Andrew Ryan

Neurons in the hypothalamic paraventricular nucleus send collaterals to the spinal cord and to the rostral ventrolateral medulla in the rat

The hypothalamic paraventricular nucleus (PVN) projects to the rostral ventrolateral medulla (RVLM) and to the intermediolateral cell column (IML) of the spinal cord. The present study determined whether the same neurons can innervate both regions. In each rat, two retrogradely-transported tracers, microspheres tagged with fluorescein or rhodamine, were injected into the left lower thoracic/upper lumbar IML (fluorescein) and into the pressor region of the left RVLM (rhodamine). In the PVN over 90% of the neurons labelled with either tracer were found ipsilateral to the injection site. Double labelled cells averaged almost one-third of the spinally-projecting cells in four of the five animals. In the remaining animal, there were few double-labelled cells. The results suggest that a population of PVN neurons innervates both the lower-thoracic/upper lumbar IML and the RVLM.

Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer

Tumour heterogeneity in primary prostate cancer is a well-established phenomenon. However, how the subclonal diversity of tumours changes during metastasis and progression to lethality is poorly understood. Here we reveal the precise direction of metastatic spread across four lethal prostate cancer patients using whole-genome and ultra-deep targeted sequencing of longitudinally collected primary and metastatic tumours. We find one case of metastatic spread to the surgical bed causing local recurrence, and another case of cross-metastatic site seeding combining with dynamic remoulding of subclonal mixtures in response to therapy. By ultra-deep sequencing end-stage blood, we detect both metastatic and primary tumour clones, even years after removal of the prostate. Analysis of mutations associated with metastasis reveals an enrichment of TP53 mutations, and additional sequencing of metastases from 19 patients demonstrates that acquisition of TP53 mutations is linked with the expansion of subclones with metastatic potential which we can detect in the blood.

SCRIB expression is deregulated in human prostate cancer, and its deficiency in mice promotes prostate neoplasia

Loss of cellular polarity is a hallmark of epithelial cancers, raising the possibility that regulators of polarity have a role in suppressing tumorigenesis. The Scribble complex is one of at least three interacting protein complexes that have a critical role in establishing and maintaining epithelial polarity. In human colorectal, breast, and endometrial cancers, expression of the Scribble complex member SCRIB is often mislocalized and deregulated. Here, we report that Scrib is indispensable for prostate homeostasis in mice. Scrib heterozygosity initiated prostate hyperplasia, while targeted biallelic Scrib loss predisposed mice to prostate intraepithelial neoplasia. Mechanistically, Scrib was shown to negatively regulate the MAPK cascade to suppress tumorigenesis. Further analysis revealed that prostate-specific loss of Scrib in mice combined with expression of an oncogenic Kras mutation promoted the progression of prostate cancer that recapitulated the human disease. The clinical significance of the work in mice was highlighted by our observation that SCRIB deregulation strongly correlated with poor survival in human prostate cancer. These data suggest that the polarity network could provide a new avenue for therapeutic intervention.

A Preclinical Xenograft Model Identifies Castration-Tolerant Cancer-Repopulating Cells in Localized Prostate Tumors

This study uses a preclinical xenograft model to reveal prostate cancer cells that exist in untreated localized disease, survive androgen withdrawal, and are potential therapeutic targets. The Enemy Within Prostate cancer is one of the most common types of cancer in men. In advanced stages, it is typically treated with medications that mimic castration, depriving the tumor of androgen stimulation. Unfortunately, these cancers eventually become castration-resistant and begin to grow even in the absence of hormonal input. What isn’t known is how these cancer cells develop the ability to survive androgen deprivation, and whether some types of stem-like castration-resistant cells are already present in prostate cancer from early stages or evolve later during the course of treatment. Now, Toivanen and colleagues shed some light on this mystery, with a report of castration-tolerant cells derived from early localized tumors that had not yet been exposed to anti-androgen therapy. The authors used primary prostate tumors from 12 men with localized cancer, implanting them in a mouse xenograft model to study the effects of androgen deprivation on the tumors’ survival. Castration of the host mice led to rapid regression, but not disappearance of the tumors. Even after a prolonged period of castration (4 weeks), some residual tumor foci persisted. When testosterone stimulation was restored in the host animals, these residual cells rebounded, regenerating masses that were histologically similar to the original tumors. This work by Toivanen et al. indicates that some prostate cancer cells can survive castration and later repopulate the tumor when androgen stimulation is available. Thus far, there is no indication that these castration-tolerant cells can proliferate in the absence of androgens, unlike the cells found in more advanced “castration-resistant” prostate cancer. Additional work will be needed to clarify whether these might be a type of prostate cancer stem cells, and what makes them different from the population of “androgen-sensitive” cancer cells that do not survive androgen depletion. Although there are many questions that must still be answered about the biology of these castration-tolerant cells, this work raises the intriguing possibility that we may eventually be able to specifically target and eradicate them, thus preventing prostate cancer recurrence in patients. A lack of clinically relevant experimental models of human prostate cancer hampers evaluation of potential therapeutic agents. Currently, androgen deprivation therapy is the gold standard treatment for advanced prostate cancer, but inevitably, a subpopulation of cancer cells survives and repopulates the tumor. Tumor cells that survive androgen withdrawal are critical therapeutic targets for more effective treatments, but current model systems cannot determine when they arise in disease progression and are unable to recapitulate variable patient response to treatment. A model system was developed in which stromal-supported xenografts from multiple patients with early-stage localized disease can be tested for response to castration. The histopathology of these xenografts mimicked the original tumors, and short-term host castration resulted in reduced proliferation and increased apoptosis in tumor cells. After 4 weeks of castration, residual populations of quiescent, stem-like tumor cells remained. Without subsequent treatment, these residual cells displayed regenerative potential, because testosterone readministration resulted in emergence of rapidly proliferating tumors. Therefore, this model may be useful for revealing potential cellular targets in prostate cancer, which exist before the onset of aggressive incurable disease. Specific eradication of these regenerative tumor cells that survive castration could then confer survival benefits for patients.

