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Supported by NHMRC Career Development Award, National Breast Cancer Foundation Novel Concept Award, and Cancer Australia.

Aim To develop a process for the production, dissemination and implementation of international evidence-based pathology cancer datasets (IPCDS). Methods The International Collaboration on Cancer Reporting (ICCR) was established in 2011 between the Pathology Colleges and Associations of the USA, UK, Canada and Australia. Cancer datasets from various organisations are harmonised and updated by internationally recognised pathologists and subjected to evidentiary and worldwide review, followed by publication in peer reviewed journals. Key international cancer organisations endorse and participate in the process. Results and Discussion Four datasets have been published and posted to the ICCR website to date. The International Agency for Research in Cancer (IARC) has partnered with ICCR to synchronise the publication of subsequent ICCR datasets with future WHO Tumour Classification volumes. The ICCR is engaging with organisations involved in tumour staging including the Union for International Cancer Control (UICC) and the American Joint Commission on Cancer (AJCC). The European Society of Pathology (ESP) joined ICCR as a founding member in 2013, bringing over 68 countries and more than one billion people under a common process. Conclusions The ICCR has developed an efficient process for the production of standardised and evidence-based IPCDS. Engagement with key international cancer and pathology organisations will foster their adoption worldwide.

Omega class glutathione transferase (GSTO) has been recently described in a number of mammalian species. We used immunohistochemistry to determine the cellular and tissue distribution of GSTO1–1 in humans. Expression of GSTO1–1 was abundant in a wide range of normal tissues, particularly liver, macrophages, glial cells, and endocrine cells. We also found nuclear staining in several types of cells, including glial cells, myoepithelial cells of the breast, neuroendocrine cells of colon, fetal myocytes, hepatocytes, biliary epithelium, ductal epithelium of the pancreas, Hoffbauer cells of the placenta, and follicular and C-cells of the thyroid. These observations and the known activity of GSTO1–1 suggest biological functions that are not shared with other GSTs. (J Histochem Cytochem 49:983–987, 2001)

The objective of this study was to determine the accuracy of image-guided fine needle aspiration cytology (FNAC) in the diagnosis of pulmonary lesions. A retrospective study was undertaken of 286 patients with 288 lesions, who underwent a total of 302 procedures. The FNAC diagnoses were reported as malignant, suspicious, atypical, benign or non-diagnostic. Subsequently the FNAC diagnoses were correlated with either the histological or clinical diagnoses. Of the 288 lesions, 64.6% were reported on FNAC as malignant, 2.1% suspicious, 2.4% atypical, 20.8% benign and 10.1% nondiagnostic. On review of the suspicious, atypical, selected benign cases and non-diagnostic FNAC by an independent pathologist there was agreement with the original FNAC diagnosis in all cases. All of 186 malignant FNAC diagnoses were confirmed malignant either clinically or on subsequent histology. Four of the six suspicious FNAC diagnoses had a malignant outcome, one patient had organising pneumonia on excision biopsy and one was lost to follow up. Six of the seven atypical FNAC diagnoses were confirmed on histology as malignant, while one lesion resolved spontaneously. Fifty-two of 60 benign FNAC diagnoses were confirmed benign either clinically or on histology. Seven of the lesions diagnosed as benign on FNAC were proven to be malignant. One patient with a benign FNAC diagnosis was lost to follow-up. Ten of the 29 non-diagnostic FNAC group were later shown on clinical or histological follow up to be malignant. This study shows that image guided FNAC for the diagnosis of malignant pulmonary lesions has a sensitivity of at least 92% and a specificity of at least 96%. It is a reliable diagnostic test although its accuracy is limited by technical difficulties in obtaining an adequate sample.

Joubert syndrome is an autosomal recessive disease characterised by hypoplasia or agenesis of the cerebellar vermis, a syndrome of episodic apnoea-hyperpnoea, rhythmic protrusion of the tongue, abnormal eye movements, hypotonia, ataxia, and psychomotor retardation. Extracerebral malformations include multicystic kidney disease, congenital hepatic fibrosis, sacral dermoid cyst and polydactyly. We report the clinical and pathological findings of a 15-year-old girl with Joubert syndrome diagnosed at autopsy. This patient had bilateral colobomata, which has not been previously described in females with Joubert syndrome.

Dichloroacetate (DCA) has recently been proposed as a novel non-toxic anticancer agent that can reverse the glycolytic phenotype in cancer cells through the inhibition of pyruvate dehydrogenase kinase. Aims and Methods Three mouse cancer cell lines (V14, 4T1 and B16) were used to examine the histological changes caused by DCA in vivo. V14 or 4T1 mammary adenocarcinoma cells were injected subcutaneously and B16 melanoma cells were injected intravenously. Mice were treated with 0.5–2.0 g/L DCA in the drinking water. Results Growth of established V14 tumours was halted by DCA treatment (28% reduction in size, 1.5 g/L for 9 days, n = 8 per group, p = 0.08). Treatment from the day of cell injection resulted in fewer tumours being established (5/8 sites in treated mice vs 16/16 untreated, p = 0.03) and complete regression of one tumour. Histological assessment found DCA treatment resulted in increased tumour-infiltrating lymphocytes (TILs) but no change in mitotic rate, fibrosis or necrosis. Tumours from the 4T1 or B16 cell lines showed no growth inhibition and no evidence of TILs, correlating with a lack of sensitivity to growth inhibition in vitro. Conclusions In V14 tumours DCA appears to prevent or delay breast cancer formation either through growth inhibition or enhanced immune function.

BACKGROUND Intradiploic hematoma of the skull was first reported in 1934. The pathogenesis of this lesion is unclear. It is a very rare benign reactive process occurring after minor head trauma, with only seven cases reported in the literature to date. CASE DESCRIPTION A 3-year-old right hand dominant male presented with a non-tender parietal scalp swelling of a 1-year duration. History included a skull fracture located in the same region 24 months before presentation. Neurological examination was unremarkable. Pathological examination after curettage of the lesion revealed features consistent with organizing hematoma. CONCLUSIONS The pathology of chronic diploic hematoma mimics aneurysmal bone cyst, giant cell tumor, giant cell reparative granuloma, fibrous dysplasia, eosinophilic granuloma, intradiploic epidermoid and dermoid cyst, cavernous hemangioma, circumscribed osteomyelitis, and tuberculous granuloma. Chronic diploic hematoma is a lesion that must be differentiated from various skull lesions both radiologically and histologically as it is amenable to treatment with a complete cure once excised.

