Chris Hemmings

National Working Group Meeting on ALK Diagnostics in Lung Cancer

The global landscape of molecular testing is rapidly changing, with the recent publication of the International Association for the Study of Lung Cancer (IASLC)/College of American Pathologists (CAP) guidelines and the ALK Atlas. The IASLC/CAP guidelines recommend that tumors from patients with non‐small cell lung cancer (NSCLC) be tested for ALK rearrangements in addition to epidermal growth factor receptor (EGFR) mutations. The spur for this recommendation is the availability of novel therapies that target these rearrangements. This article is based on coverage of a Pfizer‐sponsored National Working Group Meeting on ALK Diagnostics in Lung Cancer, held around the 15th World Lung Cancer Conference, in Sydney on October 31, 2013. It is based on the presentations given by the authors at the meeting and the discussion that ensued. The content for this article was discussed and agreed on by the authors.

Personalising pancreas cancer treatment:When tissue is the issue

The treatment of advanced pancreatic cancer has not moved much beyond single agent gemcitabine until recently when protocols such as FOLFIRINOX (fluorouracil, leucovorin, irinotecan and oxaliplatin) and nab-paclitaxel-gemcitabine have demonstrated some improved outcomes. Advances in technology especially in massively parallel genome sequencing has progressed our understanding of the biology of pancreatic cancer especially the candidate signalling pathways that are involved in tumourogenesis and disease course. This has allowed identification of potentially actionable mutations that may be targeted by new biological agents. The heterogeneity of pancreatic cancer makes tumour tissue collection important with the aim of being able to personalise therapies for the individual as opposed to a one size fits all approach to treatment of the condition. This paper reviews the developments in this area of translational research and the ongoing clinical studies that will attempt to move this into the everyday oncology practice.

Is carcinoma a mesenchymal disease? The role of the stromal microenvironment in carcinogenesis

Summary Most research into the biology of carcinoma has focused on the epithelial cells therein; the inherent assumption has been that the tumour arises from epithelial cells ‘gone bad’, and that the surrounding stroma is simply an ‘innocent bystander’. However, there is increasing evidence that there is a complex interplay between tumour cells and their surrounding microenvironment, and that the latter may be just as important in determining the development and clinical behaviour of a given tumour. Similarly, traditional oncological practice has been predominantly aimed at a perceived ideal goal of killing all the tumour epithelial cells, with only a few recently developed therapies seeking to affect other components (such as tumour vasculature); but identifying stromal factors involved in tumour growth and survival may well lead to the development of novel therapies. This review examines current understanding of the interplay between tumour epithelial cells and their microenvironment, and enumerates various stromal factors which appear to play a role in tumour progression and/or metastasis.

Objective analysis of cancer stem cell marker expression using immunohistochemistry

Analysis of immunohistochemical expression is often a subjective and semiquantitative process that can lead to the inconsistent reporting of results. To assess the effect that region selection and quantification method have on results, five different cancer stem cell markers were used in this study to compare tissue scoring with digital analysis methods that used three different tissue annotation methods. Samples of tumour and normal mucosa were used from 10 consecutive stage II colon cancer patients and stained for the putative cancer stem cell markers ALDH1, CD44v6, CD133, Lgr5 and SOX2. Tissue scoring was found to have considerably different results to digital analysis with the three different digital methods harbouring concordant results overall. However, SOX2 on normal tissue and CD133 on tumour and normal tissue produced discordant results which could be attributed to the different regions of tissue that were analysed. It is important that quantification method and selection of analysis areas are considered as part of study design to ensure that reproducible and consistent results are reported in the literature.

