Validation of biomarkers and digital image analysis in breast pathology

For women worldwide, the risk of developing breast cancer is second only to that of nonmelanoma skin cancer. Significant improvements have been made in survival over the past decades and today about 80 % of the patients survive 10 years or more after their breast cancer diagnosis. Still, far from all patients enjoy the relatively good survival indicated by statistics on breast cancer patients as one homogenous group. Improving prognostication of aggressive vs. less aggressive disease, and to separate tumors based on genetic differences for optimal treatment strategies, is therefore the focus of intensive research, including this thesis. In paper I, we compared if tumor characteristics differ depending on what method of sampling the tumor that have been used for analysis. We compared routine immunohistochemistry on surgically resected breast specimens, including stains of the Estrogen receptor alpha (ER), the Progesterone receptor (PR), Human Epidermal growth factor receptor 2 (HER2) and the proliferation-associated protein Ki67, with analysis of the same stains done on material obtained from fine needle aspiration (immunocytochemistry). We found that there were substantial differences in the expression of these biomarkers between the two methods. Thus, the same rules for interpretation of biomarkers cannot be used for immunohistochemistry and immunocytochemistry, and consequently, validation of each method should be performed individually. In paper II, we explored the scope of digital image analysis in biomarker evaluations. We scored ER, PR, HER2 and Ki67 status in several different regions of breast tumors by both manual methods and digital image analysis. The outcomes of the scoring of these biomarkers were then combined into IHC surrogate subtypes and compared to PAM50 gene expression-based subtypes as well as patient survival. All tested methods of automated digital image analysis of Ki67 outperformed manual scores in terms of sensitivity and specificity for the Luminal B subtype. Comparing digital versus manual testing concordance to all breast cancer subtypes as determined by PAM50 assays, the digital approach was superior to the manual method. The manual and digital image analysis methods matched each other in hazard ratio for all-cause mortality of patients with tumors with a “high” vs “low” Ki67 index. Manual assessments of the biomarkers ER, PR, HER2 and Ki67 were in most aspects less precise than digital image analysis. In paper III, we evolved the concept of paper I with an evaluation of the concordance of consecutive Ki67 assessments performed on fine needle aspiration cytology versus resected tumor specimens. We investigated how a status of Ki67 “low” and “high” as determined by immunohistochemistry and immunocytochemistry corresponded to overall survival, respectively. Again, Ki67-index varied when the two methods were used on the same tumors, and was prone to switch the classification between low and high proliferation. ER evaluations were discordant in 5.3 % of the tumors, which in the clinical setting would mean that 1 in 20 patients would risk being left out of beneficial endocrine treatment or being given it without benefit. Ki67 “high”, as determined by immunohistochemistry, defined as a proportion of Ki67-positive cells above the 67th percentile of the material, was significantly associated with poor overall survival and a significantly higher probability of axillary lymph node metastasis. This could not be reproduced for immunocytochemistry. In summary, this study adds to the results of paper I, in which we showed discordance between the methods. By including survival data, we now conclude that not merely are the methods discordant, but immunocytochemistry fails to provide prognostic information. Consequently, immunohistochemistry should be regarded as the superior method. In paper IV, we focused on proliferation comparing the results in the tumors’ hot spot, in the tumor periphery, and as the average proportion of Ki67-positive cells across the whole tumor section. Both manual and digital scores of Ki67 and the recently described marker for mitotic activity, PHH3, were evaluated along with mitotic counts. Their sensitivity and specificity for the gene expression based Luminal B versus A breast cancer subtypes, for the high versus low transcriptomic grade, for axillary lymph node status as well as for their prognostic value for breast cancer specific and overall survival were analyzed. Digital image analysis of Ki67 in hot spots outperformed the other markers in sensitivity and specificity both for gene expression subtypes and transcriptomic grade. In contrast to mitotic counts, tumors with high expression of Ki67, as defined by digital image analysis and high numbers of PHH3-positive cells, had significantly increased HR for all-cause mortality at 10 years from diagnosis. When we replaced the manual mitotic counts with digital image analysis of Ki67 in hot spots as the marker for proliferation when determining histological grade, the differences in estimated mean overall survival between the highest and lowest grades increased. It also added significantly more prognostic information to the classic Nottingham combined histological grade. We conclude that digital image analysis of Ki67 in hot spots might be suggested as the marker of choice for proliferative activity in breast cancer

Evaluation of PD-L1 expression in various formalin-fixed paraffin embedded tumour tissue samples using SP263, SP142 and QR1 antibody clones

