Are Clinically Insignificant Prostate Cancers Really Insignificant among Korean Men?

Purpose

We aimed to determine whether 12 core-extended biopsies of the prostate could predict insignificant prostate cancer (IPCa) in Koreans reliably enough to recommend active surveillance.

Materials and Methods

Two hundred and ninety-seven patients who underwent radical prostatectomy after 12 core-extended prostate biopsies were retrospectively reviewed. 38 cases (12.8%) were shown to be IPCa.

Results

The average age was 65.2 years, serum PSA was 5.49 ng/dL, and the PSA density was 0.11. The Gleason scores (GS) were 6 (3+3) in 31, 5 (3+2) in 4, and 4 (2+2) in 3. After radical prostatectomy, higher GS was given in 16 (42.1%), whereas lower GS was given in 1 case (2.6%), as compared with the GS obtained from biopsy. 11 (28.9%) had GS of 7 (3+4) and 5 (13.2%) had GS of 7 (4+3). 6 in GS 7 (4+3) and 1 in GS 7 (3+4) showed prostate capsule invasion and 1 in GS 7 (4+3) had seminal vesicle invasion. Prostate capsule invasion was observed in 1 with GS 6 (3+3). The rate of inaccuracy of the contemporary Epstein criteria was 42.1%. Only PSA density was a reliable indicator of clinically IPCa (odds ratio=1.384, 95% CI, 1.103 to 2.091).

Conclusion

Diagnosis of IPCa from a prostate biopsy underestimated the true nature of prostate cancer in as many as 42.1% of Koreans.     

Incidentally detected prostate cancer in cystoprostatectomies: pathological and morphometric comparison with clinically detected cancer in totally embedded specimens

There are limited data regarding the pathological features of incidentally detected prostate cancer. Examination of cystoprostatectomy specimens obtained during bladder cancer treatment affords a unique opportunity to examine incidentally detected prostate cancer and determine its relationship with clinically detected prostate cancer obtained during radical prostatectomy. We compared the pathological findings of incidentally detected prostate cancer in 132 consecutive cystoprostatectomy specimens from patients treated for bladder cancer with a consecutive series of 228 radical prostatectomy specimens from patients treated for prostate cancer. All specimens were totally embedded and whole-mounted. Karyometry was evaluated in select subsets of patients from the study groups. Incidentally detected cancer was found in 42% of cystoprostatectomy specimens, and the cancers were of lower Gleason score and lower pathological stage with fewer positive surgical margins than in clinically detected cancers in age-matched radical prostatectomies. High-grade prostatic intraepithelial neoplasia (PIN) was present in 82% of radical prostatectomy specimens, in 70% of cystoprostatectomies with incidentally detected prostate cancer, and in 54% of cystoprostatectomies without prostate cancer. Mean nuclear and nucleolar area was lower in incidentally detected cancer and PIN when compared with clinically detected cancer and PIN, respectively, similar to the results with proliferative indices. We conclude that incidentally detected cancer is less aggressive than clinically detected cancer.     

腹腔镜下精准解剖定位行前列腺癌或膀胱癌根治术降低术后苏醒延迟发生风险

目的 探讨腹腔镜下前列腺癌或膀胱癌根治术术后苏醒延迟的危险因素,为临床早期防治提供信息.方法 选择2016年9月～2019年1月期间于南通市第二人民医院行腹腔镜下前列腺癌或膀胱癌根治术治疗的327例患者作为观察对象,对患者的临床资料、手术资料进行收集,统计术后苏醒延迟发生率,经Logistic回归分析法分析苏醒延迟的危...     

Bladder cancer tissue-derived exosomes suppress ferroptosis of T24 bladder cancer cells by transporting miR-217

