Detection of Second Primary Lymphoma in Late Diffuse Large B-cell Lymphoma Recurrences.

Approximately one-third of patients with diffuse large B-cell lymphoma (DLBCL) relapse and often require salvage chemotherapy followed by autologous stem cell transplantation. In most cases, the clonal relationship between the first diagnosis and subsequent relapse is not assessed, thereby potentially missing the identification of second primary lymphoma. In this study, the clonal relationship of 59 paired DLBCL diagnoses and recurrences was established by next-generation sequencing-based detection of immunoglobulin gene rearrangements. Among 50 patients with interpretable results, 43 patients (86%) developed clonally related relapsed disease. This was observed in 100% of early recurrences (<2 years), 80% of the recurrences with an interval between 2 and 5 years, and 73% of late recurrences (≥5 years). On the other hand, 7 (14%) out of 50 patients displayed different dominant clonotypes in primary DLBCL and clinical recurrences, confirming the occurrence of second primary DLBCL; 37% of DLBCL recurrences that occurred ≥4 years after diagnosis were shown to be second primary lymphomas. The clonally unrelated cases were Epstein-Barr virus positive in 43% of the cases, whereas this was only 5% in the relapsed DLBCL cases. In conclusion, next-generation sequencing-based clonality testing in late recurrences should be considered in routine diagnostics to distinguish relapse from second primary lymphoma, as this latter group of patients with DLBCL may benefit from less-intensive treatment strategies.

Accelerated Tissue Processing With Minimal Formalin Fixation Time for 9-Gauge Vacuum-Assisted Breast Biopsy Specimens.

OBJECTIVES: Vacuum-assisted biopsy (VAB) of the breast seems unsuitable for rapid processing due to large size. We tested microwave-based acceleration. METHODS: As a proof-of-principle study, 9-gauge VAB specimens were taken from eight mastectomy specimens. Forty-two biopsy specimens were processed. Quality of H&E was evaluated in 84 slides, and estrogen receptor (ER), progesterone receptor (PR), E-cadherin, and human epidermal growth factor receptor 2 (HER2) stains were evaluated in six slides. Preoperative biopsy specimens were used as a control. RESULTS: Diagnostic quality of H&E slides was good in 87%, reasonable in 12%, and low in 1%. Quality of E-cadherin was good in 75% and reasonable in 25%. Quality of ER was good in 83% and reasonable in 17%. PR and both HER2 immunohistochemistry and fluorescence in situ hybridization were good in all slides. Quality of experimental slides was similar to control slides. CONCLUSIONS: Nine-gauge VAB specimens can be processed within 4 hours. Slides are suitable for all routine pathologic stains. This enables a same-day diagnosis.

HER2, chromosome 17 polysomy and DNA ploidy status in breast cancer; a translational study.

Breast cancer treatment depends on human epidermal growth factor receptor-2 (HER2) status, which is often determined using dual probe fluorescence in situ hybridisation (FISH). Hereby, also loss and gain of the centromere of chromosome 17 (CEP17) can be observed (HER2 is located on chromosome 17). CEP17 gain can lead to difficulty in interpretation of HER2 status, since this might represent true polysomy. With this study we investigated whether isolated polysomy is present and how this effects HER2 status in six breast cancer cell lines and 97 breast cancer cases, using HER2 FISH and immunohistochemistry, DNA ploidy assessment and multiplex ligation dependent probe amplification. We observed no isolated polysomy of chromosome 17 in any cell line. However, FISH analysis did show CEP17 gain in five of six cell lines, which reflected gains of the whole chromosome in metaphase spreads and aneuploidy with gain of multiple chromosomes in all these cases. In patients' samples, gain of CEP17 indeed correlated with aneuploidy of the tumour (91.1%; p < 0.001). Our results indicate that CEP17 gain is not due to isolated polysomy, but rather due to widespread aneuploidy with gain of multiple chromosomes. As aneuploidy is associated with poor clinical outcome, irrespective of tumour grade, this could improve future therapeutic decision making.

