Roos J. Leguit

Whole slide images for primary diagnostics in dermatopathology: a feasibility study

Background During the last decade, whole slide images (WSI) have been used in many areas of pathology such as teaching, research, digital archiving, teleconsultation and quality assurance testing. However, WSI have not regularly been used for routine diagnosis, because of the lack of validation studies. Aim To test the validity of using WSI for primary diagnosis of skin diseases. Materials and methods 100 skin biopsies and resections which had been diagnosed light microscopically one year previously were scanned at 20× magnification, and rediagnosed by six pathologists (every pathologist assessed his own cases), having the original clinical information available, but blinded to the original diagnoses. The WSI diagnoses were compared to the initial light microscopy diagnosis and classified as concordant, slightly discordant (without clinical consequences) or discordant. Results The light microscopy and the WSI based diagnosis were concordant in 94% of the cases. The light microscopy and WSI diagnosis were slightly discordant in 6% of the cases. For one of the slightly discrepant cases the WSI diagnosis was considered better, while the original diagnosis was preferred for the other five cases. There were no discordant cases with clinical or prognostic implications. Conclusion Primary histopathological diagnosis of skin biopsies and resections can be done digitally using WSI.

Morphological differentiation of severe aplastic anaemia from hypocellular refractory cytopenia of childhood: reproducibility of histopathological diagnostic criteria

Baumann I, Führer M, Behrendt S, Campr V, Csomor J, Furlan I, de Haas V, Kerndrup G, Leguit R J, De Paepe P, Noellke P, Niemeyer C & Schwarz S 
(2012) Histopathology 61, 10–17

Whole slide images for primary diagnostics of gastrointestinal tract pathology: a feasibility study

During the last decade, whole slide images have been used in many areas of pathology such as teaching, research, digital archiving, teleconsultation, and quality assurance testing. However, whole slide images have as yet not much been used for up-front diagnostics because of the lack of validation studies. The aim of this study was, therefore, to test the feasibility of whole slide images for diagnosis of gastrointestinal tract specimens, one of the largest areas of diagnostic pathology. One hundred gastrointestinal tract biopsies and resections that had been diagnosed using light microscopy 1 year before were rediagnosed on whole slide images scanned at ×20 magnification by 5 pathologists (all reassessing their own cases), having the original clinical information available but blinded to their original light microscopy diagnoses. The original light microscopy and whole slide image-based diagnoses were compared and classified as concordant, slightly discordant (without clinical consequences), and discordant. The diagnoses based on light microscopy and the whole slide image-based rediagnoses were concordant in 95% of the cases. Light microscopy and whole slide image diagnosis in the remaining 5% of cases were slightly discordant, none of these were with clinical or prognostic implications. Up-front histopathologic diagnosis of gastrointestinal biopsies and resections can be done on whole slide images.

The COX-2 promoter polymorphism -765 G > C is associated with early-onset, conventional and stump gastric cancers

COX-2 overexpression is known to be an important mechanism in gastric carcinogenesis. Previously we have found that early-onset gastric cancer has a unique COX-2 low-expressing phenotype that differs significantly from that of the frequent overexpression seen in conventional gastric cancers. To investigate whether the COX-2 –765 G>C promoter polymorphism (known to lead to a reduction of COX-2 promoter activity in the colon) may explain this difference in expression, we carried out single-nucleotide polymorphism (SNP) analysis of 241 gastric cancers, including early-onset gastric cancer, conventional gastric cancers and gastric stump cancers, as well as in 100 control patients, using real-time PCR and sequence analysis, and correlated these findings with COX-2 expression using immunohistochemistry. We found that the C allele was present in 30% of early-onset gastric cancers, 24% of conventional gastric cancer, 23% of stump cancers, in contrast to 41% in the control group. There was a statistically significant difference in the presence of the C allele in patients with gastric cancer compared with the control group (P=0.007), with the C allele being associated with protection against gastric cancer. However, there was no significant difference between the early-onset, conventional and stump gastric cancer groups. Interestingly, there was no correlation between the presence of the C allele and a difference in COX-2 expression. In summary, we show that the COX-2 –765 G allele promoter polymorphism is significantly associated with gastric cancer when compared with the normal control group, but does not appear to be related directly to COX-2 expression pattern in gastric cancer. Although early-onset gastric cancers appear to have a unique COX-2 expression pattern when compared with conventional gastric cancer, the exact mechanism by which this occurs is yet to be elucidated.