Background & objectives: Cancer cells can avoid immune destruction through the inhibitory ligand PD-L1. PD-1 is a surface cell receptor, part of the immunoglobulin family. Its ligand PD-L1 is expressed by tumour cells and stromal tumour infltrating lymphocytes (TIL). Methods: Forty-four cancer cases were included in this study (24 triple-negative breast cancers (TNBC), 10 non-small cell lung cancer (NSCLC) and 10 malignant melanoma cases). Three clones of monoclonal primary antibodies were compared: QR1 (Quartett), SP 142 and SP263 (Ventana). For visualization, ultraView Universal DAB Detection Kit from Ventana was used on an automated platform for immunohistochemical staining Ventana BenchMark GX. Results: Comparing the sensitivity of two different clones on same tissue samples from TNBC, we found that the QR1 clone gave higher percentage of positive cells than clone SP142, but there was no statistically significant difference. Comparing the sensitivity of two different clones on same tissue samples from malignant melanoma, the SP263 clone gave higher percentage of positive cells than the QR1 clone, but again the difference was not statistically significant. Comparing the sensitivity of two different clones on same tissue samples from NSCLC, we found higher percentage of positive cells using the QR1 clone in comparison with the SP142 clone, but once again, the difference was not statistically significant. Conclusion: The three different antibody clones from two manufacturers Ventana and Quartett, gave comparable results with no statistically significant difference in staining intensity/ percentage of positive tumour and/or immune cells. Therefore, different PD-L1 clones from different manufacturers can potentially be used to evaluate the PD- L1 status in different tumour tissues. Due to the serious implications of the PD-L1 analysis in further treatment decisions for cancer patients, every antibody clone, staining protocol and evaluation process should be carefully and meticulously validated

Deciphering the interplay of molecular alterations underpinning renal cell carcinoma by label-free mass spectrometry and clinical proteomics: A systems medicine approach for precision diagnosis

Renal neoplasia is the 14th most common tumor type diagnosed worldwide. With a vast heterogeneity, renal neoplasia encompasses different subtypes. 90% of the neoplasms arise from the epithelial layer of the nephron and vary from benign renal masses (renal oncocytoma, RO) to more indolent or aggressive cancers (renal cell carcinomas, RCC). As RCC subtypes, clear cell (ccRCC) subtype is the most predominant subtype, followed by papillary (pRCC) and chromophobe (chRCC). Despite the different outcomes, some overlapped histological and morphological features difficult their differentiation and diagnosis. Therefore, new approaches for a clear and accurate diagnosis are still needed. To achieve this goal, renal tissue biopsies diagnosed with ccRCC (n = 7), pRCC (n = 5), chRCC (n = 5), RO (n = 5) and normal adjacent tissue (NAT, n= 5) were enrolled in this study. As a very resourceful tool for proteome analysis and biomarker discovery, mass spectrometry (MS)-based methods were used to interrogate the proteome of each tumor in order to undisclosed differences trough which to develop faster and accurate diagnostics. The results achieved with this doctoral thesis include i) the accomplishment of an effective ultrasonic workflow to recover the proteome of optimal cutting temperature (OCT)-embedded tissues, ii) a novel analytical approach based on MALDI-MS profiling to distinguish chRCC from RO, iii) a 109-protein panel to discriminate between chRCC and RO and NAT, iv) a top 24-protein panel to diagnose ccRCC, pRCC, chRCC and RO based on absolute concentration values, v) the translation and validation of three promising biomarkers by immunohistochemical analysis, and vi) an approach for phosphopeptide enrichment. This work brings new insights into the different mechanisms underlying formation of these tumors as well as it provides valuable information to improve clinical diagnosis by opening new avenues for immunohistochemistry and mass spectrometry-based approaches

Estimating the trauma-death interval : a histological investigation of fracture healing.