1399 H&E-stained sentinel lymph node sections of breast cancer patients: the CAMELYON dataset.

Background: The presence of lymph node metastases is one of the most important factors in breast cancer prognosis. The most common way to assess regional lymph node status is the sentinel lymph node procedure. The sentinel lymph node is the most likely lymph node to contain metastasized cancer cells and is excised, histopathologically processed, and examined by a pathologist. This tedious examination process is time-consuming and can lead to small metastases being missed. However, recent advances in whole-slide imaging and machine learning have opened an avenue for analysis of digitized lymph node sections with computer algorithms. For example, convolutional neural networks, a type of machine-learning algorithm, can be used to automatically detect cancer metastases in lymph nodes with high accuracy. To train machine-learning models, large, well-curated datasets are needed. Results: We released a dataset of 1,399 annotated whole-slide images (WSIs) of lymph nodes, both with and without metastases, in 3 terabytes of data in the context of the CAMELYON16 and CAMELYON17 Grand Challenges. Slides were collected from five medical centers to cover a broad range of image appearance and staining variations. Each WSI has a slide-level label indicating whether it contains no metastases, macro-metastases, micro-metastases, or isolated tumor cells. Furthermore, for 209 WSIs, detailed hand-drawn contours for all metastases are provided. Last, open-source software tools to visualize and interact with the data have been made available. Conclusions: A unique dataset of annotated, whole-slide digital histopathology images has been provided with high potential for re-use.

Assessment of HER2 status in breast cancer biopsies is not affected by accelerated tissue processing.

AIMS: To establish whether core needle biopsy (CNB) specimens processed with an accelerated processing method with short fixation time can be used to determine accurately the human epidermal growth factor receptor 2 (HER2) status of breast cancer. METHODS AND RESULTS: A consecutive case-series from two high-volume breast clinics was created. We compared routine HER2 immunohistochemistry (IHC) assessment between accelerated processing CNB specimens and routinely processed postoperative excision specimens. Additional amplification-based testing was performed in cases with equivocal results. The formalin fixation time was less than 2 h and between 6 and 72 h, respectively. Fluorescence in-situ hybridisation and multiplex ligation-dependent probe amplification were used for amplification testing. One hundred and forty-four cases were included, 15 of which were HER2-positive on the routinely processed excision specimens. On the CNB specimens, 44 were equivocal on IHC and required an amplification-based test. Correlation between the CNB specimens and the corresponding excision specimens was high for final HER2 status, with an accuracy of 97% and a kappa of 0.85. CONCLUSIONS: HER2 status can be determined reliably on CNB specimens with accelerated processing time using standard clinical testing methods. Using this accelerated technology the minimum 6 h of formalin fixation, which current guidelines consider necessary, can be decreased safely. This allows for a complete and expedited histology-based diagnosis of breast lesions in the setting of a one-stop-shop, same-day breast clinic.

Eight-Color Multiplex Immunohistochemistry for Simultaneous Detection of Multiple Immune Checkpoint Molecules within the Tumor Microenvironment.

Therapies targeting immune checkpoint molecules CTLA-4 and PD-1/PD-L1 have advanced the field of cancer immunotherapy. New mAbs targeting different immune checkpoint molecules, such as TIM3, CD27, and OX40, are being developed and tested in clinical trials. To make educated decisions and design new combination treatment strategies, it is vital to learn more about coexpression of both inhibitory and stimulatory immune checkpoints on individual cells within the tumor microenvironment. Recent advances in multiple immunolabeling and multispectral imaging have enabled simultaneous analysis of more than three markers within a single formalin-fixed paraffin-embedded tissue section, with accurate cell discrimination and spatial information. However, multiplex immunohistochemistry with a maximized number of markers presents multiple difficulties. These include the primary Ab concentrations and order within the multiplex panel, which are of major importance for the staining result. In this article, we report on the development, optimization, and application of an eight-color multiplex immunohistochemistry panel, consisting of PD-1, PD-L1, OX40, CD27, TIM3, CD3, a tumor marker, and DAPI. This multiplex panel allows for simultaneous quantification of five different immune checkpoint molecules on individual cells within different tumor types. This analysis revealed major differences in the immune checkpoint expression patterns across tumor types and individual tumor samples. This method could ultimately, by characterizing the tumor microenvironment of patients who have been treated with different immune checkpoint modulators, form the rationale for the design of immune checkpoint-based immunotherapy in the future.

Migrating into the Tumor: a Roadmap for T Cells.

Tumors can be divided into 'hot' (T cell inflamed) or 'cold' (T cell noninflamed) according to the presence of immune cells. In this review, we discuss variables that influence T cell migration into the tumor microenvironment. Chemokines can attract T cells to the tumor site and tumor intrinsic pathways can influence the composition of local chemokines. Tumor-induced vasculature can hamper T cell migration. Other immune cells and tumor-derived molecules can block T cell proliferation and survival. It is important to better understand these mechanisms in order to target them therapeutically. Enhancing T cell infiltration may increase response rates to immunotherapy and increase survival.