Comparison of horse and rabbit antithymocyte globulin in immunosuppressive therapy for refractory cytopenia of childhood

Refractory cytopenia of childhood is the most common subtype of myelodysplastic syndrome in children. In this study, we compared the outcome of immunosuppressive therapy using horse antithymocyte globulin (n=46) with that using rabbit antithymocyte globulin (n=49) in 95 patients with refractory cytopenia of childhood and hypocellular bone marrow. The response rate at 6 months was 74% for horse antithymocyte globulin and 53% for rabbit antithymocyte globulin (P=0.04). The inferior response in the rabbit antithymocyte globulin group resulted in lower 4-year transplantation-free (69% versus 46%; P=0.003) and failure-free (58% versus 48%; P=0.04) survival rates in this group compared with those in the horse antithymocyte globulin group. However, because of successful second-line hematopoietic stem cell transplantation, overall survival was comparable between groups (91% versus 85%; P=ns). The cumulative incidence of relapse (15% versus 9%; P=ns) and clonal evolution (12% versus 4%; P=ns) at 4 years was comparable between groups. Our results suggest that the outcome of immunosuppressive therapy with rabbit antithymocyte globulin is inferior to that of horse antithymocyte globulin. Although immunosuppressive therapy is an effective therapy in selected patients with refractory cytopenia of childhood, the long-term risk of relapse or clonal evolution remains. (ClinicalTrial.gov identifiers: NCT00662090)

The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid

The gold standard for diagnosis of central nervous system lymphomas still regards a stereotactic brain biopsy, with the risk of major complications for the patient. As tumor cells can be detected in cerebrospinal fluid (CSF), CSF analysis can be used as an alternative. In this respect, mutation analysis in CSF can be of added value to other diagnostic parameters such a cytomorphology and clonality analysis. A well‐known example of targeted mutation analysis entails MYD88 p.(L265P) detection, which is present in the majority of Bing Neel syndrome and primary central nervous system lymphoma (PCNSL) patients. Unfortunately, tumor yield in CSF can be very low. Therefore, use of the highly sensitive droplet digital PCR (ddPCR) might be a suitable analysis strategy for targeted mutation detection. We analyzed 26 formalin fixed paraffin embedded (FFPE) samples (8 positive and 18 negative for MYD88 p.(L265P) mutation) by ddPCR, of which the results were compared with next generation sequencing (NGS). Subsequently, 32 CSF samples were analyzed by ddPCR. ddPCR and NGS results on FFPE material showed 100% concordance. Among the 32 CSF samples, 9 belonged to patients with lymphoplasmacytic lymphoma (LPL) and clinical suspicion of Bing Neel syndrome, and 3 belonged to patients with PCNSL. Nine of these samples tested positive for MYD88 p.(L265P) (8 LPL and 1 PCNSL). This study shows that sensitive MYD88 mutation analysis by ddPCR in CSF is highly reliable and can be applied even when DNA input is low. Therefore, ddPCR is of added value to current diagnostic parameters, especially when the available amount of DNA is limited.

Plasmacytoid dendritic cell proliferations and neoplasms involving the bone marrow : Summary of the workshop cases submitted to the 18th Meeting of the European Association for Haematopathology (EAHP) organized by the European Bone Marrow Working Group, Basel 2016

Two distinct forms of neoplasms derived from plasmacytoid dendritic cells (PDC) exist: mature PDC proliferations associated with myeloid neoplasms and blastic PDC neoplasms (BPDCN). Ten cases of PDC proliferations and neoplasms in the bone marrow have been submitted to the bone marrow workshop held at the 18th EAHP meeting. Based on observations from the submitted cases, scattered PDC (≤1% of cells) and PDC aggregates (≤10 PDC/HPF) reflect the normal bone marrow composition, while in myelodysplastic syndromes (MDS), there is a propensity for larger/more PDC aggregates (1–5% and 35 PDC/HPF). A shared PTPN11 mutation between a mature PDC proliferation and an accompanying MDS provides evidence of clonal relationship in such instances and shows that PDC are a part of the malignant clone. CD123 and CD303 should be considered backbone markers to histopathologically establish the diagnosis of BPDCN, since they are detectable in almost all cases and properly well on biopsies subjected to different fixations. Expression of some T-cell markers (e.g., CD2 and CD7 but not CD3), B-cell markers (e.g., CD79a but not CD19 and CD20), and myeloid markers (e.g., CD33 and CD117 but not myeloperoxidase) can be observed in BPDCN. Genetical data of the summarized cases corroborate the important role of chromosomal losses in BPDCN. Together with five previously reported instances, one additional workshop case with MYC rearrangement proposes that translocations of MYC may be recurrent. The frequent nature of deleterious mutations of IKZF3 and deletions of IKZF1 suggests a role for the Ikaros family proteins in BPDCN.