Tumour-infiltrating regulatory T cell density before neoadjuvant chemoradiotherapy for rectal cancer does not predict treatment response

Neoadjuvant (preoperative) chemoradiotherapy (CRT) decreases the risk of rectal cancer recurrence and reduces tumour volume prior to surgery. However, response to CRT varies considerably between individuals and factors associated with response are poorly understood. Foxp3+ regulatory T cells (Tregs) inhibit anti-tumour immunity and may limit any response to chemotherapy and radiotherapy. We have previously reported that a low density of Tregs in the tumour stroma following neoadjuvant CRT for rectal cancer is associated with improved tumour regression. Here we have examined the association between Treg density in pre-treatment diagnostic biopsy specimens and treatment response, in this same patient cohort. We aimed to determine whether pre-treatment tumour-infiltrating Treg density predicts subsequent response to neoadjuvant CRT. Foxp3+, CD8+ and CD3+ cell densities in biopsy samples from 106 patients were assessed by standard immunohistochemistry (IHC) and evaluated for their association with tumour regression grade and survival. We found no association between the density of any T cell subset pre-treatment and clinical outcome, indicating that tumour-infiltrating Treg density does not predict response to neoadjuvant CRT in rectal cancer. Taken together with the findings of the previous study, these data suggest that in the context of neoadjuvant CRT for rectal cancer, the impact of chemotherapy and/or radiotherapy on anti-tumour immunity may be more important than the state of the pre-existing local immune response.

The prognostic value of cancer stem-like cell markers SOX2 and CD133 in stage III colon cancer is modified by expression of the immune-related markers FoxP3, PD-L1 and CD3

Cancer stem-like cells are highly tumourigenic cells that can repopulate entire tumours after apparent successful treatment. Recent evidence suggests they interact with other cells in the tumour microenvironment, including immune cell subsets, to enhance their survival. The aim of this study was to determine whether the expression of immune cell markers in primary colon cancer impacts the prognostic significance of cancer stem-like cell marker expression. Immunohistochemistry was used to assess the expression of putative stem cell markers (ALDH1, CD44v6, CD133, Lgr5, SOX2) and immune cell related markers (CD3, CD8, FoxP3, PD-L1) in 104 patients with stage III colon cancer. Associations of marker expression with overall and cancer-specific survival were determined using Kaplan-Meier analysis. High SOX2 expression in the central tumour area was found to be an independent factor for poor cancer-specific survival [hazard ratio (HR) 6.19; 95% confidence interval (CI) 2.24-17.14; p=0.001]. When immune-related factors were taken into account, patients categorised as SOX2low/FoxP3high had good outcome (HR 0.164; 95%CI 0.066-0.406; p<0.0001) whereas patients categorised as SOX2high/PD-L1low had poor outcome (HR 8.992; 95%CI 3.397-23.803; p<0.0001). The prognostic value of the SOX2 cancer stem-like cell marker in colon cancer is modified by expression of immune-cell related factors FoxP3 and PD-L1.

Neoadjuvant chemoradiotherapy for rectal cancer: how important is tumour regression?

Pathological complete response following neoadjuvant chemoradiotherapy (CRT) for locally advanced rectal cancer is associated with reduced local recurrence and improved long‐term outcome. However, the prognostic value of a partial response, or of tumour regression in patients with metastatic disease, is less clear.

Morphology, molecular genetics and multidisciplinary management: soft tissue pathology in 2014 and beyond

