Deep learning-based risk stratification of preoperative breast biopsies using digital whole slide images.

BACKGROUND: Nottingham histological grade (NHG) is a well established prognostic factor in breast cancer histopathology but has a high inter-assessor variability with many tumours being classified as intermediate grade, NHG2. Here, we evaluate if DeepGrade, a previously developed model for risk stratification of resected tumour specimens, could be applied to risk-stratify tumour biopsy specimens. METHODS: A total of 11,955,755 tiles from 1169 whole slide images of preoperative biopsies from 896 patients diagnosed with breast cancer in Stockholm, Sweden, were included. DeepGrade, a deep convolutional neural network model, was applied for the prediction of low- and high-risk tumours. It was evaluated against clinically assigned grades NHG1 and NHG3 on the biopsy specimen but also against the grades assigned to the corresponding resection specimen using area under the operating curve (AUC). The prognostic value of the DeepGrade model in the biopsy setting was evaluated using time-to-event analysis. RESULTS: Based on preoperative biopsy images, the DeepGrade model predicted resected tumour cases of clinical grades NHG1 and NHG3 with an AUC of 0.908 (95% CI: 0.88; 0.93). Furthermore, out of the 432 resected clinically-assigned NHG2 tumours, 281 (65%) were classified as DeepGrade-low and 151 (35%) as DeepGrade-high. Using a multivariable Cox proportional hazards model the hazard ratio between DeepGrade low- and high-risk groups was estimated as 2.01 (95% CI: 1.06; 3.79). CONCLUSIONS: DeepGrade provided prediction of tumour grades NHG1 and NHG3 on the resection specimen using only the biopsy specimen. The results demonstrate that the DeepGrade model can provide decision support to identify high-risk tumours based on preoperative biopsies, thus improving early treatment decisions.

PIK3CA mutations in endocrine-resistant breast cancer.

Around 75% of breast cancer (BC) patients have tumors expressing the predictive biomarker estrogen receptor α (ER) and are offered endocrine therapy. One-third eventually develop endocrine resistance, a majority with retained ER expression. Mutations in the phosphatidylinositol bisphosphate 3-kinase (PI3K) catalytic subunit encoded by PIK3CA is a proposed resistance mechanism and a pharmacological target in the clinical setting. Here we explore the frequency of PIK3CA mutations in endocrine-resistant BC before and during treatment and correlate to clinical features. Patients with ER-positive (ER +), human epidermal growth factor receptor 2 (HER2)-negative primary BC with an ER + relapse within 5 years of ongoing endocrine therapy were retrospectively assessed. Tissue was collected from primary tumors (n = 58), relapse tumors (n = 54), and tumor-free lymph nodes (germline controls, n = 62). Extracted DNA was analyzed through panel sequencing. Somatic mutations were observed in 50% (31/62) of the patients, of which 29% occurred outside hotspot regions. The presence of PIK3CA mutations was significantly associated with nodal involvement and mutations were more frequent in relapse than primary tumors. Our study shows the different PIK3CA mutations in endocrine-resistant BC and their fluctuations during therapy. These results may aid investigations of response prediction, facilitating research deciphering the mechanisms of endocrine resistance.

Clinical evaluation of deep learning-based risk profiling in breast cancer histopathology and comparison to an established multigene assay.

PURPOSE: To evaluate the Stratipath Breast tool for image-based risk profiling and compare it with an established prognostic multigene assay for risk profiling in a real-world case series of estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative early breast cancer patients categorized as intermediate risk based on classic clinicopathological variables and eligible for chemotherapy. METHODS: In a case series comprising 234 invasive ER-positive/HER2-negative tumors, clinicopathological data including Prosigna results and corresponding HE-stained tissue slides were retrieved. The digitized HE slides were analysed by Stratipath Breast. RESULTS: Our findings showed that the Stratipath Breast analysis identified 49.6% of the clinically intermediate tumors as low risk and 50.4% as high risk. The Prosigna assay classified 32.5%, 47.0% and 20.5% tumors as low, intermediate and high risk, respectively. Among Prosigna intermediate-risk tumors, 47.3% were stratified as Stratipath low risk and 52.7% as high risk. In addition, 89.7% of Stratipath low-risk cases were classified as Prosigna low/intermediate risk. The overall agreement between the two tests for low-risk and high-risk groups (N = 124) was 71.0%, with a Cohen's kappa of 0.42. For both risk profiling tests, grade and Ki67 differed significantly between risk groups. CONCLUSION: The results from this clinical evaluation of image-based risk stratification shows a considerable agreement to an established gene expression assay in routine breast pathology.