Introduction Grading the extent of degenerative change in tendon is an important aspect of tendinopathy research. The Bonar score1 has been widely used for this purpose since 2004. This method provides valuable guidelines for tendinosis assessment but is not without limitations, and many groups have made modifications to the original method.2 It is common for researchers to evaluate the area of highest morphological change, however the method of defining this area, and the implications of using different methods, have not been documented. In this study, we asked whether selecting the level of greatest morphological change using different aspects of the score (degree of collagen disruption, the level of cell morphological change, or the degree of vascularity) would lead to similar or different Bonar scores. Methods Human tendon from people undergoing gluteal tendon reconstruction or hip arthroplasty was assessed in each part of this study. Two researchers experienced in using the Bonar score systematically reviewed each domain of the Bonar score while viewing tendon sections. By consensus, the appropriate level of magnification used for each domain was determined. The need for polarisation was also reviewed. Additional clauses of pathological acellularity and avascularity were added to the third. Hyper and hypo cellularity were defined, and, modification of the vascularity and cellularity scores were confirmed. Two researchers, with a third to resolve disputes evaluated 32 sections of tendon. Each domain within the Bonar score was evaluated in each of the following areas: the area of worst collagen disruption; the area scoring the highest vascularity; and thirdly the area scoring the highest tenocyte morphology change. Results A total of 84 areas of tendon were evaluated. The total Bonar score was highest in the areas of most cellular morphological (CM) changes (mean, (SD) 14.4 (1.50)) then highest collagen disruption (CD: 13.0 (2.56)), and lowest for the area of vascularity (VS) (11.6 (1.68), (regression: CD vs TM p=0.008, CM vs VS p Conclusions and recommendations This paper provides clarification of, and recommendations on the use of the commonly used Bonar score. Cellularity is best assessed using one FoV×100. In human tissue, hypo-cellularity ≤20 nuclei per HPF, Hyper-cellularity ≥30 nuclei per HPF. Normal=20 to 30 nuclei per HPF. Cell morphology was best assessed at ×200 magnification, over four FoV. Vascularity should be assessed at ×400 magnification, up to 10 FoV, and Ground substance was best assessed at ×100. When evaluating a FoV, a 20% rule should be applied. That is, if 20% of the tissue can be scored at the highest level, this score is applied to the entire FoV. To provide accurate/reliable assessment, tendon sections should be viewed by a minimum, of two researchers, simultaneously, but independently with the final score decided either by consensus, or a third assessor. These results suggest that using the area of maximum tenocyte morphological changes provides a justifiable score that is representative of the worst degree of tendinosis in the specimen. We believe our modified Bonar score provides clearer definitions of each grade and also specifies the level of magnification for each domain, producing a more robust assessment tool. Further evaluation of the Bonar score9s applicability to commonly used tendinopathy laboratory models should be carried out.

The Warburg effect occurs in 90% of tumours, where there is a high rate of glycolysis even in the presence of oxygen. This results in increased lactate production and reduced pyruvate oxidation. Dichloroacetate (DCA) is used clinically for congenital lactic acidosis and can reverse the Warburg effect by inhibiting the pyruvate dehydrogenase kinases (PDKs), activating pyruvate dehydrogenase thus promoting oxidative metabolism of pyruvate. The PDKs have different sensitivities towards DCA inhibition (PDK2>PDK4>PDK1>>PDK3). We have investigated the effects of DCA on cancer growth in vivo and in vitro, and examined the expression of the PDK isoforms to explain variation of cancer cell responses to DCA. DCA halted the growth of established V14 mouse mammary tumours in vivo, but was ineffective against 4T1 mouse mammary tumours. In 8 human cancer cell lines of breast, colon, pancreatic and prostate origin, 5 mM DCA (48 hr) inhibited growth by 5-40% in vitro. Western blotting for expression of PDK isoforms revealed that the most sensitive T-47D cells expressed PDK2 and low levels of the other PDKs, whereas less sensitive cells expressed high levels of PDK1 and/or PDK3. In siRNA knockdown (kd) experiments, PDK3-kd in MCF7 cells (low DCA sensitivity, high PDK3 levels) increased sensitivity to DCA by 30%, while DCA did not further inhibit growth of PDK2-kd T-47D cells, confirming that sensitivity to DCA growth inhibition is determined by the PDK profiles. PDK1 & 3 are up-regulated in hypoxia, which may reduce the effectiveness of DCA. To the contrary, DCA increased apoptosis in hypoxia in MCF7 cells, suggesting that DCA can enhance effects of other stressors/drugs, an area of ongoing investigation. Use of the PDK profiles to target DCA sensitive tumours will improve the outcomes of clinical trials using DCA that are currently underway Citation Format: Bevan P. Gang, Melissa Rooke, Sheenu Mishra, Jane E. Dahlstrom, Anneke C. Blackburn, Cancer Metabolism and Genetics Group. Sensitivity to dichloroacetate is determined by PDK expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5421. doi:10.1158/1538-7445.AM2013-5421 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Retinopathy of prematurity (ROP) is a vasoproliferative disorder that can lead to blindness. Red light irradiation at 670 nm wavelength promotes cellular differentiation, proliferation and wound repair. 670 nm light is believed to stimulate mitochondrial function by increasing cytochrome oxidase efficiency and ATP production. In the retina, 670 nm light protects photoreceptors from the effects of a number of noxious stimuli including toxins, light-induced and oxygen-induced degeneration. Aims To determine whether treatment with 670 nm light would promote normal vessel development in a mouse model of ROP (oxygen induced retinopathy – OIR model) and whether it would affect organ development and growth. Method C57/Bl6j mice were treated as controls. The other groups were OIR mice (75% oxygen, p7–12 days; normoxia p12–17 days) and C57/Bl6j and OIR mice that were treated with 9 J/cm2 of 670 nm light daily from p7–17. At p17 animals were sacrificed and the retinal vasculature visualised by labelling with lectin. Analysis for the presence of neovascularisation and vaso-obliteration was performed using established protocols. Weight and length measurements were recorded daily, and at p17 their organs were harvested. All organs were examined macroscopically and microscopically. Results There was a significant reduction (p Conclusions Exposure to 670 nm red light may be a novel strategy to promote normal vessel development and protect against ROP. 670 nm treatment might also prove useful in assisting pre-term infants increase body weight and reduce lung damage.