A new preoperative nomogram to predict minimal prostate cancer: Accuracy and error rates compared to other tools to select patients for active surveillance

PURPOSE We designed and fully evaluated the performance of a nomogram to identify patients with prostate cancer who may be suitable for active surveillance. MATERIALS AND METHODS We developed a nomogram to predict the probability of minimal prostate cancer (total tumor volume less than 0.5 cc, organ confined disease and no Gleason pattern 4 or 5) using preoperative data on 2,525 Australian patients who underwent radical prostatectomy. Accuracy and error rates at multiple probability cutoffs were compared with those of contemporary Epstein criteria and the Prostate Cancer Research International: Active Surveillance trial inclusion criteria when applied to these patients. High risk disease was defined as 1 or more adverse characteristics (including positive surgical margins, seminal vesicle invasion, extracapsular extension, 50% or greater Gleason pattern 4/5 and/or tumor volume 4.0 cc or greater) at radical prostatectomy. RESULTS Minimal cancer was confirmed in 152 men (6.0%) at prostatectomy. The bootstrap corrected predictive accuracy of our nomogram was 93.3% vs 89.1% and 91.0% for Prostate Cancer Research International: Active Surveillance and Epstein criteria, respectively. For men with a nomogram derived minimal cancer probability of 0% to 4.9%, 5.0% to 19.9%, 20.0% to 34.9%, 35.0% to 49.9% and 50.0% to 71.0% the rate of high risk disease was 70.8%, 37.8%, 22.4%, 9.0% and 3.8%, respectively. In contrast, the rate of high risk disease for men who met Prostate Cancer Research International: Active Surveillance and Epstein criteria were 17.1% and 13.9%, respectively. CONCLUSIONS A detailed breakdown of the expected rates of false-positive results and high risk disease associated with the nomogram derived probability of minimal cancer would provide more complete information to clinicians and patients on which to base therapeutic clinical decisions for presumed early stage prostate cancer.

Volume expansion does not activate neuronal projections from the NTS or depressor VLM to the RVLM

We investigated whether a monosynaptic connection from the nucleus tractus solitarius (NTS) or the depressor ventrolateral medulla (VLM) to the pressor region of the rostral VLM (RVLM) constituted part of the reflex pathway activated by cardiopulmonary baroreceptors. Volume expansion in the conscious rabbit, which elicits renal nerve inhibition predominantly via cardiac mechanoreceptors, was used as the stimulus. The protein Fos was used as a marker of neuronal activation. The retrogradely transported tracer rhodamine-tagged microspheres, previously injected into the pressor region of the RVLM, identified medullary neurons that projected to that region. Volume expansion significantly increased the number of Fos-positive cell nuclei in the NTS and in the depressor VLM. Neurons that projected to the RVLM were found throughout the depressor region of the VLM and in the NTS but were not activated by volume expansion. Thus, although the central reflex pathways activated by volume expansion include the NTS and the depressor region of the VLM, we could not find evidence for a monosynaptic connection between those regions and the RVLM.We investigated whether a monosynaptic connection from the nucleus tractus solitarius (NTS) or the depressor ventrolateral medulla (VLM) to the pressor region of the rostral VLM (RVLM) constituted part of the reflex pathway activated by cardiopulmonary baroreceptors. Volume expansion in the conscious rabbit, which elicits renal nerve inhibition predominantly via cardiac mechanoreceptors, was used as the stimulus. The protein Fos was used as a marker of neuronal activation. The retrogradely transported tracer rhodamine-tagged microspheres, previously injected into the pressor region of the RVLM, identified medullary neurons that projected to that region. Volume expansion significantly increased the number of Fos-positive cell nuclei in the NTS and in the depressor VLM. Neurons that projected to the RVLM were found throughout the depressor region of the VLM and in the NTS but were not activated by volume expansion. Thus, although the central reflex pathways activated by volume expansion include the NTS and the depressor region of the VLM, we could not find evidence for a monosynaptic connection between those regions and the RVLM.

Establishment of primary patient-derived xenografts of palliative TURP specimens to study castrate-resistant prostate cancer.

