Search

Merkel cell carcinoma with a sarcomatous component is very rare, with only 12 cases reported in the literature, often with overtly malignant myoid differentiation. We report a case of metastatic Merkel cell carcinosarcoma presenting in a lymph node 6 months after a diagnosis of cutaneous Merkel cell carcinoma with conventional histologic features. The metastatic lesion showed a unique biphasic appearance with admixed populations of neuroendocrine epithelial cells and fascicles of mitotically active spindle cells with mild cytological atypia. In addition to the immunomorphological features, a common molecular profile between the epithelial and mesenchymal components further supported the notion of carcinosarcoma in this case. To the best of our knowledge, a bland sarcomatous component has not been previously described in Merkel cell carcinosarcoma, which can be easily overlooked as a reactive stromal reaction microscopically.

Low-coverage whole-genome sequencing (LC-WGS) can provide insight into oncogenic molecular changes. Serum extracellular vesicles (EV) represent a novel liquid biopsy source of tumoral DNA. This study compared copy number alteration (CNA) profiles generated from LC-WGS of formalin-fixed paraffin-embedded (FFPE) tumoral DNA and EV-DNA obtained from cancer patients. Patients with squamous cell carcinoma of the base of tongue (n = 3) and cutaneous squamous cell carcinoma (n = 2) were included. LC-WGS (0.5-1X coverage) was performed on FFPE-DNA and serum EV-DNA. Similarity between CNA profiles was analysed using QDNAseq. FFPE samples had a mean CNA of 31 (range 17–50) over 1.9 × 109 (range 1.0–2.6 × 109) bp in length, and EV samples had a mean CNA value of 17 (range 7–19) over 7.6 × 108 (range 2.9–15 × 108) bp in length. A mean of 8 (range 0–21) CNA over 5.9 × 108 (range 1.6–14 × 108) bp in length was found to overlap between EV and FFPE-derived samples per patient. Although the mean correlation efficient between samples was r = 0.34 (range − .08 to 0.99), this was not statistically significant (p > 0.05). Regions of highest deletion and duplication in FFPE samples were not well reflected in the EV-DNA. Selected CNA regions in EV-associated DNA were reflective of the primary tumor, however appreciation of global CNA and areas of most significant change was lost. The utility of LC-WGS of EV-derived DNA is likely limited to molecular alterations of known interest.

AimsDetermination of the number of plasma cells in bone marrow biopsies is required for the diagnosis and ongoing evaluation of plasma cell neoplasms. We developed an automated digital enumeration platform to assess plasma cells identified by antigen expression in whole bone marrow sections in multiple myeloma, and compared it with manual assessments.MethodsBone marrow trephine biopsy specimens from 91 patients with multiple myeloma at diagnosis, remission and relapse were stained for CD138 and multiple myeloma oncogene 1 (MUM1). Manual assessment and digital quantification were performed for plasma cells in the entire trephine section. Concordance rates between manual and digital methods were evaluated for each antigen by intraclass correlation analyses (ICC) with associated Spearman’s correlations.ResultsThe digital platform counted 16 484–1 118 868 cells and the per cent CD138 and MUM1-positive plasma cells ranged from 0.05% to 93.5%. Overall concordance between digital and manual methods was 0.63 for CD138 and 0.89 for MUM1. Concordance was highest with diffuse plasma cell infiltrates (MUM1: ICC=0.90) and lowest when in microaggregates (CD138: ICC=0.13). Manual counts exceeded digital quantifications for both antigens (CD138: mean=26.4%; MUM1: mean=9.7%). Diagnostic or relapse threshold counts, as determined by CD138 manual assessments, were not reached with digital counting for 16 cases (18%).ConclusionsAutomated digital enumeration of the entire, immunohistochemically stained bone marrow biopsy section can accurately determine plasma cell burden, irrespective of pattern and extent of disease (as low as 0.05%). This increases precision over manual visual assessments which tend to overestimate plasma burden, especially for CD138, and when plasma cells are in clusters.