SummaryThe Harlequin infant represents the most severe form of nonbullous ichthyosis. Although the clinical features of infants with Harlequin ichthyosis are generally similar, histological, ultrastructural, and biochemical analyses have not shown consistent findings. An unexpected case of Harlequin ichthyosis in a female infant born at 35 wks gestation is presented. Light microscopic and ultrastructural investigations of skin biopsies are detailed. The presence of extracellular lipid material in the stratum corneum has not been described in the previously reported cases of Harlequin ichthyosis.

A healthy 4-year-old boy presented with a 4 month history of episodic pain and swelling of his left jaw which appeared to respond to antibiotics. An ultrasound, orthopantomogram (OPG), CT scan and MRI revealed a 3 cm well circumscribed lesion in the left mandible near, but not related to, his posterior molar teeth. The radiological differential diagnosis included a mandibular abscess or neoplasm such as Ewing's sarcoma, or Langerhans cell histiocytosis (LCH). A fine needle aspirate (FNA) was performed. The cytology, in conjunction with the immunohistochemistry (S100 protein and CD1a expression by the histiocyte-like cells) and electron microscopy (demonstrating Birbeck bodies) showed features characteristic of LCH. The boy was treated with an intra-lesional injection of methyl prednisolone with radiological and clinical evidence of regression of the lesion. Localised LCH is also known as eosinophilic granuloma (EG). Its pathogenesis is unknown, although recent studies suggest it is a disease that results from mononuclear phagocyte dysregulation that may be infective, autoimmune or neoplastic in origin. EG is rare, usually affecting children 5-15 years. The jaws are affected in 10-20% of cases with mandible involvement more common in adults. No consensus exists for the optimal therapy which includes curettage, intra-lesional prednisolone and chemotherapy.

While genetic testing for familial cancer susceptibility factors has excelled in recent years, the prevention options for those carrying high risk alleles have not. Altered bioenergetics has now been acknowledged as an emerging hallmark of cancer, and is believed to be a critical characteristic acquired during tumorigenesis. Several very safe drugs are available that can modify bioenergetics. Dichloroacetate (DCA) inactivates pyruvate dehydrogenase kinase, resulting in activation of pyruvate dehydrogenase, reduced lactic acid production and increased mitochondrial activity. Metformin, a type 2 diabetes treatment which activates AMPK, thereby inhibiting mTOR, has unambiguously been demonstrated to reduce the risk of many cancer types in diabetics where insulin treatments do not. We have tested these drugs as chemopreventive agents against the mammary tumours that occur in the spontaneous BALB/c-Trp53+/- mouse tumour model. In vitro studies on breast cancer cell lines indicate that DCA (1-5 mM), metformin (30-300 uM) or the combination of the two can significantly inhibit breast cancer cell growth over 4 days of treatment. These results support the possibility that DCA and metformin could prevent or delay breast cancer formation in BALB/c-Trp53+/- mice. To examine this, four groups of female BALB/c-Trp53+/- mice were given distilled water (n=75), DCA (1.5 g/L in drinking water, ~180 mg/kg/day, n=53), metformin (0.25 g/L in drinking water, ~30 mg/kg/day, n=61) or DCA + metformin (n=51) from 8 weeks of age, and monitored for tumour development over 78 weeks. The overall tumour-free survival curves were not significantly different (Kaplan-Meier analysis) between the treatment groups, suggesting that metformin does not reduce cancer risk in non-diabetics. However, the occurrence of mammary tumours in the four groups was altered. DCA reduced the number and increased the latency of mammary tumours (20.8% of DCA treated mice with mean latency of 63.8 weeks compared to 28.0% of untreated mice with mean latency of 55.0 weeks), whereas metformin had no effect (26.2% of mice, average latency 54.7 weeks). Examining the mammary tumour-free survival curves, DCA appeared to eliminate the early onset mammary tumours (latency

A 51-year-old man presented with pain on eating hard food for 2 years following extraction of the left mandibular second pre-molar and second molar teeth. The extraction was complicated by alveolar osteitis (dry socket). Initial imaging did not show any bony deficiency but subsequent imaging hinted at a possible bony defect and the underlying mandibular bone was debrided. At the time of debridement material, in retrospect, consistent with Alvogyl was noted. Light microscopy showed low grade chronic inflammation in the soft tissues secondary to refractile vegetable matter like material consistent with Alvogyl. Some of the foreign material was associated with aggregates of multinucleated giant cells and some islands of new bone. The mandibular bone present in the specimen was viable. Alvogyl is a commonly used alveolar dressing with fibres derived from Cibotium species (tree fern). According to the manufacturer (Septodont) this fibrous consistency allows adherence to the alveolus and the product is self-eliminated with assistance from the patient’s tongue movements. A very small number of studies have shown Alvogyl to retard wound healing. There are no published case reports of chronic pain due to the retention of Alvogyl and chronic inflammation.

Activation of long interspersed elements, type 1 (LINE-1 or L1) retrotransposons may be one of the mechanisms facilitating geno-mic instability of breast carcinomas. Aim To determine whether the expression of L1 proteins can serve as a useful biomarker in breast cancers. Methods L1 expression was evaluated in breast cancer cell lines and breast tissues by Western blot, immunofluorescence and immunohistochemistry. The expression of L1 proteins was determined in non-neoplastic breast tissue, fibroadenomas, ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDC), and was correlated with pathological parameters and patient prognosis. Results L1 protein expression was higher in non-invasive compared with highly metastatic breast cell lines. DCIS showed greater immunoreactivity for L1 proteins than IDC. Normal breast tissue did not express L1 protein while fibroadenomas showed low L1 expression. L1 immunostaining positively correlated with the tumour’s expression of oestrogen and progesterone receptors (p Discussion L1 protein expression is highest in DCIS supporting the notion that genomic instability occurs at an early stage of cancer development. Nuclear L1 protein overexpression in invasive cancers appears to be a predictor of poor prognosis.