Direct inhibition of STAT signaling by platinum drugs contributes to their anti-cancer activity.

Platinum-based chemotherapeutics are amongst the most powerful anti-cancer drugs. Although their exact mechanism of action is not well understood, it is thought to be mediated through covalent DNA binding. We investigated the effect of platinum-based chemotherapeutics on signaling through signal transducer and activator of transcription (STAT) proteins, which are involved in many oncogenic signaling pathways. We performed in vitro experiments in various cancer cell lines, investigating the effects of platinum chemotherapeutics on STAT phosphorylation and nuclear translocation, the expression of STAT-modulating proteins and downstream signaling pathways. Direct binding of platinum to STAT proteins was assessed using an AlphaScreen assay. Nuclear STAT3 expression was determined by immunohistochemistry and correlated with disease-free survival in retrospective cohorts of head and neck squamous cell carcinoma (HNSCC) patients treated with cisplatin-based chemoradiotherapy (n= 65) or with radiotherapy alone (n = 32). At clinically relevant concentrations, platinum compounds inhibited STAT phosphorylation, resulting in loss of constitutively activated STAT proteins in multiple distinct cancer cell lines. Platinum drugs specifically inhibited phospho-tyrosine binding to SH2 domains, thereby blocking STAT activation, and subsequently downregulating pro-survival- and anti-apoptotic- target genes. Importantly, we found that active STAT3 in tumors directly correlated with response to cisplatin-based chemoradiotherapy in HNSCC patients (p = 0.006). These findings provide insight into a novel, non-DNA-targeted mechanism of action of platinum drugs, and could be leveraged into the use of STAT expression as predictive biomarker for cisplatin chemotherapy and to potentiate other therapeutic strategies such as immunotherapy.

Brief fixation enables same-day breast cancer diagnosis with reliable assessment of hormone receptors, E-cadherin and HER2/Neu.

AIMS: Preoperative core needle biopsy (CNB) is commonly used to confirm the diagnosis of breast cancer. For treatment purposes and for determining histological type, especially in case of neoadjuvant therapy, oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) status and E-cadherin assessments are crucial. Considering the increasing demand for same-day diagnosis of breast lesions, an accelerated method of CNB processing was developed, in which the tissue fixation time is radically reduced. METHODS: To determine whether short fixation time frustrates assessment of ER, PR and E-cadherin immunohistochemistry (IHC) and HER2 fluorescence in situ hybridisation (FISH), 69 consecutive patients with 70 invasive breast carcinomas were included through the same-day diagnostics programme of breast lesions of the Radboud university medical center and the hospital Pantein. IHC for ER, PR and E-cadherin and HER2 FISH were compared between CNBs fixed for approximately 60-90 min and traditionally fixed resection specimens. RESULTS: Overall agreement between CNBs and resection specimens was 98.6% for ER (p<0.001; κ=0.93), 90.0% for PR (p<0.001; κ=0.75), 100% for E-cadherin (p<0001; κ=1.00) and 98.6% (p<0.001; κ=0.94) for HER2 FISH. Positive and negative predictive values were respectively 98.4% and 100% for ER, 95.9% and 76.2% for PR, 100% and 100% for E-cadherin and 90% and 100% for HER2 FISH. CONCLUSIONS: Hormone receptors and E-cadherin IHC and HER2 FISH are highly comparable between briefly fixed CNBs and the corresponding traditionally fixed resection specimens, and can therefore reliably be used in the daily clinical practice of same-day diagnostics of breast cancer.

Expansion of a BDCA1+CD14+ Myeloid Cell Population in Melanoma Patients May Attenuate the Efficacy of Dendritic Cell Vaccines.

The tumor microenvironment is characterized by regulatory T cells, type II macrophages, myeloid-derived suppressor cells, and other immunosuppressive cells that promote malignant progression. Here we report the identification of a novel BDCA1(+)CD14(+) population of immunosuppressive myeloid cells that are expanded in melanoma patients and are present in dendritic cell-based vaccines, where they suppress CD4(+) T cells in an antigen-specific manner. Mechanistic investigations showed that BDCA1(+)CD14(+) cells expressed high levels of the immune checkpoint molecule PD-L1 to hinder T-cell proliferation. While this BDCA1(+)CD14(+) cell population expressed markers of both BDCA1(+) dendritic cells and monocytes, analyses of function, transcriptome, and proteome established their unique nature as exploited by tumors for immune escape. We propose that targeting these cells may improve the efficacy of cancer immunotherapy. Cancer Res; 76(15); 4332-46. ©2016 AACR.