The accurate, reliable estimation of the ‘age’ of a fracture, or the time elapsed since trauma was sustained, has important implications In a variety of forensic contexts. Such information could greatly aid the forensic diagnosis of child abuse, the reconstruction of events during a violent incident such as homicide or a road traffic accident, and assist in the identification of unknown remains. Forensic fracture dating has largely relied on radiographical and histological evidence, but has lacked precision and consistency. The research presented here alms to test the hypothesis that correlations exist between the histologically- and immunohistochemically-observable phenomena at a fracture site and the known trauma-death interval of an individual. This was achieved by comparing the known trauma-death interval (TDI) to the extent of healing visible on histological slides prepared from formalin-fixed, paraffin-embedded, decalcified blocks of bone excised from the fracture site of 52 rib, skull and femur fractures from 29 individual forensic cases submitted to the Medico-Legal Centre Sheffield between 1992 and 2002. The slides were stained with haematoxylin and eosin to stain nuclei and cytoplasm, Peris’ Prussian Blue stain for haemosiderin granules, mono-clonal anti-CD68 antibody for osteoclasts, and anti-bone sialoprotein antibody as an osteoblast and osteocyte marker. Quantifiable parameters such as the percentage cover of red blood cells, of living and necrotic compact bone, and the size, abundance and dispersal of immuno-positive and inflammatory cells were examined and compared to the TDI using human observers and Scion Image histomorphometry software. Statistically significant correlations were found between TDI and the presence of haemosiderin granules later than three days post-trauma; and the dispersal and location of CD68 positive cells; as well as the estimated percentage cover of fibroblasts and red blood cells at the fracture site. Other trends and correlations were found, which contribute to the understanding of bone’s immediate responses to trauma. It is hoped that this research may aid the prediction of the time elapsed since trauma in a forensic context and broaden the scope of trauma analysis in forensic anthropology

Immunohistochemistry in Diagnostic Veterinary Pathology

Immunohistochemistry is the application of antigen-antibody interactions plus a detection system to tissue sections for the purpose of identifying and localising a given substance. Widely applied in the fields of research science and medical diagnostic pathology, immunohistochemistry is beginning to be used in diagnostic veterinary pathology and this work was performed to investigate the potential of the technique in assisting with certain histological challenges. Chapter one provides a broad technical introduction to immunohistochemistry, highlighting the strengths and pitfalls of the technique, and concludes with a literature review which focuses particularly on the rapidly expanding veterinary literature dealing with the use of immunohistochemical methods. Chapter two describes the use of specific antibodies to determine the cell of origin of thymic tumours in a variety of species. These commercially available antibodies were raised against human epitopes but were found to be effective on domestic animals. Immunohistochemistry was found to be invaluable in differentiating between thymic epithelial and lymphoid tumours. In chapter three antibodies to specific subclasses of cytokeratin were applied to canine tissues and found to be effective at localising different cytokeratins in normal canine skin and in a range of epithelial tumours. In chapter four the identification of a nuclear tumour suppressor gene protein, p53, by immunohistochemistry is described. Immunohistochemical detection of this protein indicates abnormal functional status of p53 which occupies a pivotal role in the cell's response to DNA damage. A panel of anti-human p53 antibodies were used on canine, equine and bovine tumours and very high levels of p53 protein expression were detected in equine squamous cell carcinomas

Assessing pain and inflammation in arthritis using novel imaging methods

Enhanced bone resorption is a common pathology in destructive bone diseases. Many cytokines (e.g. IL-6 and TNF-α) and chemokines (e.g. CCL2, CCL3 and CCL5) elevated in patients exert pathological roles in leukocyte migration and inflammation, but their effect on direct bone destruction remained elusive. Published research into osteoclastogenesis was carried out in co-cultures, from which osteoclast differentiation, resorption and mediator secretion data was obtained. The main objective of this thesis was to establish a working methodology for the in vitro differentiation of CD14+ve mononuclear cells into osteoclasts and to decipher the direct effects of IL-6 and CCL3 on osteoclastogenesis and bone resorption. IL-6 trans-signalling exerted an effect in both basal and pathological osteoclastogenesis, whereas its inhibition via sgp130-Fc significantly reduced osteoclast formation and bone resorption in vitro. Although not examined in vivo, the translational use of sgp130-Fc as a therapeutic for destructive bone pathology was postulated from data showing a reduction in osteoclast differentiation and resorption after stimulation with HYPER-IL-6. Secondary to IL-6 trans-signalling, it was hypothesised that the neutralisation of CCL3 in vitro and in vivo would significantly reduce osteoclast differentiation and resorption. Osteoclast number significantly reduced in the presence of anti-CCL3, but TRAP+ve cell count was unaltered, suggesting an early role of CCL3 in osteoclast multi-nucleation/fusion. Additionally, in vivo data showed a protective effect of anti-CCL3 with significantly reduced bone erosive scores and osteoclast counts thereby presenting CCL3 as a novel biomarker of disease activity in destructive bone disease. In contrast, TNF-α, CCL2, and CCL5 were shown to have no role in direct osteoclast differentiation in our monocultures. In conclusion, for the first time this work documents novel and important roles of IL-6 trans-signalling and CCL3 in increased osteoclast differentiation and bone resorption. Our data highlights the importance of IL-6 trans-signalling and provides evidence for the use of CCL3 as a predictive biomarker in destructive bone diseases

Objective localisation of oral mucosal lesions using optical coherence tomography.