Preterm infants are delivered while glomerulogenesis is ongoing and thus may be exposed to a number of insults including medications that may affect renal development. Podocytes detected in the urine are an indicator of glomerular injury in a number of renal conditions. Aims To determine whether preterm and term infants excrete podocytes in their urine and if exposure to gentamicin and indo-methacin increased podocyte excretion in their urine. Methods Preterm infants less than 33 weeks gestation had urine collected each day while receiving either gentamicin or indometha-cin and the number of casts and podocytes present in the urine were compared with preterm and term control infants urine who did not receive gentamicin or indomethacin. Results and Conclusions Forty-two neonates were included in the study. Podocytes were present in small numbers (

A 15-year-old male presented with an incidentally discovered radiopacity above an unerupted wisdom tooth of the right mandible. A biopsy 2 years prior was inconclusive. Light microscopy of the biopsy revealed proliferating cords of odontogenic epithelium with a moderately cellular mesenchymal matrix enclosing loose stellate reticulum-like areas. There was associated new enamel and dentine formation with the structure of a complex odontoma. These features are of an ameloblastic fibro-odontoma. Ameloblastic fibro-odontoma is a rare, slowing growing, mixed odontogenic neoplasm which is usually diagnosed before the age of 20. They involve the posterior mandible or maxilla and can be associated with unerupted teeth. Radiographically the ameloblastic fibroma component appears as a well defined radiolucent area with the odontomatous component appearing as irregular radiopacities. Radiological differential diagnoses include calcifying odontogenic cyst and odontoma. Ameloblastic fibro-odontomas are generally not aggressive and can be treated with surgical curettage without removal of adjacent teeth. Recurrences have been reported. The risk of developing an amelobastic fibrosarcinoma is less than for an ameloblastic fibroma. However, follow-up is recommended.

A 1-year-old infant, with a right sided unilateral cleft lip and palate, presented to her paediatric dentist for management of a tooth that had been present since birth. This tooth was hanging on by a thread of soft tissue and was situated on the left anterior medial aspect of her cleft palate. The tooth exfoliated within 2 weeks. The infant’s sister and father have type I osteogenesis impefecta which is associated with developmental defects of dentine. Macroscopically the tooth measured 5x4x3 mm and had a mottled white appearance. Light microscopy revealed features of an invaginated odontome (dens in dente, dens invaginatus, gestant odontone). The dentine was normal in appearance apart from its developmental defects. An invaginated odontome is a developmental anomaly arising as a result of enfolding of the enamel organ during the early stages of tooth development leading to an invagination of the crown (or sometimes the root) of a tooth. Maxillary permanent lateral incisors teeth are the most commonly affected. Supernumerary teeth are also more likely to have invaginations. Up to 22% of children with unilateral cleft lip or palate or both have an associated supernumerary tooth which is thought to be a result of fragmentation of the dental lamina during cleft formation.

A 76-year-old man with a smoking history of 60 pack years had a poorly differentiated squamous cell carcinoma locally excised from his tongue in 2008 with no subsequent chemoradiation. The following year he developed a right neck lump. Previous imaging of the neck had diagnosed a calcified right carotid vessel with no suspicion of malignancy. Positron emission tomography-computed tomography (PET-CT) in 2009 reported calcified lymph nodes in the right upper anterior cervical triangle, suspicious for malignancy. Fine needle aspiration cytology (FNAC) confirmed metastatic squamous cell carcinoma. A right radical neck dissection revealed metastatic squamous cell carcinoma in one of 28 lymph nodes. Calcification and heterotopic ossification with marrow formation was present in seven lymph nodes including the node involved by carcinoma. Lymph node calcification in the neck is uncommon, occurring in about 1% of enlarged nodes. It is mostly associated with past radiation therapy, old tuberculosis or healed necrotic abscesses but has occasionally been reported in the context of metastatic malignancy. The exact mechanism of heterotopic ossification in the setting of malignancy is unknown although some investigators conclude it is a result of metaplasia of fibroblasts induced by factors released by rapidly dividing cells or from osteoprogenitor stem cells lying dormant within the affected tissues.

Whilst schemes exist in the UK and North America, there is at present no internationally standardised instrument for reporting head and neck pathology. It can be difficult to assess and code information in reports across jurisdictions if there is lack of uniformity in content and presentation. Purpose The aim of this protocol is to ensure that oral cancer pathology reports are uniform in their content, contain relevant information and can be easily interpreted to direct further treatment and provide a guide for discussing prognosis. Methodology A multidisciplinary committee (comprising four pathologists, a head and neck surgeon and a radiation oncologist) was formed as part of the Cancer Services Advisory Committee of the Royal College of Pathologists of Australasia (RCPA) in 2010. Relevant stakeholders including the Australian and New Zealand Head and Neck Cancer Society will be invited to nominate a representative to review the protocol before endorsement by the RCPA. Results The group has developed the standards and guidelines to be used. The protocol is in its final draft format and is ready for review by stakeholders. Discussion It is envisaged that all oral cancer cases throughout Australasia will be reported according to this protocol, thus ensuring that all current histopathological prognosticators will be identified and included in a clinically and pathologically complete and standardised pathology report.

Tissue specimens remain important tools in medical education despite innovative imaging techniques and web based resources. Reduction in autopsies has made acquiring specimens difficult. Aim To create a surgical specimen museum as a teaching resource. Methods All surgical specimens are evaluated on accession in the department and photographed. The surgeon is contacted to obtain agreement to contact their patient. Following issue of the pathology report, a letter is sent to the patient inviting them to consent to donating their specimen. Following consent the specimen is either retained as a ‘wet’ specimen or mounted. Representative histology slides are produced. The students were surveyed as to the value of this learning resource. Results This museum was established 7 years ago. Consent has been obtained from 706/867 patients approached. The spectrum of diseases includes inflamed appendices, hydatidiform moles and other specimens which would rarely be seen at autopsy. Students rated this resource very highly and patients who have chosen to view their specimen have indicated benefit. Conclusions The spectrum of diseases represented in this museum is diverse and clinically relevant. There is widespread support from patients for the use of their surgical specimens for education. Students and patients have benefited from its establishment.

