Impact of COVID-19 pandemic on diagnostic pathology in the Netherlands.

BACKGROUND: The COVID-19 pandemic has a huge impact on healthcare provided. The nationwide pathology registry of the Netherlands, PALGA, offers an outstanding opportunity to measure this impact for diseases in which pathology examinations are involved. METHODS: Pathology specimen numbers in 2020 were compared with specimen numbers in 2019 for 5 periods of 4 weeks, representing two lockdowns and the periods in between, taking into account localization, procedure and benign versus malignant diagnosis. RESULTS: The largest decrease was seen during the first lockdown (spring 2020), when numbers of pathology reports declined up to 88% and almost all specimen types were affected. Afterwards each specimen type showed its own dynamics with a decrease during the second lockdown for some, while for others numbers remained relatively low during the whole year. Generally, for most tissue types resections, cytology and malignant diagnoses showed less decrease than biopsies and benign diagnoses. A significant but small catch-up (up to 17%) was seen for benign cervical cytology, benign resections of the lower gastro-intestinal tract, malignant skin resections and gallbladder resections. CONCLUSION: The COVID-19 pandemic has had a significant effect on pathology diagnostics in 2020. This effect was most pronounced during the first lockdown, diverse for different anatomical sites and for cytology compared with histology. The data presented here can help to assess the consequences on (public) health and provide a starting point in the discussion on how to make the best choices in times of scarce healthcare resources, considering the impact of both benign and malignant disease on quality of life.

Risk of cervical intra-epithelial neoplasia and invasive cancer of the cervix in DES daughters.

OBJECTIVE: Women exposed to diethylstilbestrol in utero (DES) have an increased risk of clear cell adenocarcinoma (CCA) of the vagina and cervix, while their risk of non-CCA invasive cervical cancer is still unclear. METHODS: We studied the risk of pre-cancerous (CIN) lesions and non-CCA invasive cervical cancer in a prospective cohort of 12,182 women with self-reported DES exposure followed from 2000 till 2008. We took screening behavior carefully into account. Incidence was obtained through linkage with the Netherlands Nationwide Pathology database (PALGA). General population data were also derived from PALGA. RESULTS: The incidence of CIN1 was increased (Standardized Incidence Ratio (SIR)=2.8, 95% Confidence Interval (CI)=2.3 to 3.4), but no increased risk was observed for CIN2+ (CIN2, CIN3 or invasive cancer) compared to the screened general population (SIR=1.1, 95% CI=0.95 to1.4). Women with DES-related malformations had increased risks of both CIN1 and CIN2+ (SIR=4.1, 95%CI=3.0 to 5.3 and SIR=1.5, 95%CI=1.1 to 2.0, respectively). For CIN2+, this risk increase was largely restricted to women with malformations who were more intensively screened. CONCLUSIONS: An increased risk of CIN1 among DES daughters was observed, especially in women with DES-related malformations, probably mainly due to screening. The risk of CIN2+ (including cancer) was not increased. However, among DES daughters with DES-related malformations a true small risk increase for non-CCA cervical cancer cannot be excluded.

First experiences of photoacoustic imaging for detection of melanoma metastases in resected human lymph nodes.

BACKGROUND AND OBJECTIVE: Excision and histological assessment of the first draining node (sentinel lymph node) is a frequently used method to assess metastatic lymph node involvement related to cutaneous melanoma. Due to the time required for accurate histological assessment, nodal status is not immediately available to the surgeon. Hence, in case histological examination shows metastases, the patient has to be recalled to perform additional lymphadenectomy. To overcome these drawbacks we studied the applicability of photoacoustic tomographic imaging as an intra-operative modality for examining the status of resected lymph nodes. MATERIALS AND METHODS: In melanoma patients undergoing lymphadectomy for metastatic disease, six suspect lymph nodes were photoacoustically (PA) imaged using multiple wavelengths. Histopathologal examination showed three nodes without tumor cells (benign nodes) and three nodes with melanoma cells (malignant nodes). PA images were compared with histology and anatomical features were analyzed. In addition, PA spectral analysis was performed on areas of increased signal intensity. RESULTS: After correlation with histopathology, multiple areas containing melanoma cells could be identified in the PA images due to their increased response. Malignant nodes showed a higher PA response and responded differently to an increase in excitation wavelength than benign nodes. In addition, differences in anatomical features between the two groups were detected. CONCLUSIONS: Photoacoustic detection of melanoma metastases based on their melanin content proves to be possible in resected human lymph nodes. The amount of PA signal and several specific anatomical features seem to provide additional characteristics for nodal analysis. However, it is as yet preliminary to designate a highly accurate parameter to distinguish between malignant and benign nodes. We expect to improve the specificity of the technique with a future implementation of an adjusted illumination scheme and depth correction for photon fluence.

