Lyndal Anderson

Lymph node metastasis of primary endometrial cancers: Associated proteins revealed by MALDI imaging

Metastasis is a crucial step of malignant progression and is the primary cause of death from endometrial cancer. However, clinicians presently face the challenge that conventional surgical‐pathological variables, such as tumour size, depth of myometrial invasion, histological grade, lymphovascular space invasion or radiological imaging are unable to predict with accuracy if the primary tumour has metastasized. In the current retrospective study, we have used primary tumour samples of endometrial cancer patients diagnosed with (n = 16) and without (n = 27) lymph node metastasis to identify potential discriminators. Using peptide matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI‐MSI), we have identified m/z values which can classify 88% of all tumours correctly. The top discriminative m/z values were identified using a combination of in situ sequencing and LC‐MS/MS from digested tumour samples. Two of the proteins identified, plectin and α‐Actin‐2, were used for validation studies using LC‐MS/MS data independent analysis (DIA) and immunohistochemistry. In summary, MALDI‐MSI has the potential to identify discriminators of metastasis using primary tumour samples.

IgG4‐related sclerosing cholangitis: the cholangiocarcinoma mimic

A 51-year-old woman presented with a 3-week history of painless obstructive jaundice on a background of diet-controlled type 2 diabetes mellitus and significant weight loss. There was no history of inflammatory bowel disease. Physical examination revealed cachexia and jaundice. Relevant blood tests showed bilirubin, 56 μmol/L; alkaline phosphatase 1069 IU/L; gamma-glutyamyltransferase, 1114 IU/L; raised carbohydrate antigen 19-9 level, 213 kU/L; and normal carcinoembryonic antigen level, 2.1 ng/mL. Ultrasound showed common hepatic duct wall thickening and second-order duct involvement bilaterally (Fig. 1). Computed tomography (CT) scan revealed intrahepatic duct dilatation and a hilar stricture suspicious for a Klatskin-type cholangiocarcinoma (CCa) (Fig. 2). Magnetic resonance cholangiopancreatography reported an irregular, enhancing soft-tissue mass at the porta hepatitis (Fig. 1). Staging laparoscopy revealed no evidence of hepatic or peritoneal metastasis. Percutaneous transhepatic cholangiogram and temporary biliary stent decompression was performed and subsequently right portal vein embolization to stimulate preoperative left lobe hypertrophy. An open extended right hepatectomy including segment 4B was undertaken. Operative findings included no demonstrable left lobe involvement or vascular invasion on intraoperative ultrasound. Extended lymphadenectomy was complicated by a pancreatic capsule injury, repaired intraoperatively. Histopathology did not confirm carcinoma of the stricture but features of immunoglobulin G4 (IgG4) autoimmune-related disease, with plasma cells in the wall of the gallbladder (>50 per high power field), with the majority being IgG4 positive. Sections of the left hepatic duct showed marked dilatation and chronic inflammation throughout the wall consisting of plasma cells and lymphocytes with scanty neutrophils and eosinophils (Fig. 3). Immunoglobulin levels were unremarkable (IgG level 11.4 g/L and IgG4 level 11.5 g/L). One week post-operatively, the patient developed abdominal distension with leucocytosis. CT scan revealed a pancreatic fistula with acute fluid collection and a reactive pancolitis, which required endo-

Serous ovarian and primary peritoneal cancers: A comparative analysis of clinico-pathological features, molecular subtypes and treatment outcome

OBJECTIVE Primary peritoneal cancer is rare and considered equivalent to stage III/IV ovarian cancer, but questions remain concerning its underlying biology, prognosis and optimal management. METHODS Clinico-pathological and treatment details of primary peritoneal (n=120) and ovarian cancer (n=635) were obtained on women recruited to the Australian Ovarian Cancer Study. Log-rank test was used to compare survival and cox proportional hazards models were fitted to obtain hazard ratios and 95% confidence intervals, both unadjusted and adjusted for age, grade, FIGO stage, residual disease and treatment with neoadjuvant chemotherapy. Molecular subtype was determined by gene expression profiling using published data. RESULTS Compared with advanced serous ovarian cancer, primary peritoneal cancer patients were older (mean age 65.5 vs. 60.2years, p<0.001), more often treated with neoadjuvant chemotherapy (38.4% vs. 11.4%, p<0.001). Gene expression profiling classified a substantially higher proportion of primary peritoneal carcinomas as C1 (mesenchymal, reactive stromal infiltration) subtype (70.6% vs. 32.1%, p=0.029), which was associated with lower complete surgical resection rate. Women with primary peritoneal cancer had significantly shorter progression-free (11.6 vs. 13.6months, p=0.007) and overall survival (31.7 vs. 39.8months, p=0.012). In multivariate analysis, residual disease and neoadjuvant chemotherapy were both independently associated with increased risk of progression and death. CONCLUSIONS Primary peritoneal cancer patients were more frequently treated with neoadjuvant chemotherapy and had inferior survival. Different tumor biology characterized by activated stromal fibrosis in primary peritoneal cancer may underlie the differences in treatment and clinical outcome.