It has been reported that miR-217 can inhibit the oncogenic activity and progression of bladder cancer (BCa) cells, but it has not been explored whether miR-217 is involved in the regulation of ferroptosis. In the present study, RNA transfection, real-time PCR, flow cytometry, Western blotting assays, immunofluorescence and ELISA were performed to explore the effects and mechanisms of miR-217 in BCa tissue-derived exosomes. We found that extracellular fluid from bladder cancer tissue promoted the growth and miR-217 expression of T24 cells and inhibited ferroptosis. MiR-217 was confirmed to inhibit ferroptosis in bladder cancer cells by RNA interference and functional assays. By cell membrane fluorescence probe (CM-Dil) labeling, inhibiting exosome secretion by GW4689 and exosome extraction, we determined that BCa tissue-derived exosomes transport miR-217 into T24 cells. Culture of T24 cells with extracellular fluid after RNA interference showed that exosomes carrying miR-217 derived from BCa tissues inhibited ferroptosis of T24 cells. We conclude that bladder cancer tissue-derived exosomes inhibit ferroptosis of T24 bladder cancer cells by transporting miR-217. The results of our study provide a new insight into the progression of bladder cancer.     

Molecular evidence that invasive adenocarcinoma can mimic prostatic intraepithelial neoplasia (PIN) and intraductal carcinoma through retrograde glandular colonization

Prostate cancer often manifests as morphologically distinct tumour foci and is frequently found adjacent to presumed precursor lesions such as high‐grade prostatic intraepithelial neoplasia (HGPIN). While there is some evidence to suggest that these lesions can be related and exist on a pathological and morphological continuum, the precise clonal and temporal relationships between precursor lesions and invasive cancers within individual tumours remain undefined. Here, we used molecular genetic, cytogenetic, and histological analyses to delineate clonal, temporal, and spatial relationships between HGPIN and cancer lesions with distinct morphological and molecular features. First, while confirming the previous finding that a substantial fraction of HGPIN lesions associated with ERG‐positive cancers share rearrangements and overexpression of ERG, we found that a significant subset of such HGPIN glands exhibit only partial positivity for ERG. This suggests that such ERG‐positive HGPIN cells either rapidly invade to form adenocarcinoma or represent cancer cells that have partially invaded the ductal and acinar space in a retrograde manner. To clarify these possibilities, we used ERG expression status and TMPRSS2–ERG genomic breakpoints as markers of clonality, and PTEN deletion status to track temporal evolution of clonally related lesions. We confirmed that morphologically distinct HGPIN and nearby invasive cancer lesions are clonally related. Further, we found that a significant fraction of ERG‐positive, PTEN‐negative HGPIN and intraductal carcinoma (IDC‐P) lesions are most likely clonally derived from adjacent PTEN‐negative adenocarcinomas, indicating that such PTEN‐negative HGPIN and IDC‐P lesions arise from, rather than give rise to, the nearby invasive adenocarcinoma. These data suggest that invasive adenocarcinoma can morphologically mimic HGPIN through retrograde colonization of benign glands with cancer cells. Similar clonal relationships were also seen for intraductal carcinoma adjacent to invasive adenocarcinoma. These findings represent a potentially undervalued indicator of pre‐existing invasive prostate cancer and have significant implications for prostate cancer diagnosis and risk stratification. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Benign glandular inclusion in obturator lymph node of a man treated for prostate carcinoma

Benign glandular inclusions in lymph nodes are extremely rare in men. Their identification is essential because it changes dramatically the prognosis and therapy of neoplasms. Described herein is the first case of benign glandular inclusion in an obturator lymph node dissected during a radical prostatectomy for treatment of prostate adenocarcinoma. A 60-year-old man underwent radical prostatectomy and obturator-hypogastric lymph node dissection for treatment of prostate adenocarcinoma. Benign glandular inclusion was found in microscopic examination. The lesion was characterized by two glandular spaces lined by a single, cuboid, benign epithelium localized in the sinus of one of four dissected lymph nodes. Immunohistochemistry showed mesothelial differentiation. Pathologists should be aware of benign glandular inclusion in obturator lymph nodes dissected during a radical prostatectomy for treatment of prostate cancer in order to avoid the incorrect diagnosis of metastatic disease.     