Remarkable effects of imatinib in a family with young onset gastrointestinal stromal tumors and cutaneous hyperpigmentation associated with a germline KIT-Trp557Arg mutation: case report and literature overview.

Gastrointestinal stromal tumors (GISTs) occur mostly sporadically. GISTs associated with a familial syndrome are very rare and are mostly wild type for KIT and platelet-derived growth factor alpha (PDGFRA). To date 35 kindreds and 8 individuals have been described with GISTs associated with germline KIT mutations. This is the third family described with a germline p.Trp557Arg mutation in exon 11 of the KIT gene. The effect of imatinib in patients harboring a germline KIT mutation has been rarely described. Moreover, in some studies imatinib treatment was withheld considering the lack of evidence for efficacy of this treatment in GIST patients harboring a germline KIT mutation. This paper describes a 52-year old patient with a de novo germline p.Trp557Arg mutation with multiple GISTs throughout the gastrointestinal tract and cutaneous hyperpigmentation. Imatinib treatment showed long-term regression of the GISTs and evident pathological response was seen after resection. Remarkably, the hyperpigmentation of the skin also diminished during imatinib treatment. Genetic screening of the family revealed the same mutation in two daughters, both with similar cutaneous hyperpigmentation. One daughter, aged 23, was diagnosed with multiple small intestine GISTs, which were resected. She was treated with adjuvant imatinib which prompted rapid regression of the cutaneous hyperpigmentation. Imatinib treatment in GIST patients harboring a germline KIT mutation shows favorable and long-term responses in both the tumor and the phenotypical hyperpigmentation.

TuBaFrost 3: regulatory and ethical issues on the exchange of residual tissue for research across Europe.

The regulatory regimes for research with residual tissue and accompanying data differ widely between countries in the European Union (EU): from specific consent to opt-out or even no consent at all. This could greatly hamper research where the exchange of tissue and accompanying data has become the gold standard, like in TubaFrost. Instead of adhering to international guidelines, which have a democratic deficit, or an attempt for a new set of possible harmonising rules, TubaFrost chose to create a coordinating rule: if tissue may legitimately be used for a certain kind of research in the country where it was taken and under whose jurisdiction the patient falls, it may also be used for such research in the country where it is sent to in the context of a scientific program even if in that other country other regulations would apply for research with residual tissue taken from patients under their jurisdiction. This coordinating rule has a sound basis in EU law in general and will solve the problems related to diverging national regulatory regimes in the case of cross national research with residual tissue.

TuBaFrost 6: virtual microscopy in virtual tumour banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated Virtual Microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting bio-repositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

TuBaFrost 1: Uniting local frozen tumour banks into a European network: an overview.

TuBaFrost is the consortium responsible for the creation of a virtual European human frozen tumour tissue bank: a collection of high quality frozen residual, accurately classified tumour tissue samples, which are stored in European cancer centres and universities. This virtual tissue bank, searchable on the internet, has rules for access and use, and a code of conduct to comply with the various legal and ethical regulations in European countries. The easy accessibility and the European scale of the bank will result in the availability of a large number of samples even of rarer tumour types. Standardisation of collection, storage and quality control throughout the network is achieved minimising inter-institutional variability. A website providing access to upload, search and request samples is a key tool of the tissue bank. The search engine makes use of virtual microscopy. An overview of the development of the European virtual frozen tissue bank infrastructure is described in this paper. The various key aspects are described in more detail in a series of articles to appear in this Journal.

TuBaFrost 2: Standardising tissue collection and quality control procedures for a European virtual frozen tissue bank network.

Tumour Bank Networking presents a great challenge for oncological research as in order to carry out large-scale, multi-centre studies with minimal intrinsic bias, each tumour bank in the network must have some fundamental similarities and be using the same standardised and validated procedures. The European Human Frozen Tumour Tissue Bank (TuBaFrost) has responded to this need by the promotion of an integrated platform of tumour banks in Europe. The operational framework for TuBaFrost has drawn upon the best practice of standard workflows and operating procedures employed by members of the TuBaFrost project and key initiatives worldwide.

TuBaFrost 4: access rules and incentives for a European tumour bank.