Evidence for lymphatic pathogenesis of endosalpingiosis

Sir, There is recent speculation and early evidence to suggest that acquired endosalpingiosis is at the benign end of a spectrum of müllerian disorders (müllerianoses) of serous differentiation that includes benign and proliferating (borderline) ovarian serous neoplasms, their ‘primary peritoneal’ counterparts and, perhaps most intriguing of all, so-called noninvasive and invasive peritoneal implants (long attributed to accompanying ovarian neoplasms), and all derived from shed tubal epithelium which implants on the visceral and parietal serosal surfaces of the female pelvis and abdomen. No mention is made of lymphatic spread but this must occur. A recently encountered case of florid chronic salpingitis, pseudocarcinomatous epithelial proliferation with lymphatic spread to the adjacent ovary and the presence of a proliferating (borderline) surface serous papillary tumour in the same ovary, supports this additional possible mechanism for development of acquired endosalpingiosis. A 33-year-old otherwise asymptomatic woman presented with secondary infertility. Pelvic ultrasound revealed a complex left adnexal mass which, at laparotomy, proved to be a thickened abnormal left fallopian tube attached by inflammatory adhesions to a multicystic ovarian mass with surface excrescences. Fine peritoneal adhesions and thickwalled cysts were also present in the pouch of Douglas and, to a lesser extent, around the right adnexa, which appeared otherwise normal. Due to the patient’s wish to retain

Evidence for lymphatic pathogenesis of endosalpingiosis: the more things change, the more they stay the same.

Plus ca change, plus c’est la meme chose. It is rapidly closing in on a century since John Sampson’s brace of seminal publications in the mid 1920s concerning the nature and origin of endometriosis and endosalpingiosis. While his terminology and pathogenetic concepts may seem a little quaint by current standards, they were the beginnings of our understanding the derivation of heterotopic endometrial and endosalpingeal tissues, i.e., beyond the anatomical limits in which they were normally found. Thus, local invasion (Sampson’s first category) would correspond to acquired adenomyosis and salpingitis isthmica nodosa. Everywhere else (outside the uterus and fallopian tubes), such ectopic but benign endometrial tissue was ascribed to processes of implantation or metastasis on the one hand or developmental misplacement on the other. Some 20 years later, Goodall added ‘endocervicosis’ to complete the mullerianosis triptych, with local invasion being what we now also think of as deep endocervical glands (deep Nabothian cysts) or cervical endocervicosis and implantation/metastasis equating to peritoneal endocervicosis. There was no proposed developmental misplacement for endocervicosis. Sampson’s original concept of mullerianosis (specifically endometriosis) was of normally sited endometrial tissue with the sometime capacity to transport itself to a variety of ectopic positions by the same mechanisms with which we are familiar in relation to malignant disease and ‘developmentally misplaced endometrial tissue’ was regarded by him as only a theoretical and unsupported pathogenetic possibility. However, others have espoused this latter concept as a valid, if not all-encompassing explanation for ectopic mullerian type tissue, the secondary mullerian system of Lauchlan, a molecular genetic explanation for which is provided by Gaetje et al. It might be only semantics which separates developmentally misplaced tissue of mullerianosis and the mullerian choristoma championed by Batt et al. and of which endocervical examples include lesions reported in the posterior wall of the bladder and small intestine. Endosalpingeal examples would include, but are not limited to, those reported in the appendix and bladder, perhaps also including cystic endosalpingiosis in a variety of intrauterine and extrauterine circumstances and when it occurs in combination with other choristomatous tissues. The parallel concepts of congenital (developmental, ‘embryologically patterned’, choristomatous) and acquired (implantational, metastatic) origins of various forms of

Extramammary paget's disease mimicking localized malignancy on cervical cytology

A case of Extramammary Pagets Disease (EMPD) involving the cervix mimicking cervical carcinoma on routine cervical cytology in a 66‐year‐old woman with a history of recurrent multifocal EMPD involving the vulva, perineum, perianal area, and rectum is reported. The patient had undergone multiple excisions and reconstructions of EMPD, however, had a benign cervical smear history and reported no vaginal bleeding. The conventional papanicolaou smear was reported as concerning for carcinoma with abundant, well preserved material illustrating highly atypical cells with anisonucleosis and dense cytoplasm and focal microacini. Gross anatomical distortion from EMPD and previous surgery precluded satisfactory outpatient colposcopic assessment. Examination under anesthesia was performed and colposcopy revealed a four quadrant high grade lesion extending into the vagina with one area suspicious for malignancy on the cervix. Directed biopsies were taken and histopathology confirmed EMPD with diffuse adenocarcinoma in situ cells infiltrating skin and mucosa of all specimens. These large pale cells were seen both a singly and in clusters of nests within the mucosa. Periodic Acid Schiff staining was positive for intracytoplasmic mucin and supportive immunohistochemistry was performed with strong reactivity in atypical cells for CK7, CEA, and Cam 5.2. There was no evidence of invasive malignancy and the patient remains under clinical surveillance. Cervical EMPD is rare but should be considered in women with abnormal cervical cytology and a history of vulval EMPD. Diagn. Cytopathol. 2016;44:931–934.