Clinicopathologic and prognostic significance of p21 (Cip1/Waf1) expression in bladder cancer

Recent studies have shown that altered expression p21 is shown to associate with tumorigenesis and tumor progression. To investigate the clinicopathological significance and prognostic value of p21 in bladder cancer (BCa). A total of 48 patients with BCa were included in this study. The correlation between p21 expression and clinicopathologic features and survival was studied. Also, a meta-analysis was performed to investigate the relationship between the p21 and BCa survival. Low p21 expression was detected both in tumor tissues compared with adjacent normal tissues. The expression of p21 was closely associated with advanced pathologic TNM stage (P = 0.001) and tumor grade (P = 0.013). Moreover, patients with low p21 expression had shorter recurrence-free survival (P = 0.016) and overall survival rates (P = 0.039). Multivariate Cox regression analysis revealed that p21 low expression was an independent prognostic factor for recurrence free survival (P = 0.03). Additionally, our meta-analysis. The available outcome data from six articles were examined. A meta-analysis of the HR indicated a significantly poor overall survival (OS, HR: 1.75, 95% CI: 1.38-2.21), recurrence free survival (RFS, HR: 1.83, 95% CI: 1.57-2.15), progression free survival (PFS, HR: 2.02, 95% CI: 1.48-2.75), and cancer specific survival (CSS, HR: 1.89, 95% CI: 1.53-2.33) in patients with low expression levels of p21. Our present results indicated that low p21 expression predicated tumor recurrence and poor prognosis in bladder cancer.     

PTEN loss in intraductal carcinoma of the prostate has low incidence in Japanese patients

Clinical and genomic features of prostate cancer (PCa) vary considerably between Asian and Western populations. PTEN loss is the most frequent abnormality in intraductal carcinoma of the prostate (IDC-P) in Western populations. However, its prevalence and significance in Asian populations have not yet been well studied. In the present study, we evaluated PTEN expression in IDC-P in a Japanese population and its association with ERG expression. This study included 45 and 59 patients with PCa with and without IDC-P, respectively, who underwent radical prostatectomy. PTEN loss was observed in 10 patients with PCa with IDC-P (22%) and nine patients with PCa without IDC-P (17%). ERG expression was relatively frequent in patients with PCa with PTEN loss, although a significant difference was not observed. The co-occurrence of PTEN loss and ERG expression was observed in four patients with PCa with IDC-P and one without IDC-P. PTEN loss and ERG expression did not affect progression-free survival, regardless of the presence of IDC-P. The frequency of PTEN loss in IDC-P is lower in Asian patients than in Western patients. Our results indicate that mechanisms underlying IDC-P in Asian populations are different from those of Western populations.     

APC mutational patterns in gastric adenocarcinoma are enriched for missense variants with associated decreased survival

Adenomatous polyposis coli (APC) mutations are causally associated with familial adenomatous polyposis (FAP) and are recurrent somatic events across numerous tumor types, including gastric adenocarcinoma. Severity of disease in FAP correlates with specific APC mutations, but the impact of given mutations on phenotype in gastric cancer is not well studied. Sequencing data from the Genomic Data Commons (GDC) demonstrate an APC mutational pattern in gastric cancer that differs dramatically from that seen in colon cancer. Exome sequencing data from APC‐mutant colon and gastric adenocarcinomas in GDC was filtered for single nucleotide variants (SNVs) using MuTect2 Variant Aggregation and Masking pipeline, Somatic Aggregation Workflow. APC mutations were found in 57/441 gastric (12.9%) and 309/433 colon adenocarcinomas (71.4%). There was a significant difference in the proportion of stopgain, frameshift, and missense mutations between tumor types(P < .00001). Colon tumors were predominated by frameshift and stopgains, comprising 47.7% and 35.7%, respectively. In contrast, 47.1% of gastric mutations were missense. Gastric tumors harboring missense mutations showed decreased overall survival relative to other mutational subtypes(P = .008). In the gastric samples, 25.9% of frameshift and stopgain mutations are in the 3′ portion of the gene, compared to 1.4% of colon samples. APC mutations demonstrate different distributions in gastric and colon adenocarcinoma, with a shift toward missense variants in gastric tumors and worse survival in gastric tumors harboring them. As different mutations confer variable degrees of protein dysfunction and resultant clinical manifestation, expanded investigation of specific mutational patterns will prove integral to future‐risk stratification strategies.     

