The long and winding road to biomarkers for immunotherapy: a retrospective analysis of samples from patients with triple-negative breast cancer treated with pembrolizumab.

BACKGROUND: Immune checkpoint blockade (ICB) in combination with chemotherapy improves outcome of patients with triple-negative breast cancer (TNBC) in metastatic and early settings. The identification of predictive biomarkers able to guide treatment decisions is challenging and currently limited to programmed death-ligand 1 (PD-L1) expression and high tumor mutational burden (TMB) in the advanced setting, with several limitations. MATERIALS AND METHODS: We carried out a retrospective analysis of clinical-pathological and molecular characteristics of tumor samples from 11 patients with advanced TNBC treated with single-agent pembrolizumab participating in two early-phase clinical trials: KEYNOTE-012 and KEYNOTE-086. Clinical, imaging, pathological [i.e. tumor-infiltrating lymphocytes (TILs), PD-L1 status], RNA sequencing, and whole-exome sequencing data were analyzed. We compared our results with publicly available transcriptomic data from TNBC cohorts from TCGA and METABRIC. RESULTS: Response to pembrolizumab was heterogeneous: two patients experienced exceptional long-lasting responses, six rapid progressions, and three relatively slower disease progression. Neither PD-L1 nor stromal TILs were significantly associated with response to treatment. Increased TMB values were observed in tumor samples from exceptional responders compared to the rest of the cohort (P = 3.4 × 10-4). Tumors from exceptional responders were enriched in adaptive and innate immune cell signatures. Expression of regulatory T-cell markers (FOXP3, CCR4, CCR8, TIGIT) was mainly observed in tumors from responders except for glycoprotein-A repetitions predominant (GARP), which was overexpressed in tumors from rapid progressors. GARP RNA expression in primary breast tumors from the public dataset was significantly associated with a worse prognosis. CONCLUSIONS: The wide spectrum of clinical responses to ICB supports that TNBC is a heterogeneous disease. Tumors with high TMB respond better to ICB. However, the optimal cut-off of 10 mutations (mut)/megabase (Mb) may not reflect the complexity of all tumor subtypes, despite its approval as a tumor-agnostic biomarker. Further studies are required to better elucidate the relevance of the tumor microenvironment and its components as potential predictive biomarkers in the context of ICB.

Genome-wide gene expression profiling to predict resistance to anthracyclines in breast cancer patients.

Validated biomarkers predictive of response/resistance to anthracyclines in breast cancer are currently lacking. The neoadjuvant Trial of Principle (TOP) study, in which patients with estrogen receptor (ER)-negative tumors were treated with anthracycline (epirubicin) monotherapy, was specifically designed to evaluate the predictive value of topoisomerase II-alpha (TOP2A) and develop a gene expression signature to identify those patients who do not benefit from anthracyclines. Here we describe in details the contents and quality controls for the gene expression and clinical data associated with the study published by Desmedt and colleagues in the Journal of Clinical Oncology in 2011 (Desmedt et al., 2011). We also provide R code to easily access the data and perform the quality controls and basic analyses relevant to this dataset.

Clinical validation of a molecular assay for intra-operative detection of metastases in breast sentinel lymph nodes.

BACKGROUND: In breast cancer patients, the status of the sentinel lymph nodes (SLNs) has been shown to accurately reflect the presence of metastases in the axillary lymph nodes (ALNs). Intra-operative SLN evaluation by frozen section histology may miss positive cases, leading to a second surgery for complete ALN dissection. Permanent section histology itself has tissue sampling limitations and is partially dependent on pathologist expertise. METHODS: A prospective study (N=78) was conducted in our institution to validate a new intra-operative molecular assay, the GeneSearch breast lymph node (BLN) assay. This assay quantifies the expression of mammaglobin and cytokeratin-19 genes using quantitative RT-PCR technology to determine SLN status. Fresh SLN sections (2 mm thick) were analyzed alternatively by BLN assay or post-operative histology (haematoxylin-eosin and immunohistochemistry). The subject was considered positive when histology revealed a focus >0.2 mm. RESULTS: BLN assay results corroborated with histologic results in 75 out of 78 patients for an overall agreement of 96%, a sensitivity of 92%, and a specificity of 97%. The positive and negative predictive values of the BLN assay were of 86% (12/14) and 98% (63/64), respectively. Interestingly, a statistically significant correlation was observed between the metastases' histologic size and both assay markers' expression levels as represented by cycle time to positivity (rho > or = 0.71, all p<0.0001). CONCLUSIONS: The performance of the BLN assay in identifying nodal metastases >0.2 mm was similar to that of permanent section histology, with the added advantages of an objective and rapid output that could be used for intra-operative decision to remove additional ALN.