Prognostic impact of HER2 biomarker levels in trastuzumab-treated early HER2-positive breast cancer.

BACKGROUND: Overexpression of human epidermal growth factor receptor 2 (HER2) caused by HER2 gene amplification is a driver in breast cancer tumorigenesis. We aimed to investigate the prognostic significance of manual scoring and digital image analysis (DIA) algorithm assessment of HER2 copy numbers and HER2/CEP17 ratios, along with ERBB2 mRNA levels among early-stage HER2-positive breast cancer patients treated with trastuzumab. METHODS: This retrospective study comprised 371 early HER2-positive breast cancer patients treated with adjuvant trastuzumab, with HER2 re-testing performed on whole tumor sections. Digitized tumor tissue slides were manually scored and assessed with uPath HER2 Dual ISH image analysis, breast algorithm. Targeted ERBB2 mRNA levels were assessed by the Xpert® Breast Cancer STRAT4 Assay. HER2 copy number and HER2/CEP17 ratio from in situ hybridization assessment, along with ERBB2 mRNA levels, were explored in relation to recurrence-free survival (RFS). RESULTS: The analysis showed that patients with tumors with the highest and lowest manually counted HER2 copy number levels had worse RFS than those with intermediate levels (HR = 2.7, CI 1.4-5.3, p = 0.003 and HR = 2.1, CI 1.1-3.9, p = 0.03, respectively). A similar trend was observed for HER2/CEP17 ratio, and the DIA algorithm confirmed the results. Moreover, patients with tumors with the highest and the lowest values of ERBB2 mRNA had a significantly worse prognosis (HR = 2.7, CI 1.4-5.1, p = 0.003 and HR = 2.8, CI 1.4-5.5, p = 0.004, respectively) compared to those with intermediate levels. CONCLUSIONS: Our findings suggest that the association between any of the three HER2 biomarkers and RFS was nonlinear. Patients with tumors with the highest levels of HER2 gene amplification or ERBB2 mRNA were associated with a worse prognosis than those with intermediate levels, which is of importance to investigate in future clinical trials studying HER2-targeted therapy.

Breast cancer patient-derived whole-tumor cell culture model for efficient drug profiling and treatment response prediction.

Breast cancer (BC) is a complex disease comprising multiple distinct subtypes with different genetic features and pathological characteristics. Although a large number of antineoplastic compounds have been approved for clinical use, patient-to-patient variability in drug response is frequently observed, highlighting the need for efficient treatment prediction for individualized therapy. Several patient-derived models have been established lately for the prediction of drug response. However, each of these models has its limitations that impede their clinical application. Here, we report that the whole-tumor cell culture (WTC) ex vivo model could be stably established from all breast tumors with a high success rate (98 out of 116), and it could reassemble the parental tumors with the endogenous microenvironment. We observed strong clinical associations and predictive values from the investigation of a broad range of BC therapies with WTCs derived from a patient cohort. The accuracy was further supported by the correlation between WTC-based test results and patients' clinical responses in a separate validation study, where the neoadjuvant treatment regimens of 15 BC patients were mimicked. Collectively, the WTC model allows us to accomplish personalized drug testing within 10 d, even for small-sized tumors, highlighting its potential for individualized BC therapy. Furthermore, coupled with genomic and transcriptomic analyses, WTC-based testing can also help to stratify specific patient groups for assignment into appropriate clinical trials, as well as validate potential biomarkers during drug development.

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

Independent Clinical Validation of the Automated Ki67 Scoring Guideline from the International Ki67 in Breast Cancer Working Group.