There is increasing use of FFPE tissues for molecular profiling. We sought to determine the effects of fixation on our ability to detect bacterial DNA in tissues. Aim To investigate the effect of FFPE, and two de-paraffinisation methods on DNA yields, bacterial DNA amplification and molecular fingerprinting. Methods Intestinal tissue and corresponding gut microflora were collected from mice and chickens. Triplicate samples from 14 animals were either snap frozen at -80°C (FS); or fixed in 10% formalin prior to processing. The FFPE samples were de-paraffinised using either the traditional method (TM) or a microwave method (MW). DNA was extracted from all tissues using a modified commercial kit protocol. DNA yields were determined, and amplification of bacterial 16S rDNA fragments compared. Denaturing gradient gel electrophoresis (DGGE) was used to generate ‘fingerprints’ of PCR amplicons to obtain an estimate of number of species of gut microbiota present. Results DNA yields from FFPE-TM and -MW tissues were 11% and 35% respectively of those from FS. 16S PCR products were obtained from 93% of FS, 7% of FFPE-TM and 57% of FFPE-MW samples. DGGE profiles showed FS yielded PCR fingerprints with the most bands (avg 22 bands) followed by FFPE-MW (avg 3 bands). Conclusion Compared to FS, FFPE tissue yields less bacterial DNA, and is less likely to enable the generation of PCR amplicons or DGGE bands for molecular profiling. The MW method yielded twice the DNA of TM. The amplification success of the 260 bp 16S rDNA fragment is poor when DNA has been extracted from FFPE, regardless of de-paraffinisation method.

A 14-month-old boy presented with a palpable swelling of his left anterior maxilla. Computed tomography (CT) scan showed a 2 cm circumscribed expansile lesion (cystic +/– soft tissue component) in the anterior maxilla. A review of the radiology showed that the lesion was separate from the dental apparatus and was of intermediate aggression. Light microscopy of the biopsy revealed an intraosseous circumscribed proliferation of bland spindle cells set in a fibromyxoid stroma with occasional extravasation of red blood cells. Immuno-histochemically, the tumour cells showed positive staining for vimentin, and both nuclear and cytoplasmic expression for β-catenin. The features were of a nodular fasciitis-like lesion that was β-catenin positive. The patient is doing well, with no clinical or radiological evidence of recurrence 1 year post-operatively. Cranial nodular fasciitis-like lesions are relatively uncommon and are thought to be a post-traumatic reactive process. A recent study has shown that a subset of these lesions have dysregulation of the Wnt/β-catenin pathway, suggesting that they maybe pathobiolo-gically related to fibromatoses. A few cases have been associated with familial adenomatous polyposis (FAP). Testing for FAP has been recommended for this child.

Aim Review new research to understand better the reported higher rates of breast cancer in the ACT, Australia. Methods Results of two studies published in 2010 were reviewed. 1,2 Rates of breast cancer and risk factors were compared with national figures. Results ACT females had a higher rate of breast cancer, with an age-standardised incidence of 129.6 cases per 100 000 women for 2002-2006, compared with the national average of 113 per 100 000. However, for most years, this higher incidence was not statistically significant. Participation in BreastScreen was similar to the national rate. Breast cancers detected by screening were likely to be smaller, have fewer positive lymph nodes and be treated by breast-conserving surgery. Breast cancer survival improved in the ACT (5-year survival 87% in 1995-1999; 92% in 2000-2004). Differences in risk factors related to socioeconomic status, fertility, maternal age and alcohol intake could place ACT women at higher risk of breast cancer. Discussion A higher incidence of breast cancer in the ACT cannot be attributed to a different rate of participation in BreastScreen. ACT females appear to have a different risk factor profile for breast cancer, which could account for the higher incidence. The declining mortality from breast cancer reflects the efficacy of radiological screening, along with adherence to nationally recommended breast cancer treatment guidelines in the ACT.

saline during pregnancy was observed; microalbuminuria was not detected in either group. Following PE induction, the pregnant WT group showed increased SBP (115mmHg to 143mmHg), cellular infiltrate within the glomeruli and cytoplasmic swelling and foot process enfacement of the visceral podocyte. All parameters in the hCD39tg group remained unchanged. Pup number and weight did not differ between the groups. Conclusion: PE was induced in mice by transfer of maternally derived Th1 polarised cells manifesting as hypertension and structural change within the glomerulus. The transgenic expression of hCD39 protects against the development of PE.

A 60-year-old man presented with an ulcerated and verrucous lesion on the lateral gingiva surrounding the lower right canine. Biopsy showed this to be a squamous cell carcinoma and the patient proceeded to a right partial mandibulectomy with selective neck dissection. Light microscopy of the mandibulectomy specimen showed a multifocal, well differentiated, keratinising squamous cell carcinoma invading into the mandible. No lymph node metastases where found in 13 lymph nodes. The carcinoma appeared to have arisen in a typical verruca vulgaris (viral wart). Immunohistos-taining for p16 (INK4a) however was weak and only focally positive. Electron microscopy failed to demonstrate any definite viral particles. Molecular testing for human papilloma virus (HPV) is yet to be performed. The tumour lacked the characteristic histological features of a verrucous carcinoma. While this case has not been proven to be HPV associated, HPV associated squamous cell carcinoma of the head and neck is now considered a distinct pathologic entity. Recent evidence supports HPV playing an independent role in tumour development and behaviour in the head and neck. HPV positive tumours are observed in younger, lighter alcohol consuming individuals and are associated with a better overall and disease-specific survival.