Correlation between topoisomerase-IIalpha gene amplification and protein expression in HER-2 amplified breast cancer.

Topoisomerase-IIalpha (topo-II) is a molecular target for topo-II inhibitors, which makes it a potential predictive marker of responsiveness to these agents. We aim to correlate topo-II gene and protein status on 103 HER-2 amplified breast cancer samples. Paraffin-embedded blocks were screened by FISH for topo-II gene amplification (topo-II: CEP17 ratio >/=1.5; triple probe by Vysis inc.) and analyzed by IHC for topo-II protein expression (continuous variable; clone KiS1) and Ki-67 (positive if >25% of stained cells; clone MIB-1). Topo-II gene amplification was observed in 36.9% (38/103) of the HER-2 amplified study population. HER-2 amplification level (e.g. copy number) was not shown to be predictive for topo-II amplification. The median percentage of topo-II positively stained cells by IHC for topo-II non-amplified and amplified cases were 5% and 10%, respectively. A weak but significant correlation was observed between topo-II gene amplification level and percentage of positively stained cells (Spearman's ranks correlation coefficient of 0.23, p=0.02), the observed correlation being higher in patients with positive staining for Ki-67. Contrary to HER-2, where gene amplification is almost always correlated with protein overexpression in breast cancer, topo-II gene amplification apparently does not always lead to protein overexpression, at least when the latter is evaluated by IHC. Other factors, specifically the tumor proliferation status, may interfere with the topo-II protein status. Although the great majority of topo-II gene aberrations occur in HER-2 positive tumors, the level of HER-2 amplification does not predict for topo-II amplification.

Correlation between complete response to anthracycline-based chemotherapy and topoisomerase II-alpha gene amplification and protein overexpression in locally advanced/metastatic breast cancer.

UNLABELLED: Anthracycline-based regimens are among the most active but also with greater risk of both acute and long-term side effects, namely cardiotoxicity. Predictive markers of response to anthracyclines are therefore essential. Topoisomerase-IIalpha (topo-II) is the target of anthracyclines and preliminary data suggest its promising role as a predictive marker of sensitivity to these drugs. After screening a population of about 350 patients with locally advanced or metastatic breast cancer, two subgroups were selected for the present analysis: a study group (31 patients), composed of 14 complete responders (CR-a) and 17 true non-responders (PD-a) to anthracycline-based CT, and a control group (28 patients), composed of 7 CR (CR-t) and 21 true non-responders (PD-t) to taxane-based CT. True non-responders were defined as progressive disease (PD) within the first three cycles of CT. Archival tumor samples of these patients were collected, biological markers evaluated and their status correlated with response to therapy. HER-2 and topo-II gene status were evaluated by FISH (Vysis multi-color probe-positivity cut-off: >/=2 ratio for HER-2 and >/=1.5 for topo-II), topo-II protein was evaluated by IHC (positivity cut-off >10%). All cases in which HER-2 gene was non-amplified did not show topo-II gene aberrations. No association was found between HER-2 gene amplification and response to anthracyclines (5/14 (36%) CR and 5/17 (29%) PD to anthracycline-based CT were HER-2+). The topo-II gene was amplified in 3/14 (21%) CR but only in 1/17 (6%) PD to anthracyclines. Amplification of the topo-II gene was seen in 1/7 (14%) CR and in 3/21 (14%) PD to a taxane-based CT. Topo-II protein was overexpressed in 6/11 (55%) CR and in 2/17 (12%) PD to anthracyclines, while in the control group, overexpression was seen in 5/7 (71%) CR and 8/20 (40%) PD. IN CONCLUSION: i) HER-2 gene amplification did not seem to be correlated with response to anthracyclines. ii) Both topo-II gene amplification and protein overexpression seem to correlate with response to anthracyclines, although other factors, such as p53 and cell proliferation, are most likely to be involved. iii) The role of combined evaluation of several relevant markers and of potential 'molecular signatures' are currently being evaluated in prospective randomized clinical trials.