Clonality analysis of multifocal papillary thyroid carcinoma by using genetic profiles

Papillary thyroid carcinoma (PTC) is the most common adult thyroid malignancy and often presents with multiple anatomically distinct foci within the thyroid, known as multifocal papillary thyroid carcinoma (MPTC). The widespread application of the next‐generation sequencing technologies in cancer genomics research provides novel insights into determining the clonal relationship between multiple tumours within the same thyroid gland. For eight MPTC patients, we performed whole‐exome sequencing and targeted region sequencing to identify the non‐synonymous point mutations and gene rearrangements of distinct and spatially separated tumour foci. Among these eight MPTCs, completely discordant mutational spectra were observed in the distinct cancerous nodules of patients MPTC1 and 5, suggesting that these nodules originated from independent precursors. In another three cases (MPTC2, 6, and 8), the distinct MPTC foci of these patients had no other shared mutations except BRAF V600E, also indicating likely independent origins. Two patients (MPTC3 and 4) shared almost identical mutational spectra amongst their separate tumour nodules, suggesting a common clonal origin. MPTC patient 7 had seven cancer foci, of which two foci shared 66.7% of mutations, while the remaining cancer foci displayed no common non‐synonymous mutations, indicating that MPTC7 has multiple independent origins accompanied by intraglandular disease dissemination. In this study, we found that 75% of MPTC cases arose as independent tumours, which supports the field cancerization hypothesis describing multiple malignant lesions. MPTC may also arise from intrathyroidal metastases from a single malignant clone, as well as multiple independent origins accompanied by intrathyroidal metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

High-level cytoplasmic cyclin D1 expression in lymph node metastases from prostate cancer independently predicts early biochemical failure and death in surgically treated patients

Fleischmann A, Rocha C, Saxer‐Sekulic N, Zlobec I, Sauter G & Thalmann G N
(2011) Histopathology 58 , 781–789
High‐level cytoplasmic cyclin D1 expression in lymph node metastases from prostate cancer independently predicts early biochemical failure and death in surgically treated patients Aims: To test the prognostic significance of cyclin D1 in nodal‐positive prostate cancer. Methods and results: Nuclear and cytoplasmic cyclin D1 expression was evaluated in 119 nodal‐positive prostate cancer patients undergoing radical prostatectomy and extended lymphadenectomy. Cyclin D1 was correlated with various tumour features and biochemical recurrence‐free survival (bRFS), disease‐specific survival (DSS) and overall survival (OS). In the metastases, high‐level cytoplasmic cyclin D1 expression independently predicted poor outcome (5‐year bRFS, 12.5% versus 26.4%, P = 0.006; 5‐year DSS, 56.3% versus 80.7%, P = 0.007; 5‐year OS, 56.3% versus 78.7%, P = 0.011). These patients had a 2.62‐fold elevated risk of dying from prostate cancer as compared with patients with low‐level cytoplasmic cyclin D1 expression (P = 0.024). All other subcellular compartments of cyclin D1 expression in primary tumours and metastases were prognostically non‐significant. Conclusions: The subcellular location of cyclin D1 expression in prostate cancer is linked to specific clinical courses. Survival stratification according to biomarker expression in metastases indicates an important role for tumour sampling from these tissues.     

Cancer/testis antigen SPATA19 is frequently expressed in benign prostatic hyperplasia and prostate cancer

Spermatogenesis‐associated 19 (SPATA19) is a cancer/testis antigen overexpressed in various cancers. However, its protein expression profile in malignant or non‐malignant tissues remains unknown. Thus, in this study, we investigated SPATA19 protein expression patterns in a panel of non‐malignant human samples and primary prostate cancer (PCa) with or without benign prostatic hyperplasia (BPH) tissues. SPATA19 was absent in all non‐malignant tissues investigated (n=14) except testis and prostate tissues. In terms of malignancies, all PCa cases were positive for SPATA19 exhibiting frequency between 20 and 100% (median 85%) with 63 (52.5%) and 57 (47.5%) cases demonstrating weak/moderate and strong intensities, respectively. Thirty‐nine PCa cases (32.5%) contained BPH, and all BPH glands were SPATA19 positive (frequency between 20 and 100%; median 90%) with 13 (33.3%) demonstrating strong SPATA19 expression. Higher SPATA19 expression (higher frequency, intensity, or H‐score) was not associated with overall survival or disease‐specific survival (DFS) in all PCa cases. However, biochemical recurrence (BR) was associated with worse DFS (p = 0.005) in this cohort of 120 patients, and cases with strong SPATA19 intensity were associated with BR (p = 0.020). In conclusion, we showed that SPATA19 protein was frequently expressed in both BPH and PCa glands, and this warrants future investigations on its pathogenic roles in the disease.     