PhDIdentification of the most representative location for biopsy is critical in establishing the definitive diagnosis of oral mucosal lesions. Currently, this process involves visual evaluation of the colour characteristics of tissue aided by topical application of contrast enhancing agents. Although, this approach is widely practiced, it remains limited by its lack of objectivity in identifying and delineating suspicious areas for biopsy. To overcome this drawback there is a need to introduce a technique that would provide macroscopic guidance based on microscopic imaging and analysis. Optical Coherence Tomography is an emerging high resolution biomedical imaging modality that can potentially be used as an in vivo tool for selection of the most appropriate site for biopsy. This thesis investigates the use of OCT for qualitative and quantitative mapping of oral mucosal lesions. Feasibility studies were performed on patient biopsy samples prior to histopathological processing using a commercial OCT microscope. Qualitative imaging results examining a variety of normal, benign, inflammatory and premalignant lesions of the oral mucosa will be presented. Furthermore, the identification and utilisation of a common quantifiable parameter in OCT and histology of images of normal and dysplastic oral epithelium will be explored thus ensuring objective and reproducible mapping of the progression of oral carcinogenesis. Finally, the selection of the most representative biopsy site of oral epithelial dysplasia would be investigated using a novel approach, scattering attenuation microscopy. It is hoped this approach may help convey more clinical meaning than the conventional visualisation of OCT images

The expression of p53 and related proteins in human breast tumours and malignant melanomas

Individuals with the Li-Fraumeni syndrome possess germline mutations in the p53 tumour suppressor gene. These individuals develop tumours at an early age and in various combinations. One combination is breast cancer and malignant melanoma. Anecdotal reports exist of a greater than expected number of melanomas in patients with sporadic breast cancer and although an association between both tumours has previously been proposed there has been no established molecular, or genetic link. The breast like the skin is developed predominantly from ectoderm and an oestrogenic environment favours the development of both tumours. In fact it has been proposed that the breast is a modified sebaceous gland. Breast cancer is defined on the basis of epidemiological, cytological and pathological lines, but no single classification reliably predicts clinical behaviour in any subset of tumours with a reliability which exceeds 50%. Similarly, prognosis in melanomas is reliably predicted by the best known Breslow's thickness measurement and Clark's levels of invasion respectively, but a subset of tumours of relatively good prognosis induce early metastasis and death. Both tumours have a poorer prognosis in males. Are there detectable similar molecular phenotypic differences in breast tumour and melanoma groups related to distinct clinical behaviour and pathological characteristics? Is there a link between sporadic breast cancer and malignant melanoma, or are there major similarities in the molecular behaviour of both tumours based on a defect in the p53 tumour suppressor gene's control of the cell cycle? The presence of the dysplastic naevus syndrome a.k.a. the atypical mole syndrome increases the risk of the development of melanoma, but the association with breast cancer, although reported has not been established. Is there a link between both conditions based on a defect in the p53 tumour suppressor gene? Sporadic breast cancer afflicts 1 in 12 women in the United Kingdom and malignant melanomas affect a young population at their prime, yet we have been unable to significantly alter the treatment outcomes for these tumours. Have we adopted a simplistic approach to their management? Should we now examine the immunohistochemical anomalies present in these tumours before we offer treatment advice? Aim: (i) To examine the immunohistochemical expression of a set of p53 related oncogenic markers in groups of with patients both breast cancer and malignant melanoma, or breast cancer and the dysplastic naevus syndrome and compare them with control groups of sporadic breast tumours (benign and malignant) of established clinical outcome. Conclusion: The overexpression of p53 in all breast tumours in patients with melanomas suggests that these tumours may harbour mutations (either germline or somatic) in the p53 gene but this can only be confirmed by sequencing the p53 gene. Clinical outcome of the distinct unrelated groups of breast tumours may be related to undetected molecular alterations which may modify, both the prognosis and the response to chemotherapeutic interventions. p53 expression in patients with TiNO tumours may be helpful in predicting early recurrence. Aim: (ii) To examine the expression of p53 protein MIB-1, Bcl-2 and the antimelanoma antibody, HMB-45 in a subset of melanomas in patients with p53 positive malignant breast tumours (n = 9) and compare them with a control group of 66 melanomas F = 46, M = 20. Conclusion: p53 expression in sporadic melanomas is commoner in individuals who also express the protein in the dermo-epidermal junction. The MIB-1 score in the dermo-epidermal junction and Bcl-2 expression in malignant melanomas may be of prognostic significance and suggests an underlying defect in the apoptotic pathways in these tumours. (Abstract shortened.