Comparison of topoisomerase-IIalpha gene status between primary breast cancer and corresponding distant metastatic sites.

BACKGROUND: Topoisomerase-IIalpha (topo-IIalpha) is a key enzyme in DNA replication and a molecular target for anticancer drugs called topoisomerase-II inhibitors, such as anthracyclines. Its value as a predictive marker of responsiveness to these cytotoxic drugs is currently being evaluated with promising results. However, even in the metastatic setting, the choice of treatment is based on the biologic characteristics of the primary tumor. Few data are available regarding the expression of biological markers between the primary tumor and the corresponding distant metastases. METHODS: Topo-IIalpha gene status was evaluated in 29 breast cancer patients in which a primary tumor sample and a corresponding metastatic sample were both available. Fluorescent in situ hybridization (FISH) with the Vysis triple probe (Vysis multi-color topo-IIalpha spectrum orange, Her-2 spectrum green and CEP17 spectrum aqua probe) was used, which allowed the concomitant evaluation of HER-2 gene status. RESULTS: As previously reported, topo-IIalpha gene aberrations are always associated with HER-2 gene amplification; indeed no topo-IIalpha gene aberrations have been observed in the HER-2 negative tumors. Conversely, 38.5% (five patients) of the HER-2 positive primary breast tumors (13 patients) were topo-IIalpha amplified, while 61.5% (eight patients) had a normal topo-IIalpha gene. No topo-IIalpha gene deletion was found in our series. Topo-IIalpha gene amplification in the primary tumor was always associated with amplification in the corresponding metastases, and no metastases with topo-IIalpha gene amplification were seen without amplification in the primary tumor. Furthermore, the amplification level of topo-IIalpha (i.e., ratio topo-IIalpha:CEP17) remained unchanged in primary and metastatic sites. CONCLUSION: Despite the low number of patients, our results seem to indicate that topo-IIalpha gene status evaluation in the primary breast tumor accurately reflects its status in the corresponding distant metastases.

Comparison of HER-2 status between primary breast cancer and corresponding distant metastatic sites.

BACKGROUND: The humanized anti-HER-2 monoclonal antibody trastuzumab (Herceptin) is a new treatment modality for metastatic breast cancer, the efficacy of which is directly correlated with the HER-2 status of the tumour, evaluated either by immunohistochemistry (IHC) and/or by fluorescence in situ hybridisation (FISH). This analysis is generally performed on the primary tumour. There are few data regarding the HER-2 status in the corresponding distant metastases. METHODS: HER-2 status in 107 patients with a primary breast tumour and at least one distant metastatic lesion was analysed by IHC and FISH. RESULTS: We found similar levels of amplification (25% and 24%) and overexpression (13% and 19%) of HER-2 in primary and metastatic samples, respectively. Among paired primary/metastatic tumours, six (6%) showed discordance by HercepTest(TM) (n = 100): all six cases showed greater Her-2 overexpression in the metastatic tissue. By FISH (n = 68), five (7%) cases were discordant: two cases were amplified in the primary tumour but not in the metastasis, and three samples showed amplification in the metastasis but not in the primary. Finally, we analysed HER-2 status in different metastatic lesions from 17 patients that had at least two distant metastatic sites. Discordance between different sites from the same patient was 18% by IHC and 19% by FISH. CONCLUSIONS: Between the paired primary tumour and distant metastatic lesions, 94% and 93% of samples had concordant HER-2 status when analysed by IHC or FISH, respectively. These results do not support routine determination of HER-2 on metastatic sites, particularly when FISH results from the primary tumour are available.

Reliability of the tissue microarray based FISH for evaluation of the HER-2 oncogene in breast carcinoma.