T-cell Landscape in a Primary Melanoma Predicts the Survival of Patients with Metastatic Disease after Their Treatment with Dendritic Cell Vaccines.

Tumor-infiltrating lymphocytes appear to be a predictor of survival in many cancers, including cutaneous melanoma. We applied automated multispectral imaging to determine whether density and distribution of T cells within primary cutaneous melanoma tissue correlate with survival of metastatic melanoma patients after dendritic cell (DC) vaccination. CD3(+) T cell infiltration in primary tumors from 77 metastatic melanoma patients was quantified using the ratio of intratumoral versus peritumoral T-cell densities (I/P ratio). Patients with longer survival after DC vaccination had stronger T-cell infiltration than patients with shorter survival in a discovery cohort of 19 patients (P = 0.000026) and a validation cohort of 39 patients (P = 0.000016). I/P ratio was the strongest predictor of survival in a multivariate analysis including M substage and serum lactate dehydrogenase level. To evaluate I/P ratio as a predictive biomarker, we analyzed 19 chemotherapy-treated patients. Longer survival times of DC-vaccinated compared with chemotherapy-treated patients was observed for high (P = 0.000566), but not low (P = 0.154) I/P ratios. In conclusion, T-cell infiltration into primary melanoma is a strong predictor of survival after DC vaccination in metastatic melanoma patients who, on average, started this therapy several years after primary tumor resection. The infiltration remains predictive even after adjustment for late-stage prognostic markers. Our findings suggest that the I/P ratio is a potential predictive biomarker for treatment selection. Cancer Res; 76(12); 3496-506. ©2016 AACR.

Brief fixation does not hamper the reliability of Ki67 analysis in breast cancer core-needle biopsies: a double-centre study.

AIMS: Immunohistochemical assessment of Ki67 expression in core-needle biopsies (CNBs) is increasingly playing a role in therapeutic decision-making for breast cancer patients. Within the framework of same-day diagnosis of breast lesions, fixation times are markedly decreased. We therefore attempted to validate Ki67 analysis in briefly fixed breast cancer CNBs. METHODS AND RESULTS: CNBs of 136 consecutive patients with invasive breast cancer diagnosed through the same-day diagnosis programme of both the University Medical Centre Utrecht (UMCU) and the Radboud University Medical Centre (RUMC) were included. CNBs were fixed in formaldehyde for approximately 45 min at the UMCU and for between 60 and 90 min at the RUMC. Immunohistochemistry for Ki67 expression was compared between the briefly fixed CNBs and conventionally fixed resection specimens of the same tumour. The overall agreement between CNBs and resections was 122 of 142 (85.9%) (κ = 0.71; 95% CI 0.60-0.83). Positive and negative predictive values were 79.7% (95% CI 67-88%) and 91.0% (95% CI 82-96%), respectively. CONCLUSIONS: Overall agreement for Ki67 expression was good between briefly fixed CNBs and conventionally fixed resection specimens, and within the range of studies comparing conventionally fixed CNBs and resections.

The molecular background of mucinous carcinoma beyond MUC2.

The increasing interest of the oncology community in tumour classification and prediction of outcome to targeted therapies has put emphasis on an improved identification of tumour types. Colorectal mucinous adenocarcinoma (MC) is a subtype that is characterized by the presence of abundant extracellular mucin that comprises at least 50% of the tumour volume and is found in 10-15% of colorectal cancer patients. MC development is poorly understood, however, the distinct clinical and pathological presentation of MC suggests a deviant development and molecular background. In this review we identify common molecular and genetic alterations in colorectal MC. MC is characterized by a high rate of MUC2 expression. Mutation rates in the therapeutically important RAS/RAF/MAPK and PI3K/AKT pathways are significantly higher in MC compared with non-mucinous adenocarcinoma. Furthermore, mucinous adenocarcinoma shows higher rates of microsatellite instability and is more frequently of the CpG island methylator phenotype. Although the majority of MCs arise from the large intestine, this subtype also develops in other organs, such as the stomach, pancreas, biliary tract, ovary, breast and lung. We compared findings from colorectal MC with tumour characteristics of MCs from other organs. In these organs, MCs show different mutation rates in the RAS/RAF/MAPK and PI3K/AKT pathways as well, but a common mucinous pathway cannot be identified. Identification of conditions and molecular aberrations that are associated with MC generates insight into the aetiology of this subtype and improves understanding of resistance to therapies.