The Change of Prostate Cancer Treatment in Korea: 5 Year Analysis of a Single Institution

Purpose

We analyzed changes to the trends of prostate cancer management and patient characteristics at a single institute during 5 years due to the significant increase of the prostate cancer incidence and the robotic equipment in Korea.

Materials and Methods

Prostate cancer patient data from a single institute recorded from 2006 to 2010 were analyzed. Cancer stage, initial treatment modalities, and the pattern of outpatient clinical management were reviewed.

Results

Between 2006 and 2010, 386 prostate cancers were newly diagnosed at the institute. The proportion of localized and locally advanced cancer cases increased from 67% in 2006 to 79% in 2010 respectively. Among the treatment choices during follow-up in the out-patients clinic, the proportion of radical prostatectomies increased from 43% in 2006 to 62% in 2010. In contrast, the proportion of hormone therapies decreased from 58% to 37%. For initial treatment choice, radical prostatectomy was chosen for 59% of the patients who were newly diagnosed with cancer during the study period. However, hormone therapy alone was administrated as a primary therapy to 26%. Analysis of the radical prostatectomy subgroup showed that a robot-assisted technique was used in 83% of the patients, and the remaining 17% underwent an open radical prostatectomy.

Conclusion

As the prostate cancer incidence increased in Korea, the proportion of localized and locally advanced cancer also increased. In addition, the main treatment modality changed from non-surgical treatment to radical prostatectomy.     

Multiregion human bladder cancer sequencing reveals tumour evolution,bladder cancer phenotypes and implications for targeted therapy

We present an evolutionary analysis of the relative time of genetic events underlying tumorigenesis in human bladder cancers from 10 whole cystectomy specimens using multiregional whole-exome sequencing. We timed bladder cancer drivers, mutational signatures, ploidy and copy number alterations, provided evidence for kataegis and correlated alterations with tumour areas and histological phenotypes. We found that: (1) heterogeneous tumour areas/phenotypes had distinct driver mutations, (2) papillary-invasive tumours divided early into two parallel evolving branches and (3) parallel evolution of subclonal driver mutations occurred. APOBEC mutational signatures were found to be very early events, active in carcinoma in situ, and often remained a dominant source of mutations throughout tumour evolution. Genetic progression from carcinoma in situ followed driver mutations in NA13/FAT1, ZBTB7B or EP300/USP28/KMT2D. Our results point towards a more diverse mutational trajectory of bladder tumorigenesis and underpin the importance of timing of mutational processes and clonal architecture in bladder cancer as important aspects for successful prognostication and therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

偶发性前列腺癌5例临床病理分析

目的探讨偶发性前列腺腺癌的发生率、临床及病理特点。方法收集40例复旦大学附属肿瘤医院因膀胱癌而行膀胱前列腺根治切除标本中偶然发现的前列腺腺癌5例,观察并分析其临床表现、病理学特点。结果临床表现:40例膀胱前列腺根治切除标本中有5例合并前列腺腺癌,发生率为12.5%(5/40)。患者平均年龄66岁(63～70岁)。4例表现为肉眼血尿,1例为镜下血尿。术前血PSA水平仅1例为6.55 ng/ml(参考值4.0 ng/ml),另外4例PSA水平均<4.0 ng/ml。病理检查:5例均为腺泡源性腺癌,4例Gleason分级评分均为6(3+3),1例Gleason分级评分为7(4+3)。临床病理TNM分期:3例为T1a,2例为T1b。结论前列腺癌可与尿路上皮癌合并发生,往往血PSA水平正常或稍高于正常。Gleason评分较低,癌灶常较局限,如取材不充分可能会漏诊。     

Collagenous micronodules in prostate cancer revisited: are they solely associated with Gleason pattern 3 adenocarcinomas?