A 38-year-old woman presented to her dentist after she noticed a painful lump in her right gum and her right submandibular region for a few weeks. Radiology showed a large radiolucent area in the right mandible. An initial biopsy showed inflammatory changes. A second biopsy was performed 5 months later as the swelling had not resolved with antibiotics. Light microscopy and immunophenotyping (immunohistochemistry and flow cytometry) of the second biopsy showed features of a diffuse large B cell lymphoma, NOS (WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues 2008) with kappa light chain restriction. Staging showed the lymphoma was confined to the mandible (Stage 1E). The patient was treated with combination immunother-apy and chemotherapy. Primary extranodal lymphomas of the head and neck are relatively uncommon. Non-Hodgkin lymphoma (NHL) of the mandible accounts for only 0.6% of isolated NHL and 8% of all tumours in this bone. Owing to their low frequency and to their non-specific symptoms, primary non-Hodgkin lymphomas of the mandible may be misdiagnosed causing a delay in diagnosis.

Dichloroacetate (DCA) has recently been proposed as a novel and relatively non-toxic anticancer agent that can reverse the glycolytic phenotype in cancer cells through the inhibition of pyruvate dehydrogenase kinase. Over 90% of all tumours show an increased glycolysis and this response could contribute to tumour progression, metastasis and resistance to various chemotherapy agents. Reversing the glycolytic phenotype could potentially slow down tumour growth, induce apoptosis and delay metastasis and is therefore a potential anti-cancer strategy. Aims and Methods Rat breast adenocarcinoma cell lines were used to examine the effect of DCA both in vitroand in vivo. Results The growth of several breast cancer cell lines (MCF-7, T47D, 13762 MAT and V14 cells) was found to be inhibited by DCA in vitro.Further examination of 13762 MAT breast adenocarcinoma cells found that reversal of the glycolytic phenotype by DCA correlated with the inhibition of proliferation without any increase in cell death. This was despite a small but significant increase in caspase 3/7 activity, which may sensitise cancer cells to other apoptotic triggers. In vivo,DCA caused a 58% reduction in the number of lung metastases observed macroscopically after injection of 13762 MAT cells into the tail vein of rats (p = 0.0001, n>9 per group). The lesions in the DCA treated rats developed less tumoral necrosis, had a higher mitotic count and a greater lymphocytic infiltration than the control group. Conclusion These results demonstrate the anti-cancer potential of DCA and the reversing the glycolytic phenotype.

Demonstration of dysplasia on histology is the best current indicator of risk of developing cancer in Barrett’s oesophagus. Aim To determine if cellular DNA determined by flow cytometry can be used in stratifying patients at greater risk of developing high grade dysplasia/adenocarcinoma (HGD/AC). Methods 165 oesophageal biopsies [40 no dysplasia, 50 indefinite for dysplasia (ID), 55 low grade dysplasia (LGD), 10 HGD and 10 AC) from 108 patients were analysed by flow cytometry using formalin fixed paraffin embedded tissue. Results Cellular DNA content showed a strong relationship with grade of histological dysplasia, with higher grades more likely to show abnormal ploidy (χ 2 =81.2, df=3, p p ≤ 0.0001). LGD was more likely to show abnormal ploidy (18%, 10/55) compared to no dysplasia ( p = 0.06), but ID (10% abnormal ploidy, 5/50) were similar to no dysplasia ( p = 0.38). Six patients with initially no dysplasia/ ID and diploid DNA developed HGD/AC over 2–7 years with concomitant development of aneuploidy. Conclusions Abnormal ploidy on flow cytometry is strongly associated with worsening dysplasia. However, HGD/AC can still develop in patients when initial biopsies show diploid DNA and no dysplasia.

Background Procarcinogenic effects of the non-steroidal anti-inflammatory drug (NSAID) sulindac have been described in the mouse proximal colon. The possible role of hypoxia inducible factors (HIF) in this process is poorly understood. Aim To study histological effects of sulindac in HIF1α knockout mice. Methods Mice were bred with a specific homozygous HIF1α deletion in the colonic epithelium (n = 16) with their genotype controls that express HIF1α in the colon (n = 19). They were given a diet containing 320 ppm sulindac for 20 weeks and the dietary controls (n = 17) received mouse chow without sulindac. Biopsies from proximal to distal mouse colons were examined by light microscopy. The average number of biopsies analysed per mouse was 7.9 for the test and 8.5 for the control genotype. SPSS (Version 11) and StatXact 8 software were used for statistical analysis. Results Sulindac exposure was associated with acute and chronic inflammation. Neovascularisation, apoptosis, necrosis and fibrosis were not present. Neoplastic transformation was only seen in the mice receiving the sulindac diet (p Conclusion HIF1α may have a pro-inflammatory role in sulindac-induced carcinogenesis in the mouse proximal colon.

Background Tissue factor (TF) is thought to play an important role in cancer biology including cancer progression, angiogenesis and metastasis. TF may also be a prognostic marker and a potential therapeutic target. Vascular endothelial growth factor (VEGF) is known to have a role in tumour angiogenesis. Aim To perform a correlation study for the expression of TF and VEGF in breast cancers assessed by immunohistochemistry. Method Paraffin embedded tissue from 132 representative breast cancers (grade 1–3), 29 fibroadenomas and 22 normal breast sections were examined using a three point intensity scale. TF and VEGF staining was scored in the cytoplasm and the nucleus of both stromal and tumour cells in the breast cancer specimens. Results Breast cancers show stronger TF stromal cyoplasmic expression (p = 0.0021), stronger stromal nuclear staining (p = 0.0003), and stronger total TF staining (p = 0.0002) than non-neoplastic breast tissue. There was no significant difference in VEGFR expression between disease groups for stromal cytoplasmic staining but some difference in cytoplasmic staining of cancer cells of different grades (p = 0.02). Stromal nuclear staining for TF, but not VEGFR expression, correlated with patient survival. Conclusion Stromal cell TF expression is overexpressed in breast cancer and this correlates with patient survival.