AIMS: Tumour tissue microarray allows the analysis of hundreds of tumour samples simultaneously on a single microscope slide. However, the extremely small tissue samples taken from the original tissue may not always be representative of the entire tumour. METHODS: The reliability of this new technology was investigated by analysing HER-2 oncogene amplification by fluorescence in situ hybridisation (FISH) from representative slides of the whole tumour and small tissue core biopsies from 29 invasive breast tumours. RESULTS: The tissue microarray method had high accuracy; in only one of 29 cases (3.4%; 95% confidence interval, 0% to 10%) were the results discordant with whole tumour analysis. CONCLUSION: Tumour microarray is a highly reliable method for analysing HER-2 oncogene amplification by FISH in human breast tumours.

Expression of p53 in preneoplastic and early neoplastic bronchial lesions.

p53 alteration has been reported to be an early event in bronchial carcinogenesis. Our study purpose was to determine the rate of p53 expression in the various preneoplastic and early neoplastic bronchial lesions obtained by biopsy during fluorescence bronchoscopy and to analyse its association with patients characteristics. Various stages of preneoplastic lesions as well as radio-occult lung cancer were studied in biopsies obtained by fluorescence bronchoscopy. We assessed the expression of p53 by immunohistochemistry using monoclonal antibody clone DO7. The p53 expression was considered as positive if > or = 1% of cells were positive and the level of positivity was expressed in percentage of positive cells. Fourteen patients were included in each category of preneoplastic lesions. At the threshold of 1% of positive cells p53 expression was observed in 28.5% of the patients with a histologically normal epithelium. This number of positive patients increased with the severity of preneoplastic lesions and reached 100% in the mild dysplasia. The mean rates of p53 positive cells for normal epithelium, hyperplasia, metaplasia, mild and severe dysplasia, carcinoma in situ and invasive radio-occult carcinoma were respectively 0.9, 3.4, 9.1, 20.5, 50.2, 34.7 and 42.5%. There was no statistically significant correlation between p53 expression and patient characteristics such as sex, age, smoking habits and indication for fluorescence bronchoscopy. The alteration of p53 expression in patients with high risk of lung cancer was an early event: this abnormality increased with the severity of the lesions, without significant correlation with patient characteristics.

HER-2 and topo-isomerase IIalpha as predictive markers in a population of node-positive breast cancer patients randomly treated with adjuvant CMF or epirubicin plus cyclophosphamide.

BACKGROUND: The predictive role of HER-2 in node-positive breast cancer patients receiving CMF or an anthracycline-based adjuvant therapy remains unclear. In addition, topo-isomerase II alpha (topo IIalpha), as the cellular target of anthracyclines, might have value as a predictive marker. PATIENTS AND METHODS: Four hundred eighty-one archival primary tumor samples were collected among 777 patients entered into a multicenter phase III trial comparing classical CMF with epirubicin cyclophosphamide (HEC) as adjuvant therapy of node-positive breast cancer. HER-2 was evaluated by immunohistochemistry (IHC) using different antibodies (Abs). Topo IIalpha was evaluated by IHC using the Ab KiS 1. In each subgroup of patients identified by HER-2 and topo IIalpha, adjusted hazard ratios for event-free survival (EFS) and the corresponding 95% confidence intervals have been calculated for the different study comparisons. An interaction test has been performed to investigate the role of HER-2 and topo IIalpha as predictive markers. RESULTS: When HER-2 was evaluated by CB-11 and 4D5 mAbs, the EFS adjusted hazard ratios (HR) for the main study comparison HEC vs. CMF were: HER-2 positive: 0.33 (95% confidence interval (95% CI): 0.09 1.27, P = 0.08), HER-2 negative: 1.16 (95%, CI: 0.71-1.90, P = 0.56); the P-value for the interaction test was 0.10. When HER-2 was evaluated by TAB-250 + pAbl Abs, the adjusted HR for the same comparison were: HER-2 positive: 1.06 (95% CI: 0.45-2.52, P = 0.90), HER-2 negative: 0.99 (95% CI: 0.58-1.68, P = 0.97); the P-value for the interaction test was 0.84. With regard to topo IIalpha, the adjusted HR for the EFS comparison HEC vs. CMF were: topo IIalpha positive: 0.66 (95% CI: 0.32-1.36, P = 0.25), topo IIalpha negative: 1.26 (95% CI: 0.63-2.50, P = 0.51); the P-value for the interaction test was 0.13. CONCLUSIONS: This study suggests that in node-positive breast cancer patients randomly treated with CMF or an epirubicin-based regimen, the predictive value of HER-2 may vary according to the Abs used in the immunohistochemistry assay. In addition, the study supports the concept that topo IIalpha might be involved in the determination of tumor responsiveness to an anthracycline-based adjuvant therapy.