When designing infrastructure for a networked virtual tumour bank (samples remain at the collector institutes and sample data are collected in a searchable central database), it is apparent that this can only function properly after developing an adequate set of rules for use and access. These rules must include sufficient incentives for the tissue sample collectors to remain active within the network and maintain sufficient sample levels in the local bank. These requirements resulted in a key TuBaFrost rule, stating that the custodianship of the samples remains under the authority of the local collector. As a consequence, the samples and the decision to issue the samples to a requestor are not transferred to a large organisation but instead remain with the collector, thus allowing autonomous negotiation between collector and requestor, potential co-authorship in publications or compensation for collection and processing costs. Furthermore, it realises a streamlined cost effective network, ensuring tissue visibility and accessibility thereby improving the availability of large amounts of samples of highly specific or rare tumour types as well as providing contact opportunities for collaboration between scientists with cutting edge technology and tissue collectors. With this general purpose in mind, the rules and responsibilities for collectors, requestors and central office were generated.

TuBaFrost 5: multifunctional central database application for a European tumor bank.

Developing a tissue bank database has become more than just logically arranging data in tables combined with a search engine. Current demand for high quality samples and data, and the ever-changing legal and ethical regulations mean that the application must reflect TuBaFrost rules and protocols for the collection, exchange and use of tissue. To ensure continuation and extension of the TuBaFrost European tissue bank, the custodianship of the samples, and hence the decision over whether to issue samples to requestors, remains with the local collecting centre. The database application described in this article has been developed to facilitate this open structure virtual tissue bank model serving a large group. It encompasses many key tasks, without the requirement for personnel, hence minimising operational costs. The Internet-accessible database application enables search, selection and request submission for requestors, whereas collectors can upload and edit their collection. Communication between requestor and involved collectors is started with automatically generated e-mails.

TuBaFrost: European virtual tumor tissue banking.

TuBaFrost is a consortium responsible for the task to create a virtual European human frozen tumor tissue bank, composed of high quality frozen tumor tissue collections with corresponding accurate diagnosis stored in European cancer centers and universities, searchable on the Internet, providing rules for access and use and a code of conduct to comply with the various legal and ethical regulations in European countries. Such infrastructure would enlarge tissue availability and accessibility in large amounts of specified or even rare tumor samples. Design of an infrastructure for European residual tissue banking with the described characteristics, clear focus points emerge that can be broken down in dedicated subjects: (1) standardization and quality assurance (QA) to avoid inter-institute quality variation; (2) law and ethics enabling exchange of tissue samples possible between institutes in the different European countries, where law and ethics are characterized by a strong variability; (3) rules for access, with sufficient incentives for collectors; (4) central database application containing innovations on search and selection procedures; (5) support when needed with histology images; and (6) Internet access to search and upload, with in addition a solid website giving proper information on the procedures, intentions and activities not only to the scientific community, but also to the general public. One consortium decision, part of the incentives for collectors, had major impact on the infrastructure; custodianship over the tissues as well as the tissues stay with the collector institute. Resulting in specimens that are not given to an organization, taking decisions on participation of requests, but instead the local collected tissues stay very easy to access by the collector and allows autonomous negotiation between collector and requestor on cooperation, coauthorship in publication or compensation in costs. Thereby, improving availability of large amounts of high quality samples of a highly specified or rare tumor types and contact opportunities for cooperation with other institutes.

Virtual microscopy in virtual tumor banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated virtual microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting biorepositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

Antithrombin and its inherited deficiency states.

Antithrombin is the primary inhibitor of thrombin that also inhibits many of the other activated serine proteinases involved in blood coagulation. A hypercoagulable state occurs when a deficiency of antithrombin exists in plasma; the deficiency may be either inherited or acquired. This failure to regulate adequately the activity of coagulation proteinases can, with additional provocation, result in clot formation and in the clinical presentation of thromboembolic disease. The structure and function of antithrombin, nature and heterogeneity of the molecular defects in the antithrombin gene associated with inherited antithrombin deficiency, prevalence and the natural history of inherited antithrombin deficiency are all reviewed here.

Mortality and causes of death in inherited antithrombin deficiency.