This special edition of Pathology focuses on a relatively uncommon but nonetheless fascinating branch of pathology. Soft tissue tumours (with the exception of lipoma) are collectively infrequent and, comprising over 50 distinct histotypes, individually rare. Whilst sarcoma accounts for only around 2% 1 of cancers worldwide, these tumours result in significant mortality and morbidity, both during treatment and in longterm survivors following radical treatment (the latter disproportionately so in the paediatric population, in whom second malignancies are a real threat). Occurring in all age groups and anatomical locations, soft tissue tumours will come across the desk of most surgical pathologists from time to time, but because of their relative rarity and the sometimes bewildering array of diagnostic possibilities, they are often challenging for those whose experience in this area is limited. Some guidance in the pathology reporting of soft tissue sarcoma has been offered by the Royal College of Pathologists of Australasia (RCPA), under the umbrella of the Structured Pathology Reporting project (now part of the International Collaboration on Cancer Reporting, ICCR). The first edition of the protocol for soft tissue tumour resections was published in 2011 and can be accessed online; the evidence on which those recommendations were based was previously canvassed in this journal. Ongoing efforts to integrate morphological, immunohistochemical and molecular genetic information result in more-orless constant revision and reclassification of these tumours, including the characterisation of new entities (such as the recently described epithelioid inflammatory myofibroblastic sarcoma) and the disappearance of old ones (such as MFH); reclassification of lesions (such as myofibroma, now classified as a pericytic/perivascular lesion); recognition that apparently separate entities may be related, and that hybrids may occur (such as giant cell fibroblastoma and dermatofibrosarcoma protruberans, or haemosiderotic fibrolipomatous tumour and myxoinflammatory fibroblastic sarcoma); and recognition that lesions previously thought to be reactive (such as nodular fasciitis) may in fact be neoplastic. In this issue Vickie Jo and Chris Fletcher summarise the significant changes in the recently published 4th edition of the WHO classification, including new immunohistochemical markers which may assist with diagnosis, as well as elaborating new information which has come to light since that classification appeared (highlighting soft tissue pathology as a rapidly evolving area, and perhaps providing some hints as to likely changes that will appear in the 5th edition!). Many of these conceptual advances have occurred in light of molecular genetic analyses which have enhanced our understanding of sarcomagenesis. But a word of caution is offered by Cristina Antonescu and Paola dal Cin, who discuss the increasing recognition of

A cautionary note regarding detection of PD-L1 expression by tumour-associated macrophages

We read with interest the recent paper by Gottlieb et al. (2017) in which the authors evaluated Programmed Death Ligand-1 (PD-L1) expression by tumour cells and tumour-associated macrophages (TAMs) in primary high grade serous ovarian cancer (HGSOC) specimens and in matched metastases. The expression of immune checkpoint molecules in the local tumour environment, particularly of PD-L1, is currently a topic of much research focus. However, there are some technical issues surrounding the immunohistochemical detection of PD-L1 that other researchers who are considering performing this assay should be aware of. These are especially important in the context of TAM PDL1 expression. As the authors mention in their discussion, there are now several commercially available anti-human PD-L1 antibodies, and there has been much debate over the reproducibility of staining using different clones. This is particularly truewith regard to the staining of tumour-associated immune cells (Hirsch et al., 2017). We recently performed an in-house evaluation of four antibodies (E1L3N, Cell Signaling Technology; 28-8, Abcam; 22C3, Dako and SP263, Roche) for detection of PD-L1 in formalin-fixed paraffin-embedded colorectal tissue, and found significant variation in staining intensity and in sensitivity to alterations in the staining protocol, such as choice of antigen retrieval solution and/ or secondary detection reagents (Anyaegbu et al., in press). During our evaluation, we noticed considerable non-specific staining of macrophages, present when an isotype control was used in place of the primary antibody. Background staining of macrophages is a common and widely recognised phenomenon due to their high level of Fc receptor expression. However, we found it to be particularly pronounced with the protocols required to obtain optimal PD-L1 staining, despite the use of blocking reagents, since signal amplification using polymer detection methods is usually required. We have found this to be the case with HGSOC samples as well as colorectal cancer. Cellular localisation of ‘true’ PD-L1 staining is important to differentiate from non-specific or background staining. The non-specific staining of macrophages have a granular cytoplasmic appearance, lacking themembranous staining we judge to be PD-L1-specific. This finding is consistent with other publications and guidelines released for interpretation (PD-L1 IHC 22C3 pharmDx Interpretation Manual US Version, 2015; Kerr et al., 2015; Roach et al., 2016; Webb et al., 2016). In our experience, non-specific granular cytoplasmic staining can also co-exist with true membranous staining and PD-L1 expression should be recognised in these.While Gottlieb et al. do specify that they assessedmembranous PD-L1 staining of tumour cells and TAM, it is difficult to see the staining pattern in the relatively low magnification image provided. Also, these figures cannot demonstrate direct evidence of PD-L1 expression by CD68 TAM as there are not of serial sections. We do not mean to suggest that the results presented by Gottlieb et al are not valid. The extent of TAM PD-L1 expression observed in