Ki67 is an important biomarker with prognostic and potential predictive value in breast cancer. However, the lack of standardization hinders its clinical applicability. In this study, we aimed to investigate the reproducibility among pathologists following the guidelines of the International Ki67 in Breast Cancer Working Group (IKWG) for Ki67 scoring and to evaluate the prognostic potential of this platform in an independent cohort. Four algorithms were independently built by four pathologists based on our study cohort using an open-source digital image analysis (DIA) platform (QuPath) following the detailed guideline of the IKWG. The algorithms were applied on an ER+ breast cancer study cohort of 157 patients with 15 years of follow-up. The reference Ki67 score was obtained by a DIA algorithm trained on a subset of the study cohort. Intraclass correlation coefficient (ICC) was used to measure reproducibility. High interobserver reliability was reached with an ICC of 0.938 (CI: 0.920-0.952) among the algorithms and the reference standard. Comparing each machine-read score against relapse-free survival, the hazard ratios were similar (2.593-4.165) and showed independent prognostic potential (p ≤ 0.018, for all comparisons). In conclusion, we demonstrate high reproducibility and independent prognostic potential using the IKWG DIA instructions to score Ki67 in breast cancer. A prospective study is needed to assess the clinical utility of the IKWG DIA Ki67 instructions.

Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies.

Microscopy analysis of tumour samples is commonly performed on fixed, thinly sectioned and protein-labelled tissues. However, these examinations do not reveal the intricate three-dimensional structures of tumours, nor enable the detection of aberrant transcripts. Here, we report a method, which we name DIIFCO (for diagnosing in situ immunofluorescence-labelled cleared oncosamples), for the multimodal volumetric imaging of RNAs and proteins in intact tumour volumes and organoids. We used DIIFCO to spatially profile the expression of diverse coding RNAs and non-coding RNAs at the single-cell resolution in a variety of cancer tissues. Quantitative single-cell analysis revealed spatial niches of cancer stem-like cells, and showed that the niches were present at a higher density in triple-negative breast cancer tissue. The improved molecular phenotyping and histopathological diagnosis of cancers may lead to new insights into the biology of tumours of patients.

Prognostic potential of automated Ki67 evaluation in breast cancer: different hot spot definitions versus true global score.

PURPOSE: The proliferation-associated biomarker Ki67 has potential utility in breast cancer, including aiding decisions based on prognosis, but has unacceptable inter- and intralaboratory variability. The aim of this study was to compare the prognostic potential for Ki67 hot spot scoring and global scoring using different digital image analysis (DIA) platforms. METHODS: An ER+/HER2- breast cancer cohort (n = 139) with whole slide images of sequential sections stained for hematoxylin-eosin, pancytokeratin and Ki67, was analyzed using two DIA platforms. For hot spot analysis virtual dual staining was applied, aligning pancytokeratin and Ki67 images and 22 hot spot algorithms with different features were designed. For global Ki67 scoring an automated QuPath algorithm was applied on Ki67-stained whole slide images. Clinicopathological data included overall survival (OS) and recurrence-free survival (RFS) along with PAM50 molecular subtypes. RESULTS: We show significant variations in Ki67 hot spot scoring depending on number of included tumor cells, hot spot size, shape and location. The higher the number of scored tumor cells, the higher the reproducibility of Ki67 proliferation values. Hot spot scoring had greater prognostic potential for RFS in high versus low Ki67 subgroups (hazard ratio (HR) 6.88, CI 2.07-22.87, p = 0.002), compared to global scoring (HR 3.13, CI 1.41-6.96, p = 0.005). Regarding OS, global scoring (HR 7.46, CI 2.46-22.58, p < 0.001) was slightly better than hot spot scoring (HR 6.93, CI 1.61-29.91, p = 0.009). In adjusted multivariate analysis, only global scoring was an independent prognostic marker for both RFS and OS. In addition, global Ki67-based surrogate subtypes reached higher concordance with PAM50 molecular subtype for luminal A and B tumors (66.3% concordance rate, κ = 0.345), than using hot spot scoring (55.8% concordance rate, κ = 0.250). CONCLUSIONS: We conclude that the automated global Ki67 scoring is feasible and shows clinical validity, which, however, needs to be confirmed in a larger cohort before clinical implementation.

Re-testing of predictive biomarkers on surgical breast cancer specimens is clinically relevant.