To assess the contribution of inherited antithrombin deficiency to mortality, we investigated the causes of death in 14 families with inherited antithrombin deficiency. Between 1830 and 1994, 86 of 266 family members who had a probability of 0.5 or more for heterozygosity died. The causes of death were obtained for 58 of 66 deaths occurring between 1940 and 1994. Standardized mortality ratios (SMR) were calculated using mortality rates from the general population adjusted for age, sex and calendar period. The overall SMR was 0.90 from 1830 to 1994 (95% C.I. 0.72-1.11). From 1940 until 1994 44 men and 22 women died (SMR = 1.09, 95% C.I. 0.84-1.39; SMR men = 1.20, 95% C.I. 0.87-1.61; SMR women = 0.92, 95% C.I. 0.58-1.39). No excess mortality compared to the general population was found for cancer (14 deaths) or circulatory diseases (28 deaths). A slightly increased mortality caused by respiratory diseases (7 deaths, SMR = 1.68, 95% C.I. 0.68-3.47) seemed due to pneumonia (4 deaths, SMR = 2.86, 95% C.I. 0.78-7.32). Venous thromboembolic complications were listed once in association with a risk situation, and one other death could be attributed to fatal pulmonary embolism. Cerebral hemorrhages were listed three times. It could not be verified whether these hemorrhages were related to anticoagulant therapy; the frequency was slightly higher than the expected population figure (SMR = 1.49, 95% C.I. 0.31-4.36). The mean age of death for all causes was 64 years; the two fatal thromboembolic episodes occurred at age 20 and 30 years. The data show that antithrombin deficiency is associated with a normal survival and a low risk of fatal thromboembolic events. The use of long-term anticoagulant treatment in asymptomatic individuals should be considered carefully in view of the greater risk of fatal bleeding associated with long-term anticoagulant prophylaxis.

Factor V Leiden (FV R506Q) in families with inherited antithrombin deficiency.

We investigated the presence of the gene mutation of factor V, FV R506Q or factor V Leiden, responsible for activated protein C resistance, in DNA samples of 127 probands and 188 relatives from 128 families with antithrombin deficiency. The factor V mutation was identified in 18 families. Nine families were available to assess the mode of inheritance and the clinical relevance of combined defects. The factor V and antithrombin genes both map to chromosome 1. Segregation of the defects on opposite chromosomes was observed in three families. Co-segregation with both defects on the same chromosome was demonstrated in four families. In one family a de novo mutation of the antithrombin gene and in another a crossing-over event were the most likely explanations for the observed inheritance patterns. In six families with type I or II antithrombin deficiency (reactive site or pleiotropic effect), 11 of the 12 individuals with both antithrombin deficiency and the factor V mutation developed thrombosis. The median age of their first thrombotic episode was 16 years (range 0-19); this is low compared with a median age of onset of 26 years (range 20-49) in 15 of 30 carriers with only a defect in the antithrombin gene. One of five subjects with only factor V mutation experienced thrombosis at 40 years of age. In three families with type II heparin binding site deficiencies, two of six subjects with combined defects experienced thrombosis; one was homozygous for the heparin binding defect. Our results show that, when thrombosis occurs at a young age in antithrombin deficiency, the factor V mutation is a likely additional risk factor. Co-segregation of mutations in the antithrombin and factor V genes provides a molecular explanation for severe thrombosis in several generations. The findings support that combinations of genetic risk factors underly differences in thrombotic risk in families with thrombophilia.

No efficacy of annual gynaecological screening in BRCA1/2 mutation carriers; an observational follow-up study.

BRCA1/2 mutation carriers are offered gynaecological screening with the intention to reduce mortality by detecting ovarian cancer at an early stage. We examined compliance and efficacy of gynaecological screening in BRCA1/2 mutation carriers. In this multicentre, observational, follow-up study we examined medical record data of a consecutive series of 888 BRCA1/2 mutation carriers who started annual screening with transvaginal ultrasonography and serum CA125 between 1993 and 2005. The women were annually screened for 75% of their total period of follow-up. Compliance decreased with longer follow-up. Five of the 10 incident cancers were interval tumours, diagnosed in women with a normal screening result within 3-10 months before diagnosis. No difference in stage distribution between incident screen-detected and interval tumours was found. Eight of the 10 incident cancers were stage III/IV (80%). Cancers diagnosed in unscreened family members had a similar stage distribution (77% in stage III/IV). The observed number of cases detected during screening was not significantly higher than expected (Standardized Incidence Ratio (SIR): 1.5, 95% confidence interval: 0.7-2.8). For the subgroup that was fully compliant to annual screening, a similar SIR was found (1.6, 95% confidence interval: 0.5-3.6). Despite annual gynaecological screening, a high proportion of ovarian cancers in BRCA1/2 carriers are interval cancers and the large majority of all cancers are diagnosed in advanced stages. Therefore, it is unlikely that annual screening will reduce mortality from ovarian cancer in BRCA1/2 mutation carriers.