Fresh patient specimens of castrate‐resistant prostate cancer (CRPC) are invaluable for studying tumor heterogeneity and responses to current treatments. They can be used for primary patient‐derived xenografts (PDXs) or serially transplantable PDXs, but only a small proportion of samples grow successfully. To improve the efficiency and quality of PDXs, we investigated the factors that determine the initial engraftment of patient tissues derived from TURP specimens.

The polarity protein Scrib mediates epidermal development and exerts a tumor suppressive function during skin carcinogenesis

BackgroundThe establishment and maintenance of polarity is vital for embryonic development and loss of polarity is a frequent characteristic of epithelial cancers, however the underlying molecular mechanisms remain unclear. Here, we identify a novel role for the polarity protein Scrib as a mediator of epidermal permeability barrier acquisition, skeletal morphogenesis, and as a potent tumor suppressor in cutaneous carcinogenesis.MethodsTo explore the role of Scrib during epidermal development, we compared the permeability of toluidine blue dye in wild-type, Scrib heterozygous and Scrib KO embryonic epidermis at E16.5, E17.5 and E18.5. Mouse embryos were stained with alcian blue and alizarin red for skeletal analysis. To establish whether Scrib plays a tumor suppressive role during skin tumorigenesis and/or progression, we evaluated an autochthonous mouse model of skin carcinogenesis in the context of Scrib loss. We utilised Cre-LoxP technology to conditionally deplete Scrib in adult epidermis, since Scrib KO embryos are neonatal lethal.ResultsWe establish that Scrib perturbs keratinocyte maturation during embryonic development, causing impaired epidermal barrier formation, and that Scrib is required for skeletal morphogenesis in mice. Analysis of conditional transgenic mice deficient for Scrib specifically within the epidermis revealed no skin pathologies, indicating that Scrib is dispensable for normal adult epidermal homeostasis. Nevertheless, bi-allelic loss of Scrib significantly enhanced tumor multiplicity and progression in an autochthonous model of epidermal carcinogenesis in vivo, demonstrating Scrib is an epidermal tumor suppressor. Mechanistically, we show that apoptosis is the critical effector of Scrib tumor suppressor activity during skin carcinogenesis and provide new insight into the function of polarity proteins during DNA damage repair.ConclusionsFor the first time, we provide genetic evidence of a unique link between skin carcinogenesis and loss of the epithelial polarity regulator Scrib, emphasizing that Scrib exerts a wide-spread tumor suppressive function in epithelia.

Magnetic resonance imaging for prostate cancer: Comparative studies including radical prostatectomy specimens and template transperineal biopsy

Purpose Multiparametric magnetic resonance imaging (mpMRI) is an emerging technique aiming to improve upon the diagnostic sensitivity of prostate biopsy. Because of variance in interpretation and application of techniques, results may vary. There is likely a learning curve to establish consistent reporting of mpMRI. This study aims to review current literature supporting the diagnostic utility of mpMRI when compared with radical prostatectomy (RP) and template transperineal biopsy (TTPB) specimens. Methods MEDLINE and PubMed database searches were conducted identifying relevant literature related to comparison of mpMRI with RP or TTPB histology. Results Data suggest that compared with RP and TTPB specimens, the sensitivity of mpMRI for prostate cancer (PCa) detection is 80–90% and the specificity for suspicious lesions is between 50% and 90%. Conclusions mpMRI has an increasing role for PCa diagnosis, staging, and directing management toward improving patient outcomes. Its sensitivity and specificity when compared with RP and TTPB specimens are less than what some expect, possibly reflecting a learning curve for the technique of mpMRI.

Microscopic assessment of fresh prostate tumour specimens yields significantly increased rates of correctly annotated samples for downstream analysis

Aims: To assess if performing frozen sections of tissue biopsies from fresh radical prostatectomy specimens, prior to tissue banking, could improve the identification of the banked samples compared to standard fresh tumour banking procedures. Methods: Tissue biopsies banked from 332 fresh prostatectomy specimens were assessed for accuracy of diagnosis, comparing two separate methods of tumour identification: one in which tumour was identified in the gross specimen by visual inspection (n = 155) and one in which rapid frozen sectioning was applied (n = 177). The associations with correct tumour annotation and clinicopathological variables, including age, pre-operative prostate specific antigen (PSA) levels, pathological Gleason score, pathological T stage, tumour volume and surgical margins, were examined using univariable and multivariable binary logistic regression models. Results: For the gross visual inspection cohort the rate of correctly identifying and banking specimens containing prostate cancer was 69%. For the cohort assessed with rapid frozen sections, 94% of banked specimens actually had cancer. On multivariable analysis, we found that only frozen sectioning and tumour volume variables were independent predictors of correctly banked tumour specimens whilst all other routinely reported pathological variables had no influence on the success rates of fresh prostate tumour banking. Conclusion: The success rate for correctly banking fresh prostate tumour specimens is directly related to the tumour volume. Frozen section scrutiny of prostate samples is recommended to prevent misclassification of the banked material.