We investigated whether a unique immune response was instigated with the development of oral tongue squamous cell carcinomas (OTSCC), with/without nodal involvement, with/without recurrent metastatic disease, or within tumor involved nodes. One hundred and ten formalin‐fixed paraffin‐embedded samples were collected from a retrospective cohort of 67 OTSCC patients and 10 non‐cancerous tongue samples. Targets including CD4, CD8, FOXP3, PD‐L1, and PD‐1 were analyzed by immunohistochemistry. The Nanostring PanCancer Immune Profiling Panel was used for gene expression profiling. Data were externally validated in the The Cancer Genome Atlas (TCGA) head and neck (HNSCC), melanoma and lung squamous cell carcinoma (LSCC) cohorts. A 24‐immune gene signature was identified that discriminated more aggressive OTSCC cases, and although not prognostic in HNSCC was associated with survival in other TCGA cohorts (improved survival for melanoma, P, Oral tongue squamous cell carcinomas (OTSCC) exhibit primary immune failure to control carcinogenesis demonstrated by the absence of an immune response to the development of nodal metastases or recurrent disease. Proinflammatory immune gene expression is counteracted by immunosuppressive signaling mediated by FOXP3 and PD‐L1 expression. A gene signature was identified that was not prognostic in HNSCC but was significantly associated with survival in other cancers, demonstrating histotype‐dependent prognostic effects of immune regulation.

Simple Summary Approximately 50% of patients with uveal melanoma will develop incurable metastatic disease. This can be predicted, but requires a biopsy of the eye, which outside of centres of excellence, are not routinely performed. Given that uveal melanoma spreads through the blood, utilising circulating tumour cells from the blood might provide an alternative minimally invasive method to avoid the biopsy. However, the clinical application hinges on the detection rate of circulating tumour cells. Herein we assessed markers to improve the capture and detection of uveal melanoma circulating tumour cells and found that they could be detected in 86% of patients. We further found that ≥3 circulating tumour cells was significantly associated with worse survival. Abstract (1) Background: The stratification of uveal melanoma (UM) patients into prognostic groups is critical for patient management and for directing patients towards clinical trials. Current classification is based on clinicopathological and molecular features of the tumour. Analysis of circulating tumour cells (CTCs) has been proposed as a tool to avoid invasive biopsy of the primary tumour. However, the clinical utility of such liquid biopsy depends on the detection rate of CTCs. (2) Methods: The expression of melanoma, melanocyte, and stem cell markers was tested in a primary tissue microarray (TMA) and UM cell lines. Markers found to be highly expressed in primary UM were used to either immunomagnetically isolate or immunostain UM CTCs prior to treatment of the primary lesion. (3) Results: TMA and cell lines had heterogeneous expression of common melanoma, melanocyte, and stem cell markers. A multi-marker panel of immunomagnetic beads enabled isolation of CTCs in 37/43 (86%) patients with UM. Detection of three or more CTCs using the multi-marker panel, but not MCSP alone, was a significant predictor of shorter progression free (p = 0.040) and overall (p = 0.022) survival. (4) Conclusions: The multi-marker immunomagnetic isolation protocol enabled the detection of CTCs in most primary UM patients. Overall, our results suggest that a multi-marker approach could be a powerful tool for CTC separation for non-invasive prognostication of UM.

Purpose: This study compares the detection sensitivity of two separate liquid biopsy sources, cell-free (cf) DNA/RNA and extracellular vesicle (EV)-associated DNA/RNA (EV-DNA/RNA), to identify circulating Human Papilloma Virus (HPV) DNA/RNA in plasma obtained from patients with oropharyngeal squamous cell carcinoma (OPCSCC). We also report on the longitudinal changes observed in HPV-DNA levels in response to treatment. Experimental design: A prospective study was conducted that included 22 patients with locally advanced disease and six patients with metastatic OPCSCC. Twenty-three patients had HPV-related OPCSCC defined by p16 immunohistochemistry. Levels of circulating HPV-DNA and HPV-RNA from plasma-derived cf-DNA/RNA and EV-DNA/RNA were quantified using digital droplet PCR. Results: Circulating HPV-DNA was detected with higher sensitivity in cf-DNA compared to EV-DNA at 91% vs. 42% (p = p = 0.0019). In the locally advanced cohort, 100% (n = 16) of HPV-OPCSCC patients demonstrated a reduction in circulating HPV-DNA levels in cf-DNA following curative treatment, with 81% of patients demonstrating complete clearance to undetectable levels. However, in metastatic HPV-OPCSCC patients (n = 4), HPV-DNA levels did not correlate with treatment response. Conclusion: Our study demonstrates that although HPV-DNA/RNA can be detected in EV associated DNA/RNA, cf-DNA/RNA is the more sensitive liquid biopsy medium. As circulating HPV-DNA levels were found to only correlate with treatment response in the locally advanced but not metastatic setting in our small cohort of patients, the use of HPV-DNA as a dynamic biomarker to monitor treatment response requires further evaluation.

