the risk of tumor misclassification.

Neuroendocrine differentiation in breast carcinoma: clinicopathological features and outcome.

AIMS: Primary neuroendocrine (NE) breast carcinoma (BC) is an entity with a wide range of prevalence and poorly defined clinical behaviour. We evaluated the prevalence, clinicopathological features and clinical outcome of NEBC. METHODS AND RESULTS: Immunohistochemical staining for synaptophysin and chromogranin A was performed on whole sections from 1232 consecutive cases of invasive BC. We divided NEBC into focal (10-49% positive cells) and diffuse (≥50% positive cells) and compared the outcome of patients with NEBC with strictly matched non-NEBC. A total of 128 BC showed NE differentiation (10.4%): 84 diffuse (6.8%) and 44 focal (3.6%). NE differentiation showed a significant association with T4 stage (P = 0.001), solid-papillary and mucinous histotype (P < 0.0001), G2 grading (P = 0.002), positive oestrogen receptor (ER) (P = 0.003) and progesterone receptor (PR) (P = 0.002). Almost 90% of NEBC were ER(+) /HER2(-) and more than half ER(+) /HER2(-) /Ki67≥14%. Kaplan-Meier analysis revealed that patients with NEBC showed worse disease-free survival (DFS) (P = 0.04) compared to matched non-NEBC. We did not find significant differences regarding clinicopathological features, DFS and CSS between diffuse and focal neuroendocrine BC. CONCLUSIONS: This study demonstrates that NEBC represents 7-10% of invasive BC and that NE differentiation does not affect the prognosis of BC in terms of CSS.

Monosomy of chromosome 17 in breast cancer during interpretation of HER2 gene amplification.

Monosomy of chromosome 17 may affect the assessment of HER2 amplification. Notably, the prevalence ranges from 1% up to 49% due to lack of consensus in recognition. We sought to investigate the impact of monosomy of chromosome 17 to interpretation of HER2 gene status. 201 breast carcinoma were reviewed for HER2 gene amplification and chromosome 17 status. FISH analysis was performed by using double probes (LSI/CEP). Absolute gene copy number was also scored per each probe. HER2 FISH test was repeated on serial tissue sections, ranging in thickness from 3 to 20 µm. Ratio was scored and subsequently corrected by monosomy after gold control test using the aCGH method to overcome false interpretation due to artefactual nuclear truncation. HER2 immunotests was performed on all cases. 26/201 cases were amplified (13%). Single signals per CEP17 were revealed in 7/201 (3.5%) cases. Five out of 7 cases appeared monosomic with aCGH (overall, 5/201, 2.5%) and evidenced single signals in >60% of nuclei after second-look on FISH when matching both techniques. Among 5, one case showed amplification with a pattern 7/1 (HER2/CEP17>2) of copies (3+ at immunotest); three cases revealed single signals per both probes (LSI/CEP=1) and one case revealed a 3:1 ratio; all last 4 cases showed 0/1+ immunoscore. We concluded that: 1) monosomy of chromosome 17 may be observed in 2.5% of breast carcinoma; 2) monosomy of chromosome 17 due to biological reasons rather than nuclear truncation was observed when using the cut-off of 60% of nuclei harboring single signals; 3) the skewing of the ratio due to single centromeric 17 probe may lead to false positive evaluation; 4) breast carcinomas showing a 3:1 ratio (HER2/CEP17) usually show negative 0/1+ immunoscore and <6 gene copy number at FISH.

Progesterone receptor status and clinical outcome in breast cancer patients with estrogen receptor-positive locoregional recurrence.

AIMS AND BACKGROUND: The aim of this retrospective multicenter study was to evaluate the impact of progesterone receptor (PgR) loss on locoregional recurrence in patients with estrogen receptor (ER)-positive primary breast cancer and ER-positive locoregional recurrence. PATIENTS AND METHODS: Eight Italian oncology centers collected data from consecutive patients with ER-positive breast cancer and a subsequent ER-positive locoregional recurrence. RESULTS: Data were available for 265 patients diagnosed with breast cancer between 1990 and 2009. Median metastasis-free survival was 111 months in patients with PgR-positive primary tumors and locoregional recurrence (PgRpos), 38 months in patients with PgR-negative primary tumors and locoregional recurrence (PgRneg), and 63 months in patients with PgR-positive primary tumors and PgR-negative locoregional recurrence (PgRloss). In multivariate analysis, PgR status was independently associated with metastasis-free survival, with a hazard ratio of 2.84 (95% CI 1.34-6.00) for PgRneg compared with PgRpos, and 2.93 (95% CI: 1.51-5.70) for PgRloss compared with PgRpos. CONCLUSIONS: PgR absence was found to be a negative prognostic factor in breast cancer patients with ER-positive locoregional recurrence. Thus, PgR status could be a biological marker in ER-positive recurrent breast cancer.

P16 but not retinoblastoma expression is related to clinical outcome in no-special-type triple-negative breast carcinomas.