Rapidly fatal HTLV-1-associated T-cell leukemia/lymphoma in a patient with SLE

Background. A 57-year-old Afro-Jamaican woman with an 8-year history of systemic lupus erythematosus, including lupus nephritis, was admitted to hospital with intractable back pain accompanied by fever and severe malaise. At the time of presentation she was receiving immunosuppressive treatment with glucocorticoids and azathioprine. She also had gout, hypertension and type II diabetes.Investigations. Physical and neurological examination and laboratory analyses, including biochemical, hematological and electrophoresis tests, X-ray of the lumbar spine, pelvis and chest, mammography, MRI of the lumbar spine, thoracic and abdominal CT, and biopsy of a peripheral lymph node and bone marrow with immunohistochemistry and serology for human T-cell lymphotrophic virus (HTLV) 1 and 2.Diagnosis. HTLV-1-associated acute adult T-cell leukemia/lymphoma with bone marrow infiltration and hypercalcemia. Reaching the correct diagnosis was difficult and only possible through close collaboration with the pathologist and with consideration of the patients ethnic and geographical background.Management. Chemotherapy with high-dose prednisone and adjusted doses of cyclophosphamide and doxorubicin. The patient developed tumor lysis syndrome and died 3 weeks after the diagnosis was made.

Post radiation skin tumors: basal cell carcinomas, squamous cell carcinomas and angiosarcomas. A review of this late effect of radiotherapy

This review gives an overview of radiotherapy-induced malignant skin tumors as described in the present medical literature. Basal cell carcinomas are the most frequent post-radiation malignant skin tumors; however, specific incidence ratios are few and show ratios of 2%. Squamous cell carcinomas are briefly discussed, followed by post-radiation sarcomas. Most cases of post-radiation cutaneous sarcomas are angiosarcoma, malignant fibrous histiocytoma, leiomyosarcoma and fibrosarcoma. In cases of radiotherapy for breast cancer, angiosarcomas are the most frequently found malignant sarcomas worldwide (incidence 0.5%) in the irradiated area. We present 192 cases of angiosarcomas after radiotherapy for breast cancer. Also, the atypical vascular lesion, a benign vascular skin lesion occurring after radiotherapy, and the important differential diagnosis of angiosarcoma will be presented and discussed. Other skin tumors supposedly related to radiotherapy are occasionally published and summarized in this review. Because most radiation-induced malignant tumors occur many years after the initiation of radiotherapy and incidences are low, we suggest good instruction of patients regarding self control of the skin rather than a yearly follow-up.

Bone Marrow Microvascular and Neuropathic Alterations in Critical Limb Ischemia Patients

Rationale: The impact of severe cardiovascular disease and critical limb ischemia (CLI) on the bone marrow (BM) is largely unknown. Objective: To investigate microvascular and neuropathic changes in BM of patients with CLI. Methods and Results: BM biopsies were obtained from patients with CLI (n=33) included in the Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra-arterial Supplementation (JUVENTAS) trial (NCT00371371) and controls (n=12). We performed immunohistochemistry and histomorphometry of the BM to assess microvascular density and to evaluate pan-neuronal and sympathetic innervation, which is involved in progenitor cell mobilization. Microvascular density was reduced significantly in CLI compared with controls (P=0.01), as was sympathetic (P=0.047) and pan-neuronal innervation (P=0.006). No differences in microvascular density and sympathetic or pan-neuronal innervation were observed between patients with CLI with and without diabetes mellitus. Conclusions: CLI is associated with BM microvascular and neuropathic changes, both in patients with and without diabetes mellitus.