The assumption that intestinal metaplasia is a prerequisite for intraepithelial neoplasia/dysplasia and adenocarcinoma in the distal esophagus has been challenged by observations of adenocarcinoma without associated intestinal metaplasia. This study describes our experience of intestinal metaplasia in association with early Barrett neoplasia in distal esophagus and gastroesophageal junction. We reviewed the first endoscopic mucosal resection of 139 patients with biopsy-proven neoplasia. In index endoscopic mucosal resection, 110/139 (79%) cases showed intestinal metaplasia. Seven had intestinal metaplasia on prior biopsy specimens and three had intestinal metaplasia in subsequent specimens, totaling 120/139 (86%) patients showing intestinal metaplasia at some point supporting the theory of sampling error for absence of intestinal metaplasia in some cases. Those without intestinal metaplasia (13%) were enriched for higher stage disease (T1a Stolte m2 or above) supporting the assertion of obliteration of intestinal metaplasia by the advancing carcinoma. All cases of intraepithelial neoplasia and T1a Stolte m1 carcinomas had intestinal metaplasia (42/42). The average density of columnar-lined mucosa showing goblet cells was significantly less in shorter segments compared to those ≥3 cm (0.31 vs 0.51, P=0.0304). Cases where segments measured less than 1 cm were seen in a higher proportion of females and also tended to lack intestinal metaplasia. We conclude that early Barrett neoplasia is always associated with intestinal metaplasia; absence of intestinal metaplasia is attributable to sampling error or obliteration of residual intestinal metaplasia by neoplasia and those with segments less than 1 cm show atypical features for Barrett-related disease (absent intestinal metaplasia and female gender), supporting that gastroesophageal junction adenocarcinomas are heterogeneous.

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders characterized by well-known driver mutations [1]. The mutational status of Janus kinase 2 (JAK2), calretic...

There is increasing recognition of circulating tumour DNA (ctDNA) as a non-invasive alternative to tumour tissue for the molecular characterisation and monitoring of disease. Recent evidence suggests that cancer-associated changes can also be detected in the DNA contained within extracellular vesicles (EVs). As yet, there has been limited investigation into the relationship between EV DNA and ctDNA, and no studies have examined the EV DNA of breast cancer patients. The aim of this study was to use low-pass whole-genome sequencing to identify copy number variants (CNVs) in serial samples of both ctDNA and EV DNA from a patient with breast cancer. Of the 52 CNVs identified in tumour DNA, 36 (69%) were detected in at least one ctDNA sample and 13 (25%) in at least one EV DNA sample. The number of detectable variants in ctDNA and EV DNA increased over the natural history of the patient&rsquo, s disease, which was associated with progression to cerebral metastases. This case study demonstrates that, while CNVs are detectable in patient EV DNA, ctDNA has greater sensitivity than EV DNA for serial monitoring of breast cancer.