Evaluation of HER2, p53, bcl-2, topoisomerase II-alpha, heat shock proteins 27 and 70 in primary breast cancer and metastatic ipsilateral axillary lymph nodes.

BACKGROUND: Breast cancer is a heterogeneous disease. Predictive biological markers (BM) of responsiveness to therapy need to be identified. Evaluation of BM is mainly done at the primary site. However, in the adjuvant therapy of breast cancer, the main goal is control of micrometastases. It is still unknown whether heterogeneity in the expression of BM between the primary site and its micrometastases exists. OBJECTIVE: To evaluate the expression of some BM with potential predictive value from the primary breast cancer site and metastatic ipsilateral axillary lymph nodes. PATIENTS AND METHODS: Focality (percentage of positive cells) and intensity staining scores were evaluated for each marker. Freshly cut sections (4 microm) from embedded blocks of breast cancer fixed in formalin or bouin were put onto superfrost slides (Menzel-Gläser). Protein expression was evaluated immunohistochemically (IHC) using monoclonal antibodies against: topo II-alpha (clone KiS1, 1 microg/ml, Roche) with a trypsine pre-treatment (P); HSP27 (clone G3.1, 1/60, Biogenex), HSP70 (clone BRM.22, 1/80, Biogenex) and HER2 (clone CB11, 1/40, Novocastra; without P); p53 (clone D07, 1/750, Dako) and bcl-2 (clone 124, 1/60, Dako) with citrate buffer as P. RESULTS: Overall, the percentage of discordant marker status in the primary tumour and its metastatic lymph nodes was 2% for HER2, 6% for p53, 15% for bcl-2, 19% for topoisomerase II-alpha, 24% for HSP27 and 30% for HSP70. For the subgroup of patients with positive BM in the primary tumour, the percentage of discordance was 6% for HER2, 7% for p53, 14% for bcl-2, 19% for HSP70, 21% for topoisomerase II-alpha and 36% for HSP27. For the subgroup of patients with positive BM in the lymph nodes, the percentage of discordance was 9% for bcl-2, 15% for HER2 and p53, 21% for topoisomerase II-alpha, 22% for HSP27 and 25% for HSP70. CONCLUSIONS: 1) No biological marker had 100% concordant results. 2) Although some discordant cases might be explained by the limitations of the IHC technique, future studies aiming to evaluate the predictive value of BM in the adjuvant therapy of breast cancer should take into account a possible difference in BM expression between the primary and the metastatic sites.

Trisomy 6 in Merkel cell carcinoma: a recurrent chromosomal aberration.

UNLABELLED: We retrospectively investigated 17 cases of primary and metastasizing Merkel cell carcinomas (MCC) from 14 patients using chromosomal in-situ hybridization (CISH) to study the occurrence of trisomy 6 in these lesions. METHODS AND RESULTS: Histological diagnosis on all tumour samples was obtained on haematoxylin and eosin stained sections. Immunohistochemistry was performed with antibodies against pancytokeratin (CAM 5.2), cytokeratin 20 (CK20), MIC2 antigen (CD99), neuron-specific enolase (NSE), and chromogranin A (chrA). Sections (4 microm) of the paraffin-embedded tumours were analysed with alpha-satellite centromeric probes for chromosome 6 or 17 using CISH. The signal was amplified by the Tyramide Signal Amplification (TSA) assay. Immunohistochemically, the tumours showed the same general epithelial neuro-endocrine pattern: 11/13 expressed cytokeratin 20, and 47% exhibited trisomy 6, with no significant difference between primary and metastatic lesions. Incomplete follow-up data did not allow us to establish a prognostic value of trisomy 6, however, this aberration might be an additional diagnostic tool in distinguishing MCC from other small round blue cell tumours. CONCLUSIONS: CISH seems to be a promising adjunctive method to diagnose Merkel cell carcinoma. Trisomy 6 should be investigated more closely in these cases, as has been done for chromosomes 1 and 11. Of particular interest would be identification of modifications in proto-oncogene(s) located on chromosome 6.

Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples.

Determination of HER-2/neu oncogene amplification has become necessary for selection of breast cancer patients for trastuzumab (Herceptin) therapy. Fluorescence in situ hybridization (FISH) is currently regarded as a gold standard method for detecting HER-2/neu amplification, but it is not very practical for routine histopathological laboratories. We evaluated a new modification of in situ hybridization, the chromogenic in situ hybridization (CISH), which enables detection of HER-2/neu gene copies with conventional peroxidase reaction. Archival formalin-fixed paraffin-embedded tumor tissue sections were pretreated (by heating in a microwave oven and using enzyme digestion) and hybridized with a digoxigenin-labeled DNA probe. The probe was detected with anti-digoxigenin fluorescein, anti-fluorescein peroxidase, and diaminobenzidine. Gene copies visualized by CISH could be easily distinguished with a x40 objective in hematoxylin-stained tissue sections. HER-2/neu amplification typically appeared as large peroxidase-positive intranuclear gene copy clusters. CISH and FISH (according to Vysis, made from frozen pulverized tumor samples) correlated well in a series of 157 breast cancers (kappa coefficient, 0.81). The few different classifications were mostly because of low-level amplifications by FISH that were negative by CISH and immunohistochemistry with monoclonal antibody CB-11. We conclude that CISH, using conventional bright-field microscopy in evaluation, is a useful alternative for determination of HER-2/neu amplification in paraffin-embedded tumor samples, especially for confirming the immunohistochemical staining results.

Sensitivity of HER-2/neu antibodies in archival tissue samples of invasive breast carcinomas. Correlation with oncogene amplification in 160 cases.

Overexpression and amplification of the HER-2 oncogene in patients with breast cancer has correlated with early onset of metastasis, resistance to hormonal therapy and some forms of chemotherapy, and shortened survival. Therefore, evaluation of this putative prognostic or predictive factor seems critical. Because different antibodies are used for the detection of the 185-kd HER-2 oncoprotein, we studied the sensitivity of 3 frequently used antibodies. Immunohistochemistry results were correlated with gene amplification level as assessed by fluorescence in situ hybridization. Protein overexpression was found in 17.2% and 12.5% of cases using antibodies against the external (TAB250) and internal (CB11) domains of the protein, respectively, and in 38.0% of cases using a rabbit polyclonal antibody. Fluorescence in situ hybridization was successful in all 160 tumors, and amplification was found in 37 tumors (23.1%). The monoclonal antibody TAB250 had the lowest misclassification rate, 9.6% (sensitivity, 67%; specificity, 97.5%).

Tumor-infiltrating dendritic cells in adenocarcinomas of the breast: a study of 143 neoplasms with a correlation to usual prognostic factors and to clinical outcome.

Dendritic cells (DC) are the most potent antigen-presenting cells, and induce antigen-specific immune responses. Infiltration of tumors by DC is thought to reflect the interaction between the host immune system and tumor cells. Tumor-infiltrating DC (TIDC) are believed to evolve into tumor-antigen pulsed cells and then to migrate to local lymph nodes, where they activate anti-tumor immune responses. Indirect clinical evidence supporting this theory is provided by studies showing that high TIDC densities are associated with favorable prognosis in some tumor types. In the present study, we evaluated 143 primary breast adenocarcinomas for the presence of DC, using immunohistochemistry with the anti-S100 protein antibody. We analyzed the relationship between the degree of infiltration by S100+ TIDC and the usual prognostic factors and clinical outcome. The results show that 42% of breast adenocarcinomas contain S100 TIDC. The number of S100+ TIDC varies according to the grade of tumors as follows: GRIII > GRII > GRI. A relationship is also found between S100+ TIDC and tumor size, lymph-node involvement, estrogen/progesterone receptor status and age. However, the presence of S100+ TIDC, even at the highest density, was not correlated with metastasis-free survival or overall survival.