Gene-gene and gene-environment interactions determine risk of thrombosis in families with inherited antithrombin deficiency.

To analyze inherited antithrombin deficiency as a risk factor for venous thromboembolism in various conditions with regard to the presence or absence of additional genetic or acquired risk factors, we compared 48 antithrombin-deficient individuals with 44 nondeficient individuals of 14 selected families with inherited antithrombin deficiency. The incidence of venous thromboembolism for antithrombin deficient individuals was 20 times higher than among nondeficient individuals (1.1% v 0.05% per year). At the age of 50 years, greater than 50% of antithrombin-deficient individuals had experienced thrombosis compared with 5% of nondeficient individuals. Additional genetic risk factors, Factor V Leiden and PT20210A, were found in more than half of these selected families. The effect of exposure to 2 genetic defects was a 5-fold increased incidence (4.6% per year; 95% confidence interval [CI], 1.9% to 11.1%). Acquired risk factors were often present, determining the onset of thrombosis. The incidence among those with exposure to antithrombin deficiency and an acquired risk factor was increased 20-fold (20.3% per year; 95% CI, 12.0% to 34.3%). In conclusion, in these thrombophilia families, the genetic and environmental factors interact to bring about venous thrombosis. Inherited antithrombin deficiency proves to be a prominent risk factor for venous thromboembolism. The increased risks among those with exposure to acquired risk factors should be considered and adequate prophylactic anticoagulant therapy in high-risk situations seems indicated in selected families with inherited antithrombin deficiency.

A longitudinal cross-over study of serum cholesterol and lipoproteins in rabbits fed on semi-purified diets containing either casein or soya-bean protein.

1. Two groups, each of six rabbits, were fed on semi-purified diets containing either 400 g casein or 400 g soya-bean protein/kg for 20 d and then the diets of the two groups were crossed-over. 2. Just before the cross-over, the serum cholesterol concentration (mean +/- SE) was 3068 +/- 592 and 800 +/- 143 mg/l for the groups fed on casein and soya-bean protein respectively. 3. Changes in the serum cholesterol concentration were observed 1 d after crossing-over the diets. By 10 d, the cholesterol levels in the two groups had also crossed-over. 4. The changes in serum cholesterol level after the cross-over were reflected in the very-low-density lipoproteins ( VLDL) and low-density lipoproteins (LDL). 5. Lipoprotein protein concentrations in the LDL changed in the same way as cholesterol. In the VLDL however, the protein concentration decreased in both groups after the change in diet. 6. The cholesterol: protein values for the LDL and VLDL markedly increased in the rabbits changed from the soya-bean-protein diet to the casein diet, reaching a maximum 2 d after the cross-over. In the animals switched from casein to soya-bean protein, the values progressively declined. 7. The source of dietary protein exerts a rapid effect on the composition of both the VLDL and LDL which is proposed to be attributed to changes in the number and size of lipoprotein particles.

Hereditary antithrombin deficiency: heterogeneity of the molecular basis and mortality in Dutch families.

We studied the molecular basis and genetic heterogeneity of hereditary antithrombin (III) deficiency in nine Dutch families. Polymerase chain reaction (PCR) amplification and direct sequencing of all antithrombin gene exons and flanking intronic regions identified mutations in eight families. Given the opportunity to correlate the molecular basis with survival, we addressed the relevance of molecular defects to mortality in inherited antithrombin deficiency. The defects included single nucleotide deletions (7671 del G, 7768-69 del G) and insertions (5501 ins A, 2463 G-->TC) that lead to frameshifts, a single base substitution [5381 C-->T (129Arg-->stop)] leading to a premature termination codon, and single base substitutions resulting in amino acid substitutions [2652 A-->C (63Tyr-->Ser), 13380 T-->C (421Ile-->Thr), and 13407 G-->T (430Cys-->Phe)]. All affected individuals were heterozygous for the defects. Previously we found in Dutch families that antithrombin deficiency did not lead to higher mortality compared with the general population. In accordance with these findings, we observed no excess mortality in the nine families [Observed:Expected, 52:52.6; standardised mortality ratio (SMR) 1.0, 95% confidence interval (CI), 0.7-1.3]. Our findings confirmed a considerable genetic heterogeneity underlying antithrombin deficiency. We therefore concluded that the lack of excess mortality in these families is not caused by a Dutch mild defect. We suggest that the longevity is not affected by molecular defects in the antithrombin gene and hypothesize that differences in mortality or natural history between families most likely result from other (genetic) risk factors.