Aims Megakaryocyte expansion in myeloproliferative neoplasms (MPNs) is due to uncontrolled proliferation accompanied by dysregulation of proapoptotic and antiapoptotic mechanisms. Here we have investigated the intrinsic and extrinsic apoptotic pathways of megakaryocytes in human MPNs to further define the mechanisms involved. Methods The megakaryocytic expression of proapoptotic caspase-8, caspase-9, Diablo, p53 and antiapoptotic survivin proteins was investigated in bone marrow specimens of the MPNs (n=145) and controls (n=15) using immunohistochemistry. The megakaryocyte percentage positivity was assessed by light microscopy and correlated with the MPN entity, JAK2 V617F / CALR mutation status and platelet count. Results The proportion of megakaryocytes in the MPNs expressing caspase-8, caspase-9, Diablo, survivin and p53 was significantly greater than controls. A greater proportion of myeloproliferative megakaryocytes expressed survivin relative to its reciprocal inhibitor, Diablo. Differences were seen between myelofibrosis, polycythaemia vera and essential thrombocythaemia for caspase-9 and p53. CALR -mutated cases had greater megakaryocyte p53 positivity compared to those with the JAK2 V617F mutation. Proapoptotic caspase-9 expression showed a positive correlation with platelet count, which was most marked in myelofibrosis and CALR -mutated cases. Conclusions Disruptions targeting the intrinsic apoptotic cascade promote megakaryocyte hyperplasia and thrombocytosis in the MPNs. There is progressive dysfunction of apoptosis as evidenced by the marked reduction in proapoptotic caspase-9 and accumulation of p53 in myelofibrosis. The dysfunction of caspase-9, which is necessary for proplatelet formation, may be the mechanism for the excess thrombocytosis associated with CALR mutations. Survivin seems to be the key protein mediating the megakaryocyte survival signature in the MPNs and is a potential therapeutic target.

BackgroundTransforming growth factor α (TGFα) is a peptide growth factor known to be expressed in normal haemopoiesis. It is also expressed in a range of epithelial neoplasms but has not been assessed in haemopoietic malignancies. We have performed an immunohistochemical evaluation of TGFα in acute and chronic myeloid malignancies.MethodsTGFα expression was semiquantitatively assessed in 69 normal bone marrow trephines and 157 cases of myeloid malignancy using an immunohistochemical approach.ResultsBlast cells of myeloid origin in acute myeloid leukaemia (AML), myelodysplasia and accelerated and blast phases of chronic myeloid leukaemia (CML) were TGFα positive. In acute promyelocytic leukaemia the neoplastic cells had significantly weaker TGFα expression than seen in other forms of AML. The blast cells in CML-accelerated and blast phases were positive with similar expression to AML.ConclusionsTGFα is expressed in neoplastic myeloblasts and could, therefore, be used as blast cell biomarker in diagnostic haematopathology. In addition, TGFα immunohistochemistry may be of use in identifying a therapeutic target.

There is increasing interest in p53 immunohistochemistry as an adjunct to haematoxylin and eosin (HE) assessment for dysplasia in oesophageal Barrett's mucosa; however, published information on the patterns of staining remains scant. Here, we present descriptions of normal and aberrant p53 staining in non-neoplastic and dysplastic Barrett's mucosa in endoscopic mucosal resections. A retrospective series of archival endoscopic mucosal resections for biopsy proven dysplasia at our institution were retrieved for this study, comprising 28 sections from 23 patients. p53 immunohistochemistry was performed using an in-house optimised protocol and the staining pattern assessed in HE confirmed non-neoplastic, dysplastic and neoplastic areas of Barrett's mucosa with regard to individual cell intensity and location of positive cells with respect to gland microanatomy. In non-neoplastic epithelium, normal p53 staining was weak, heterogenous and localised to the crypts. In dysplastic epithelium, p53 over-expression was seen which was of moderate to strong intensity in either a crypt predominant location or diffuse involving crypt and surface epithelium. The crypt predominant pattern was observed more commonly in low grade dysplasia while the diffuse pattern was more commonly seen in high grade dysplasia. In a minority of cases, there was complete loss of p53 staining in dysplastic epithelium and contiguous neoplasia (null phenotype). p53 immuno-expression in non-neoplastic and dysplastic Barrett's mucosa is distinctive when interpreted with regard to cell intensity and gland microanatomy. We propose that these staining patterns may assist in the interpretation of dysplasia in endoscopic biopsies of Barrett's mucosa.