PURPOSE: The accuracy of predictive and prognostic biomarker assessment in breast cancer is paramount since these guide therapy decisions. The aim was to investigate the concordance of biomarkers and immunohistochemical (IHC)-based surrogate tumor subtypes between core needle biopsies (CNB) and consecutive paired breast cancer surgical resections. METHODS: This retrospective study comprised two cohorts of patients with primary breast cancer diagnosed between 2016 and 2017: one treated with primary surgery (n = 526) and one with neoadjuvant chemotherapy (NAC) (n = 216). The agreement between preoperative CNB and paired tumor specimens regarding the assessment of biomarkers and surrogate tumor subtypes was evaluated in both cohorts. RESULTS: In the primary surgery cohort, the concordance rates and kappa values for estrogen receptor (ER), progesterone receptor (PR) and Ki67 were 98.6% (κ = 0.917), 89.3% (κ = 0.725) and 78.8% (κ = 0.529), respectively. Importantly, human epidermal growth factor receptor 2 (HER2) IHC assessment showed only moderate agreement (κ = 0.462). HER2 status combining IHC and in situ hybridization was discordant in 3.6% of cases, potentially impacting on indications for HER2-targeted therapy. The concordance rate for IHC-based surrogate tumor subtypes was only 73.2-78.3%. Generally lower concordance rates for ER, PR and HER2 were observed in the NAC cohort. Here, HER2 status was discordant in 7.4%. CONCLUSIONS: The agreement of HER2 and Ki67 between CNB and paired surgical specimen in primary breast cancer is insufficient. Limited agreement of surrogate tumor subtypes indicates a significant clinical value of biomarker re-testing on surgical specimens.

Prognostic value of Ki67 analysed by cytology or histology in primary breast cancer.

AIMS: The accuracy of biomarker assessment in breast pathology is vital for therapy decisions. The therapy predictive and prognostic biomarkers oestrogen receptor (ER), progesterone receptor, HER2 and Ki67 may act as surrogates to gene expression profiling of breast cancer. The aims of this study were to investigate the concordance of consecutive biomarker assessment by immunocytochemistry on preoperative fine-needle aspiration cytology versus immunohistochemistry (IHC) on the corresponding resected breast tumours. Further, to investigate the concordance with molecular subtype and correlation to stage and outcome. METHODS: Two retrospective cohorts comprising 385 breast tumours with clinicopathological data including gene expression-based subtype and up to 10-year overall survival data were evaluated. RESULTS: In both cohorts, we identified a substantial variation in Ki67 index between cytology and histology and a switch between low and high proliferation within the same tumour in 121/360 cases. ER evaluations were discordant in only 1.5% of the tumours. From cohort 2, gene expression data with PAM50 subtype were used to correlate surrogate subtypes. IHC-based surrogate classification could identify the correct molecular subtype in 60% and 64% of patients by cytology (n=63) and surgical resections (n=73), respectively. Furthermore, high Ki67 in surgical resections but not in cytology was associated with poor overall survival and higher probability for axillary lymph node metastasis. CONCLUSIONS: This study shows considerable differences in the prognostic value of Ki67 but not ER in breast cancer depending on the diagnostic method. Furthermore, our findings show that both methods are insufficient in predicting true molecular subtypes.

Digital image analysis of Ki67 in hot spots is superior to both manual Ki67 and mitotic counts in breast cancer.

AIMS: During pathological examination of breast tumours, proliferative activity is routinely evaluated by a count of mitoses. Adding immunohistochemical stains of Ki67 provides extra prognostic and predictive information. However, the currently used methods for these evaluations suffer from imperfect reproducibility. It is still unclear whether analysis of Ki67 should be performed in hot spots, in the tumour periphery, or as an average of the whole tumour section. The aim of this study was to compare the clinical relevance of mitoses, Ki67 and phosphohistone H3 in two cohorts of primary breast cancer specimens (total n = 294). METHODS AND RESULTS: Both manual and digital image analysis scores were evaluated for sensitivity and specificity for luminal B versus A subtype as defined by PAM50 gene expression assays, for high versus low transcriptomic grade, for axillary lymph node status, and for prognostic value in terms of prediction of overall and relapse-free survival. Digital image analysis of Ki67 outperformed the other markers, especially in hot spots. Tumours with high Ki67 expression and high numbers of phosphohistone H3-positive cells had significantly increased hazard ratios for all-cause mortality within 10 years from diagnosis. Replacing manual mitotic counts with digital image analysis of Ki67 in hot spots increased the differences in overall survival between the highest and lowest histological grades, and added significant prognostic information. CONCLUSIONS: Digital image analysis of Ki67 in hot spots is the marker of choice for routine analysis of proliferation in breast cancer.

Digital image analysis in breast pathology-from image processing techniques to artificial intelligence.