A 33-year-old pregnant woman, G1P0 at 30 weeks gestation, presented to her obstetrician with abdominal tightness associated with fever. On examination, she was febrile (388C) and her full blood count showed leukocytosis with neutrophi-lia. Abdominal ultrasound examination revealed a foetal death in utero (FDIU). She had a spontaneous vaginal delivery. The liquor was heavily meconium stained. The autopsy examination found a mildly macerated male foetus with features suggestive of oligohydramnios. The placental disc was small (241 g) and showed multiple yellow cream lesions (3–18 mm) on the maternal surface. There was absent coiling of the umbilical cord. Multiple microbiological swabs from the placenta, foetal abdomen and pleura, as well as culture of foetal blood, all showed a heavy growth of Listeria mono-cytogenes . Light microscopy showed vasculitis and funisitis of the umbilical cord as well as grade 3 (of 3) acute chorioamnionitis. Multiple microabscesses and necrotising granulomata were identified in the placental disc, liver and lung. Gram stains confirmed the presence of extracellular Gram positive bacilli within the placenta and liver. Thus there was microbiological and histological evidence of disseminated granulomatous septicaemia, a known and almost always fatal outcome for this infection. Listeriosis is an uncommon and perhaps under-diagnosed cause of FDIU. It frequently occurs in the third trimester secondary to a decline in cell-mediated immunity. Symptomatic pregnant women frequently present with a febrile episode, as in our case, following a history of ingestion of contaminated food, e.g., raw soft cheese, raw vegetables, meat or sausage. Treatment and follow up of this patient is crucial as persistent vaginal colonisation by Listeria may cause subsequent abortions or stillbirths in future pregnancies.

(2000). TEST AND TEACH Number One Hundred and Two: Part 2. Pathology: Vol. 32, No. 3, pp. 223-224.

e11517 Background: The causes of variation in breast cancer survival remain uncertain. Care provided to women with breast cancer may vary in relation to both the care setting and characteristics of the clinicians. Methods: To compare the outcomes of management of breast cancer treated in rural and metropolitan centers, a prospective audit of breast cancer in a region of Australia was undertaken. Over a nine-year observation period 2102 women with invasive breast cancer underwent potentially curative surgery. Treatments received, including systemic adjuvant therapy, were compared to contemporary guideline-based indicators. Breast cancer specific mortality was analyzed using Cox proportional hazards models. Results: Overall agreement of received treatment with the indicators was high. Women treated within rural centers were, however, much less likely to receive post operative radiotherapy after breast conserving surgery (86.4% vs. 97.0%; p No significant financial relationships to disclose.

A 70-year-old male presented to his general practitioner after he noticed an ulcerated lump on the right anterior aspect of his tongue. Eighteen months prior to this presentation a total nephectomy had been performed for the management of a Fuhrman Grade 2 conventional clear cell renal cell carcinoma of the lower pole of the right kidney. The patient was known to have widespread metastases at the time of this presentation. On examination the nodule measured 10 mm across and a metastasis was suspected. In view of the clinical findings, and discomfort to the patient, an excisional biopsy was performed. Light microscopy showed features of metastatic renal cell carcinoma associated with an ulcerated overlying pyogenic granuloma-like lesion. On immunohistochemistry, the tumour cells expressed EMA, vimentin and CD10 with similar morphology to the primary renal carcinoma. Tongue metastasis of renal cell carcinoma is very rare but usually is a manifestation of wide spread disease. The tumour may masquerade clinically and microscopically as a pyogenic granuloma. Metastasis to the tongue may also be the initial site of presentation of renal cell carcinoma and so should be considered in the microscopic differential diagnosis of a clear cell malignancy in the tongue.

Diffuse Large B-cell Lymphoma (DLBCL) is the commonest subtype of Non-Hodgkin Lymphoma (NHL). Staging bone marrow (BM) biopsies in NHL are conventionally examined using histology alone, although the use of immunophenotyping to aid diagnosis is increasing. This study addresses the clinical impact of routine use of flow cytometry (FL) and immunohistochemistry (IHC) in 156 histologically proven DLBCL cases at initial diagnosis. The median age of the patient cohort was 62 years (range 20–86 years) and the gender ratio was 1.53 in favor of males. Nine patients were treated with palliative intent and were excluded from all survival analyses. The rest were treated with anthracycline based chemotherapy regimens; 36 patients received concomitant Rituximab. Median overall survival (OS) was 6.1 years (95% CI: 3.8, 8.4). Histological involvement was noted in 30 cases (19.2 %) using standardized Cheson criteria [Cheson BD, Horning SJ, Coiffier B et al. J Clin Oncol1999; 17:1244]. FL and IHC (using T-cell, B-cell, and light chain markers) detected an additional 17 cases (10.9%) each with 4 cases detected by both methods. Thirty (19.2 %) patients were upstaged to stage IV with the use of these investigations (stage I: 6, stage II: 12, stage III: 12). Kaplan Meier curves demonstrated that positivity on FL, IHC and both (FL + IHC) resulted in a significantly worse OS and progression free survival (PFS) compared to negative cases. Cox proportional hazards models were used to determine the additional benefit of FL and IHC in predicting OS over histological involvement, after adjusting for age, performance status and use of Rituximab. The first analysis compared FL with histology and showed that FL added significant independent prognostic value [Histology: HR = 2.1, 95% CI 1.0, 4.3, p=0.05; FL: HR = 2.0, 95% CI 1.0, 3.8, p=0.04]. The second analysis compared histology with IHC and showed that the two had co linearity in their prediction of survival with IHC adding significantly more than histology alone [Histology: HR = 1.3, 95% CI 0.5, 3.0, p=0.6; IHC: HR = 2.3, 95% CI 1.1, 5.0, p = 0.03]. In the final model, we examined the interactive effect of FL and IHC over and above histological diagnosis and found the interaction to be the stronger predictor of OS [Histology: HR = 1.8, 95% CI 0.8, 3.7, p =0.1; FL/IHC: HR = 2.7, 95% CI 1.2, 6.2, p=0.02]. In conclusion, our results show that FL and IHC performed routinely can improve detection of BM involvement in DLBCL and upstage ~20 % patients to stage IV disease. We found that FL added significant independent prognostic value while IHC showed greater co linearity with routine histological diagnosis. The interactive effect of FL/IHC had additional benefit over routine histology in predicting survival. These results suggest that staging BM in DLBCL should have FL and IHC performed routinely at initial diagnosis.