Myeloproliferative neoplasms (MPNs) are a group of related clonal hemopoietic stem cell disorders associated with hyperproliferation of myeloid cells. They are driven by mutations in the hemopoietic stem cell, most notably JAK2V617F, CALR, and MPL. Clinically, they have the propensity to progress to myelofibrosis and transform to acute myeloid leukemia. Megakaryocytic hyperplasia with abnormal features are characteristic, and it is thought that these cells stimulate and drive fibrotic progression. The biological defects underpinning this remain to be explained. In this study we examined the megakaryocyte genome in 12 patients with MPNs to determine whether there are somatic variants and whether there is any association with marrow fibrosis. We performed targeted next-generation sequencing for 120 genes associated with myeloid neoplasms on megakaryocytes isolated from aspirated bone marrow. Ten of the 12 patients had genomic defects in megakaryocytes that were not present in nonmegakaryocytic hemopoietic marrow cells from the same patient. The greatest allelic burden was in patients with increased reticulin deposition. The megakaryocyte-unique mutations were predominantly in genes that regulate chromatin remodeling, chromosome alignment, and stability. These findings show that genomic abnormalities are present in megakaryocytes in MPNs and that these appear to be associated with progression to bone marrow fibrosis.

e14523 Background: Low-coverage whole genome sequencing (LC-WGS) of tumors provides valuable insight into molecular changes driving oncogenesis. A novel liquid biopsy source of tumor DNA for analysis is from extracellular vesicles (EV) obtained from blood. This study compared copy number alteration (CNA) profiles generated from LC-WGS of Formalin-Fixed Paraffin-Embedded (FFPE) DNA and EV-associated DNA in cancer patients. Methods: Three metastatic base of tongue (BOT), two of which were Human Papillomavirus-related (HPV+) and two metastatic cutaneous squamous cell carcinoma (cSCC) patients were included. EV were isolated using ultracentrifugation from patients’ plasma. DNA was extracted from FFPE tumor tissue and EV. LC-WGS aiming for 0.5-1X coverage was performed using a validated method. CNA profiles were generated using the QDNAseq package, with gains defined as a log2 ratio ≥0.15 and losses < -0.15. Results: CNA profiles of FFPE samples from BOT patients demonstrated significant variation regardless of HPV status, with a mean of 20 regions containing 125 CNA (amplifications or deletions) per sample. The cSCC FFPE samples demonstrated a mean of 23 regions containing 189 CNA per sample. Overall, EV-associated DNA CNA profiles had limited similarity with primary FFPE. EV-associated DNA showed a lower number of CNA regions and CNA, with a mean of 3.7 CNA regions and 11.4 CNA for BOT samples and 6.5 regions and 15.9 CNA for cSCC samples. The HPV-BOT sample showed 2/3 EV-CNA regions matched corresponding FFPE-CNA profile (n = 15). The two HPV+ samples were less consistent with only 1/3 and 1/5 EV-CNA regions matching FFPE-CNA profiles (n = 39 and n = 6, respectively). The two cSCC cases showed more consistency with 4/6 and 6/7 EV-CNA regions matching FFPE-CNA profiles (n = 21 and n = 25, respectively). In the matched CNA regions, the mean CNA in EV-associated DNA was 7.5 and in FFPE-DNA was 97. Conclusions: Although selected EV-associated DNA CNA regions reflected the primary tumors, these were limited in number and did not globally reflect the FFPE derived CNA profiles.

The human epidermal growth factor receptor 2 (HER2) status in breast cancer is important for prognostic prediction and the determination of optimal treatment. Current methods rely on protein expression, as determined by immunohistochemistry (IHC), as well as gene amplification as determined by in situ hybridisation (ISH). We explored whether quantitative droplet digital PCR (ddPCR) can be used for the detection and absolute quantitation of HER2 mRNA. Digital droplet PCR (ddPCR) was performed for HER2 mRNA on 178 formalin-fixed paraffin-embedded (FFPE) breast cancer specimens. HER2 positive, equivocal and negative cases as defined by standard criteria were included and both core biopsies and tissue sections were assessed. HER2 positive cases contained significantly higher levels of HER2 mRNA (169–1,000,000 copies/µl) by ddPCR compared with equivocal (112–139 copies/µl, p = 0.025) and negative cases (6.2–644 copies/µl. p