Triple-negative breast carcinomas represent a tumor group of pivotal clinical importance given the lack of target therapies. The prognostic significance of triple-negative breast carcinomas remains unclear because of their histological and molecular heterogeneity. Currently, neither prognostic nor predictive factors are available for these tumors. Retinoblastoma (Rb) pathway loss has been linked to clinical outcome in various cancer types, including breast cancer. We investigated the association between Rb and p16 protein expression and clinical outcome in no-special-type triple-negative breast carcinomas. Immunohistochemical staining for Rb, p16, p53 and CK5 was carried out on a section from archival specimens of 117 no-special-type triple-negative breast carcinomas. Immunopositive p16 (p16+) and immunonegative Rb (Rb-) staining were seen in 49.5% and in 24.8% of tumors, respectively. There was an inverse correlation between p16+ and Rb- (P<0.001). P16+ was correlated with G3 grade (P<0.001), high Ki-67 (P=0.03), p53 overexpression (P<0.001) and CK5 immunopositivity (P=0.01). Rb- was not associated with any clinicopathologic variable. Follow-up and therapy data were available in 95 patients. In 20 patients treated with surgery only, neither p16+ nor Rb- immunostaining were associated with disease-free survival and overall survival. In 75 patients treated with adjuvant chemotherapy, p16+ was associated with good response to therapy with significant increased disease-free survival (P=0.001) and showed a trend towards a statistical significance for increased overall survival (P=0.056); Rb- were not associated with disease-free survival and overall survival. In multivariate analysis, p16+ was independently associated with disease-free and overall survival, with a hazard ratio of 0.18 (95% CI: 0.06-0.51; P=0.001) and 0.21 (95% CI: 0.06-0.74; P=0.015), respectively. In patients with no-special-type triple-negative breast carcinomas, p16+ is related to good response to adjuvant chemotherapy and can be considered the best surrogate marker for Rb pathway loss.

The identification of a small but significant subset of patients still targetable with anti-HER2 inhibitors when affected by triple negative breast carcinoma.

PURPOSE: Triple (ER-, PR-, HER2-) negative breast carcinoma lack targeted therapies, making this group of tumors difficult to treat. By definition, the lack of HER2 expression means a case scoring 0 or 1+ after immunophenotypical analysis and makes the patients avoiding therapeutical chances with anti-HER2 inhibitors. We sought to recruit from a group of triple negative breast carcinoma, patients eligible for effective personalized targeted therapy with anti-HER therapies on the basis of their HER2 gene status. METHODS: 135 patients diagnosed with IHC triple negative breast carcinoma were studied. Whole tissue sections were used for in situ hybridization analysis. RESULTS: 8/100 (8 %) of ductal-type triple negative breast carcinoma presented Her-2/neu gene amplification versus 2/35 (5.7 %) non-ductal triple negative breast carcinoma. Three cases showed a ratio 2.5. One case showed Her-2/neu heterogeneous gene amplification, ratio 2.3. The other six showed from 7 to 8 absolute Her-2/neu gene copy number. Two cases staged pT1c, and eight cases staged pT2. Eight cases graded G3 and two cases G2. CONCLUSION: (1) Eight percentage of ductal and 5.7 % non-ductal-type triple negative breast carcinoma present Her-2/neu gene amplification, (2) the standard diagnostic flowchart "do not FISH in 0-1+ (HER2-) breast carcinoma" should be replaced by "do FISH in triple (ER-, PR-, HER2-) negative breast carcinoma," to avoid loss of therapeutical chances in a cohort of such a patients, (3) we demonstrated the identification of a small but significant subset of patients targetable with anti-HER2 inhibitors, giving patients affected by (ex)triple negative breast carcinoma new personalized therapeutical chances.

Comparison of hormonal receptor and HER-2 status between breast primary tumours and relapsing tumours: clinical implications of progesterone receptor loss.

Differences in hormone receptor and HER-2 status between primary tumour and corresponding relapse could have a substantial impact on clinical management of patients. The aim of this study was to evaluate change in expression of hormone receptors and HER-2 status between primary tumour and corresponding local recurrence or distant metastasis. We analysed 140 primary tumours and related recurrent or metastatic samples. Hormone receptors status was evaluated by immunohistochemistry, while HER-2 status by immunohistochemistry and silver in situ hybridisation. A change in HER-2 was rare; 3.7% of cases by immunohistochemistry and only 0.7% by silver in situ hybridisation analysis. A change in estrogen and progesterone receptors was seen in 6.4% and 21.4% of cases, respectively. Estrogen receptor change was not affected by adjuvant therapy, whereas progesterone receptor was influenced by adjuvant chemotherapy associated to hormone therapy (P = 0.0005). A change in progesterone receptor was more frequent in distant metastases than in local recurrences (P = 0.03). In the setting of estrogen receptor positive tumours, patients with progesterone receptor loss in local recurrence had a statistically significant lower median metastasis free survival compared to others patients; progesterone receptor positive, 112 months; progesterone receptor negative, 24 months (P = 0.005). A change between primary tumour and corresponding relapse is frequent for progesterone receptor, infrequent for estrogen receptor and rare for HER-2. In cases with changes in HER-2, it is worthwhile reassessing HER-2 status with both immunohistochemistry and in situ hybridisation analysis. Progesterone receptor loss seems to be influenced by therapy and to correlate with a worse prognosis.