Fetuses and children are not ‘little adults’. Their diseases frequently require additional knowledge to that used in adult pathology and also a multidisciplinary approach in their diagnosis. This special theme issue of Pathology provides an update on uncommon and common, but troublesome, aspects of perinatal and paediatric pathology. The senior authors in this issue are recognised experts in their area both from Australasia and overseas and we would like to thank them for agreeing to write for this special theme edition knowing they are already very heavily committed in so many ways. The breadth of topics shows the vibrancy of pathology. Galambos and deMello, for example, describe the molecular processes in pulmonary development and how dysregulation can lead to clinical manifestations. Pathology is global. Peres and his colleagues have written on their experience of infectious diseases in a developing country. They rightly warn that while pathologists in developing countries are aware of these diseases, those in industrialised countries may not be but should be ready to expect the unexpected with increased tourism and immigration. That pathology is medicine is amply demonstrated by the following reviews: Mackay and Monagle illustrate the key role that pathologists play in the diagnosis and management of perinatal and early childhood stroke. In the paper on eosinophilic oesophagitis, Chow and his colleagues show the importance of pathology in refining a clinical entity and in triaging management. Lam and Ng show the cross-over between disciplines with the use of biochemical markers for diagnosing neonatal sepsis. Timely diagnosis reduces antimicrobial therapy and neonatal intensive care costs. The cost burden of inheritable diseases is immense and Wilcken and Wiley outline how newborn screening has reduced this burden and hint at the use of new technologies in the future to expand screening further. The unexplained stillbirth remains a diagnostic challenge and Rawlinson and colleagues pose the question, and the veracity of the evidence, of the role of viruses and other infections in stillbirth. In documenting the pathology of paediatric renal tumours, Vujanic and Charles show the interplay between pathology and oncology. They outline clearly the current differences between the North American and European approaches to initial treatment and diagnosis. Vlychou and Athanasou provide a comprehensive review of bone tumours in children, with valuable radiological-pathological correlation. If there are still those who doubt the value of autopsies, Encha-Ravazi and her colleagues show that neuropathological evaluation, including the use of molecular probes, is important for genetic counselling. The importance of effective communication with our clinical colleagues is discussed by Gordijn and her colleagues in relation to placental lesions that may recur in a subsequent pregnancy. Our sincere thanks to the reviewers who provided detailed and constructive comments which made our editorial task so much easier. Thank you to Soon Lee, Editor of Pathology, for the opportunity to produce this special theme issue. Finally, we thank Belinda Neill in the Editorial Office at the College for keeping us on target. Her efficiency and organisational skill need to be acknowledged.

10602 Background: The AJCC/UICC staging system categorises tumour size in multifocal and multicentric breast cancer (MMBC) using the largest dimension of the largest focus of tumour. This under-estimates the total tumour burden and therefore may under-estimate the potential of these tumours to metastasise. This study tests the hypothesis that an aggregate measurement of tumour size in MMBC is more closely related to nodal status than the size of the largest focus. Methods: This prospective cohort study involved women with ipsilateral invasive breast cancer with known nodal status using data from the ACT & SENSW BCTG database from July 1997 to June 2004. Pathology reports were reviewed to obtain tumour size in the unifocal group and nodal status in all women. The histopathology of MMBC cases were reviewed measuring all tumour foci that were recorded as: (1) the diameter of the largest deposit (LD), (2) the aggregate diameter (AD) of all deposits, and (3) a calculation of the aggregate volume (AV) using the largest dimension of each focus. The MMBC dimensions were compared with unifocal cancers and against nodal status using a multivariate logistic regression analysis and compared against each other using a stepwise method to determine which method is most predictive of nodal involvement. Results: 795 women met the criteria for study entry: 139 (17.5%) had MMBC and 656 (82.5%) had unifocal disease. When adjusting for tumour size method of measurement in MMBC, there was strong agreement between the AD MMBC and unifocal groups in terms of nodal status (p=0.7). Conversely, there were statistically significant differences in nodal status using LD (p=0.008) and AV (p=0.02) measurements in MMBC when compared to the unifocal group. Within the MMBC group the aggregated diameter measure was most significantly related to nodal status (p=0.001). Conclusions: The results of this study confirm that the current tumour staging system in MMBC under-estimates the potential for nodal spread and that aggregate diameter is more clinically useful. Nodal status is used in this study as a surrogate for tumour behaviour. Further studies are underway to assess relationship of aggregate size of MMBC to relapse and survival. No significant financial relationships to disclose.

Background: Pathological assessment of the placenta provides a method of auditing clinical judgement by clinicopathological examination.

Background This study was designed to assess the motivations of senior medical clinicians to teach medical students. This understanding could improve the recruitment and retention of important clinical teachers. Methods The study group was 101 senior medical clinicians registered on a teaching list for a medical school teaching hospital (The Canberra Hospital, ACT, Australia). Their motivations to teach medical students were assessed applying Q methodology. Results Of the 75 participants, 18 (24%) were female and 57 (76%) were male. The age distribution was as follows: 30–40 years = 16 participants (21.3%), 41–55 years = 46 participants (61.3%) and >55 years = 13 participants (17.3%). Most participants (n = 48, 64%) were staff specialists and 27 (36%) were visiting medical officers. Half of the participants were internists (n = 39, 52%), 12 (16%) were surgeons, and 24 (32%) were other sub-specialists. Of the 26 senior clinicians that did not participate, two were women; 15 were visiting medical officers and 11 were staff specialists; 16 were internists, 9 were surgeons and there was one other sub-specialist. The majority of these non-participating clinicians fell in the 41–55 year age group. The participating clinicians were moderately homogenous in their responses. Factor analysis produced 4 factors: one summarising positive motivations for teaching and three capturing impediments for teaching. The main factors influencing motivation to teach medical students were intrinsic issues such as altruism, intellectual satisfaction, personal skills and truth seeking. The reasons for not teaching included no strong involvement in course design, a heavy clinical load or feeling it was a waste of time. Conclusion This study provides some insights into factors that may be utilised in the design of teaching programs that meet teacher motivations and ultimately enhance the effectiveness of the medical teaching workforce.