Annexin A2 and alpha actinin 4 expression correlates with metastatic potential of primary endometrial cancer.

The prediction of lymph node metastasis using clinic-pathological data and molecular information from endometrial cancers lacks accuracy and is therefore currently not routinely used in patient management. Consequently, although only a small percentage of patients with endometrial cancers suffer from metastasis, the majority undergo radical surgery including removal of pelvic lymph nodes. Upon analysis of publically available data and published research, we compiled a list of 60 proteins having the potential to display differential abundance between primary endometrial cancers with versus those without lymph node metastasis. Using data dependent acquisition LC-ESI-MS/MS we were able to detect 23 of these proteins in endometrial cancers, and using data independent LC-ESI-MS/MS the differential abundance of five of those proteins was observed. The localization of the differentially expressed proteins, was visualized using peptide MALDI MSI in whole tissue sections as well as tissue microarrays of 43 patients. The proteins identified were further validated by immunohistochemistry. Our data indicate that annexin A2 protein level is upregulated, whereas annexin A1 and α actinin 4 expression are downregulated in tumours with lymph node metastasis compared to those without lymphatic spread. Moreover, our analysis confirmed the potential of these markers, to be included in a statistical model for prediction of lymph node metastasis. The predictive model using highly ranked m/z values identified by MALDI MSI showed significantly higher predictive accuracy than the model using immunohistochemistry data. In summary, using publicly available data and complementary proteomics approaches, we were able to improve the prediction model for lymph node metastasis in EC.

A novel role for the HLH protein Inhibitor of Differentiation 4 (ID4) in the DNA damage response in basal-like breast cancer.

Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of Differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC, through unknown mechanisms. Here, we have defined a molecular mechanism of action for ID4 in BLBC and the related disease highgrade serous ovarian cancer (HGSOV), by combining RIME proteomic analysis and ChIP-Seq mapping of genomic binding sites. Remarkably, these studies have revealed novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage and regulating DNA damage signalling. Clinical analysis demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair pathways. These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOV.

A novel cause of postmenopausal bleeding in an immunosuppressed patient

Malakoplakia is a rare histiocytic disease first described in 1902 by Michaelis and Gutmann. It is associated with host immunocompromise including chronic inflammatory conditions, infectious conditions or malnutrition. Here, we report the case of uterine malakoplakia as a rare cause of postmenopausal bleeding in an immunocompromised patient.

Intraoperative Frozen Section of Ovarian Tumors: A 6-Year Review of Performance and Potential Pitfalls in an Australian Tertiary Referral Center.

Objectives Intraoperative frozen section (IFS) offers a rapid test to guide the extent of surgery, which is essential for optimal treatment of ovarian cancer. This study evaluated the diagnostic performance and influence of IFS in the surgical management of ovarian tumors. Methods A retrospective review was conducted of IFS of adnexal lesions from 2008 to 2013, with diagnoses classified as benign, borderline, or malignant. The diagnostic performance of IFS was calculated, with a focus on primary epithelial tumors. In discordant cases, it was determined whether the results of the IFS influenced the nature of the primary surgery. Results There were 277 consecutive cases over the study period. The overall sensitivity for diagnosing malignant disease was 75.9% and the specificity was 100%. With a benign IFS result, there was a 6.25% (9/144) chance that the final diagnosis would be malignant, and a 7.6% (11/144) chance that the final diagnosis would be borderline, resulting in the potential for understaging. The predictive values for benign, borderline, and malignant IFS results were 86.1%, 66.6%, and 100%, respectively. For a borderline IFS result, there was a 33.3% chance that the final diagnosis would be malignant disease, and this was higher in older patients (53.3%). There were no instances of overdiagnosis in this series. Of 37 cases underdiagnosed, 19 received incomplete primary staging surgery guided by the IFS, and most of these were mucinous tumors. Conclusions Intraoperative frozen section is most valuable for its high specificity in diagnosing malignancy. It should be interpreted with caution in borderline tumors, particularly in older patients and in mucinous tumors. Overdiagnosis did not occur in this series; however, in younger patients, the limitations of IFS must be considered before surgery that would result in loss of fertility.