Breast cancer is the most common malignant disease in women worldwide. In recent decades, earlier diagnosis and better adjuvant therapy have substantially improved patient outcome. Diagnosis by histopathology has proven to be instrumental to guide breast cancer treatment, but new challenges have emerged as our increasing understanding of cancer over the years has revealed its complex nature. As patient demand for personalized breast cancer therapy grows, we face an urgent need for more precise biomarker assessment and more accurate histopathologic breast cancer diagnosis to make better therapy decisions. The digitization of pathology data has opened the door to faster, more reproducible, and more precise diagnoses through computerized image analysis. Software to assist diagnostic breast pathology through image processing techniques have been around for years. But recent breakthroughs in artificial intelligence (AI) promise to fundamentally change the way we detect and treat breast cancer in the near future. Machine learning, a subfield of AI that applies statistical methods to learn from data, has seen an explosion of interest in recent years because of its ability to recognize patterns in data with less need for human instruction. One technique in particular, known as deep learning, has produced groundbreaking results in many important problems including image classification and speech recognition. In this review, we will cover the use of AI and deep learning in diagnostic breast pathology, and other recent developments in digital image analysis.

Waiting times for cancer patients in Sweden: A nationwide population-based study.

AIMS: The reported long waiting times for cancer patients have mostly been related to prognostic outcome and less to patient-related experience to outcome. We assessed waiting times for patients with cancer of the breast, prostate, colon or rectum in Sweden. METHODS: The median time from referral to start of treatment was assessed using data from clinical cancer registers for patients who received curative treatment during 2011, 2012 and 2013. RESULTS: The median overall waiting time in different counties ranged from 7 to 28 days for breast cancer, from 117 to 280 days for prostate cancer, from 27 to 64 days for colon cancer and from 48 to 80 days for rectal cancer. For the entire nation, the median time from referral to start of treatment remained unchanged from 2011 to 2013 for each cancer diagnosis. CONCLUSIONS: Large variations were found in waiting times between different counties in Sweden and between different types of cancer. The long waiting times identified in this study emphasize the need to improve national programmes for more rapid diagnosis and treatment.

Frontline Science: Multiple cathepsins promote inflammasome-independent, particle-induced cell death during NLRP3-dependent IL-1ß activation.

Sterile particles cause several chronic, inflammatory diseases, characterized by repeating cycles of particle phagocytosis and inflammatory cell death. Recent studies have proposed that these processes are driven by the NLRP3 inflammasome, a platform activated by phagocytosed particles, which controls both caspase-1-dependent cell death (pyroptosis) and mature IL-1ß secretion. After phagocytosis, particles can disrupt lysosomes, and inhibitor studies have suggested that the resulting release of a lysosomal protease-cathepsin B-into the cytosol somehow activates NLRP3. However, using primary murine macrophages, we found that particle-induced cell death occurs independent of NLRP3/caspase-1 and depends instead on multiple, redundant cathepsins. In contrast, nigericin, a soluble activator of NLRP3 inflammasomes, induced cell death that was dependent on the NLRP3. Interestingly, nigericin-induced cell death depended partly on a single cathepsin, cathepsin X. By inhibiting or silencing multiple cathepsins in macrophages, several key proinflammatory events induced by sterile particles are blocked, including cell death, pro-IL-1ß production, and IL-1ß secretion. These data suggest that cathepsins might be potential therapeutic targets in particulate-mediated inflammatory disease. In support of this concept, we find that a broad-spectrum cathepsin inhibitor can suppress particle-induced IL-1-dependent peritonitis.

Multiple Cathepsins Promote Pro-IL-1ß Synthesis and NLRP3-Mediated IL-1ß Activation.