Collagenous micronodules (CMs) are microscopic stromal nodular eosinophilic fibrillar collagen deposition of uncertain histogenesis seen in prostatic adenocarcinoma. Per the 2005 International Society of Urologic Pathology (ISUP) consensus conference, they are categorized as Gleason pattern 3. This study analyzes morphological and clinical features of CMs from a large series of radical prostatectomies. Hematoxylin and eosin stained slides for 129 radical prostatectomies for adenocarcinoma of prostate with CMs and for 93 prostatic adenocarcinoma cases without CMs as comparison were examined out of a total of 667 cases performed from January 2010 to December 2011 at Houston Methodist Hospital. CMs were identified in 19% of all radical prostatectomies (129/667 cases). Almost all tumors with CMs were located in the peripheral zone (98%) as single or multiple foci of prostatic cancer glands. The vast majority of cases (96%) were identified in association with mucinous secretion. A cribriform Gleason pattern 4 was associated in 86 cases (67%). The CMs were associated with glomerulation (42%) and amphophilic luminal secretion (59%). 88 cases (68%) showed tumor foci with Gleason pattern ≥ 4 in close association with CMs. Multivariate analysis revealed CMs of the prostatic adenocarcinoma are closely related to mucinous secretion, cribriform growth pattern, and Gleason pattern 4. This study suggests that CMs are more frequently associated with Gleason pattern 4 cancer warranting morphologic reappraisal of CMs, rather than the consensus assignment of Gleason pattern 3.     

Assessment of circulating tumor cells (CTCs) in prostate cancer patients with low‐volume tumors

The objective of this study was to assess the incidence of circulating tumor cells (CTCs) in prostate cancer patients with low‐volume tumors (less than 0.5 cc) after radical prostatectomy (RP). Blood samples were collected from 64 RP patients to assess the incidence of CTCs following RP. The specimens were processed by whole‐mount section. Clinicopathological data (e.g. patient age, race, specimen weight, tumor volume, grade, stage and surgical margin status) and follow‐up PSA data were compared to CTC status. Of the 64 RP patients, nine had ‘low‐volume prostate cancer’. Seven of these patients had detectable levels of CTCs. In two of the seven patients with detectable CTCs, PSA elevation was also observed. Isolation and detection of circulating epithelial cells is possible in low‐volume prostate cancer patients. In the setting of low‐volume prostate cancer, CTCs may be associated with the presence of detectable PSA levels. However, the detection of CTCs did not predict PSA failure.     

Role of MIF1/MIF2/CD74 interactions in bladder cancer

Macrophage migration inhibitory factor (MIF1) is a pleiotropic cytokine involved in inflammation and cancer. Genetic knockout of Mif1 in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) model of bladder cancer (BCa) resulted in stage arrest at non-muscle-invasive disease in prior studies. Small-molecule inhibition of MIF1 reduced cancer-associated outcomes, but it did not fully recapitulate genetic models. D-dopachrome tautomerase (gene symbol DDT), commonly referred to as MIF2, is a functional homolog of MIF1, and both MIF1 and MIF2 can bind the cell surface receptor CD74 on multiple cell types to initiate a signaling cascade. It has been proposed that this interaction mediates part of the protumorigenic effects of MIF1 and MIF2 and may explain the discordance in prior studies. We hypothesized that MIF2 functions redundantly with MIF1 in BCa development and progression. The Cancer Genome Atlas (TCGA) analysis indicated MIF and DDT expression were increased in BCa patients compared to control. 4-Iodopyridine (4-IPP), a combined MIF1/MIF2 inhibitor, was more efficacious than ISO-1, a MIF1-only inhibitor, in preventing cellular proliferation in BCa cell lines. To evaluate these findings in vivo, wild-type (WT) and Mif1^−/− animals were exposed to 0.05% BBN in drinking water for 16 weeks to initiate tumorigenesis and then evaluated over the subsequent 4 weeks for tumor formation and progression in the presence or absence of 4-IPP. 4-IPP reduced bladder weights in WT animals and bladder weights/tumor stage in Mif1^−/− animals. To determine whether MIF1/MIF2 functioned through CD74 in BCa, WT or Cd74^−/− animals were used in the same BBN model. Although these animals were partially protected against BBN-induced BCa, 4-IPP did not enhance this effect. In conclusion, our data suggest that MIF2 mechanistically functions in a similar protumorigenic manner to MIF1, and this is at least partially through CD74. Dual inhibition of MIF homologs is more efficacious at reducing tumor burden in this model of BCa. © 2022 The Pathological Society of Great Britain and Ireland.