Sterile particles induce robust inflammatory responses that underlie the pathogenesis of diseases like silicosis, gout, and atherosclerosis. A key cytokine mediating this response is IL-1ß. The generation of bioactive IL-1ß by sterile particles is mediated by the NOD-like receptor containing a pyrin domain 3 (NLRP3) inflammasome, although exactly how this occurs is incompletely resolved. Prior studies have found that the cathepsin B inhibitor, Ca074Me, suppresses this response, supporting a model whereby ingested particles disrupt lysosomes and release cathepsin B into the cytosol, somehow activating NLRP3. However, reports that cathepsin B-deficient macrophages have no defect in particle-induced IL-1ß generation have questioned cathepsin B's involvement. In this study, we examine the hypothesis that multiple redundant cathepsins (not just cathepsin B) mediate this process by evaluating IL-1ß generation in murine macrophages, singly or multiply deficient in cathepsins B, L, C, S and X. Using an activity-based probe, we measure specific cathepsin activity in living cells, documenting compensatory changes in cathepsin-deficient cells, and Ca074Me's dose-dependent cathepsin inhibition profile is analyzed in parallel with its suppression of particle-induced IL-1ß secretion. Also, we evaluate endogenous cathepsin inhibitors cystatins C and B. Surprisingly, we find that multiple redundant cathepsins, inhibited by Ca074Me and cystatins, promote pro-IL-1ß synthesis, and to our knowledge, we provide the first evidence that cathepsin X plays a nonredundant role in nonparticulate NLRP3 activation. Finally, we find cathepsin inhibitors selectively block particle-induced NLRP3 activation, independently of suppressing pro-IL-1ß synthesis. Altogether, we demonstrate that both small molecule and endogenous cathepsin inhibitors suppress particle-induced IL-1ß secretion, implicating roles for multiple cathepsins in both pro-IL-1ß synthesis and NLRP3 activation.

The role of mindfulness skills in terms of distress tolerance: a pilot test among adult daily smokers.

BACKGROUND AND OBJECTIVES: Low distress tolerance is associated with difficulties quitting smoking. Mindfulness is associated with improved cessation outcomes and may be one method by which to increase distress tolerance. The purpose of the current study was to examine the relationship between mindfulness skills and distress tolerance among regular smokers. METHODS: Daily smokers (n=125; Mage=37.5, 70% male) completed self-report measures assessing smoking and emotions. RESULTS: After controlling for age, gender, and nicotine dependence, and education the mindfulness skills of acting with awareness and accepting without judgment significantly predicted distress tolerance. DISCUSSION AND CONCLUSIONS: For smokers, being able to pay attention to present moment vents and accept negative events without judgment is associated with a greater ability to withstand such events. SCIENTIFIC SIGNIFICANCE: These findings suggest that mindfulness-based approaches to smoking cessation may be effective because of improvements in distress tolerance. However, future prospective and laboratory-based studies are needed to better understand the mindfulness-distress tolerance link among smokers.

Drug discovery for neglected tropical diseases at the Sandler Center.

The Sandler Center's approach to target-based drug discovery for neglected tropical diseases is to focus on parasite targets that are homologous to human targets being actively investigated in the pharmaceutical industry. In this way we attempt to use both the know-how and actual chemical matter from other drug-development efforts to jump start the discovery process for neglected tropical diseases. Our approach is akin to drug repurposing, except that we seek to repurpose leads rather than drugs. Medicinal chemistry can then be applied to optimize the leads specifically for the desired antiparasitic indication.

Cruzain : the path from target validation to the clinic.

Cruzain is the major papain-like cysteine protease of Trypanosoma cruzi, the etiological agent causing Chagas' disease in humans in South America. Cruzain is indispensable for the survival and propagation of this protozoan parasite and therefore, it has attracted considerable interest as a potential drug target. This chapter charts the path from the initial identification of this proteases activity and its validation as a bone fide drug target to the arduous task of the discovery of an inhibitor targeting this protease and finally the path towards the clinic.

Waldenström's macroglobulinemia and nephrotic syndrome with membranous nephropathy.

Renal complications of Waldenström's macroglobulinemia (WM) are rarely observed. Nephrotic syndrome in association with WM has most often been secondary to amyloidosis. This article reports a case of WM with nephrotic syndrome as a result of membranous nephropathy with immunoglobulin M (IgM) deposition. A 44-year-old male diagnosed with WM 4 years previously, presented with heavy proteinuria (7.8 g/24 h). Kidney biopsy revealed expanded mesangium, thickened capillary loops and epimembranous spikes, with no significant interstitial inflammation or thickened tubular basement membranes. Immunofluorescence examination demonstrated strong granular staining of IgM and λ chains, with weaker C3 and C1q staining. Electron microscopy showed many subepithelial dense deposits, and fewer large subendothelial dense deposits. Treatment was directed at the patient's WM with maintenance rituximab and fludarabine. Subsequently, decreases were seen in both the patient's serum IgM and serum viscosity. With therapy for WM and the addition of an angiotensin receptor blocker, the patient's proteinuria also improved, from 7.8 g to 4.8 g/24 h. The patient continued to follow up with his hematologist and in 2009 creatinine was 1 mg/dl (76.26 ?mol/l), with a 24 h urine protein excretion of 0.159 g.