Frequency of subclinical interstitial lung disease in COVID-19 autopsy cases: potential risk factors of severe pneumonia.

Risk factors of severe coronavirus disease 2019 (COVID-19) have been previously reported; however, histological risk factors have not been defined thus far. The aim of this study was to clarify subclinical hidden interstitial lung disease (ILD) as a risk factor of severe pneumonia associated with COVID-19. We carefully examined autopsied lungs and chest computed tomography scanning (CT) images from patients with COVID-19 for interstitial lesions and then analyzed their relationship with disease severity. Among the autopsy series, subclinical ILD was found in 13/27 cases (48%) in the COVID-19 group, and in contrast, 8/65 (12%) in the control autopsy group (p = 0.0006; Fisher's exact test). We reviewed CT images from the COVID-19 autopsy cases and verified that subclinical ILD was histologically detectable in the CT images. Then, we retrospectively examined CT images from another series of COVID-19 cases in the Yokohama, Japan area between February-August 2020 for interstitial lesions and analyzed the relationship to the severity of COVID-19 pneumonia. Interstitial lesion was more frequently found in the group with the moderate II/severe disease than in the moderate I/mild disease (severity was evaluated according to the COVID-19 severity classification system of the Ministry of Health, Labor, and Welfare [Japan]) (moderate II/severe, 11/15, 73.3% versus moderate I/mild, 108/245, 44.1%; Fisher exact test, p = 0.0333). In conclusion, it was suggested that subclinical ILD could be an important risk factor for severe COVID-19 pneumonia. A benefit of these findings could be the development of a risk assessment system using high resolution CT images for fatal COVID-19 pneumonia.

High prevalence of upper lung field pulmonary fibrosis radiologically consistent with pleuroparenchymal fibroelastosis in patients with round atelectasis.

BACKGROUND: Upper-lung field pulmonary fibrosis (upper-PF), radiologically consistent with pleuroparenchymal fibroelastosis (PPFE), was reported to develop in patients with a history of asbestos exposure and tuberculous pleurisy, indicating that chronic pleuritis is correlated with upper-PF development. Round atelectasis reportedly emerges after chronic pleuritis. This study aimed to clarify the association between round atelectasis and upper-PF. METHODS: We examined the radiological reports of all consecutive patients with round atelectasis between 2006 and 2018 and investigated the incidence of upper-PF development. RESULTS: Among 85 patients with round atelectasis, 21 patients (24.7%) were confirmed to finally develop upper-PF lesions. Upper-PF was diagnosed after round atelectasis recognition in more than half of the patients (13/21, 61.9%), whereas upper-PF and round atelectasis were simultaneously detected in the remaining 8 patients. At the time of round atelectasis detection, almost all patients (19/21, 90.5%) had diffuse pleural thickening and round atelectasis was commonly observed in non-upper lobes of 19 patients (90.5%). Fourteen patients had round atelectasis in unilateral lung, and the remaining 7 patients had round atelectasis in bilateral lungs. Among all 14 patients with unilateral round atelectasis, upper-PF developed on the same (n = 11) or both sides (n = 3). Thus, upper-PF emerged on the same side where round atelectasis was present (14/14, 100%). The autopsy of one patient revealed a thickened parietal-visceral pleura suggestive of chronic pleuritis. Subpleural fibroelastosis was also observed. CONCLUSIONS: Upper-PF occasionally develops on the same side of round atelectasis. Upper-PF may develop as a sequela of chronic pleuritis.

A case of lung carcinoma with a unique biphasic feature: Implications for histogenesis of "fake mucoepidermoid carcinoma" developing in the peripheral lung.

We present a case of lung carcinoma with a unique biphasic feature. The patient was a 67-year-old male smoker with idiopathic pulmonary fibrosis (IPF). A subpleural tumor in the left lower lobe, embedded in fibrotic tissue, was resected. Histologically, the tumor consisted of major and minor components of mucoepidermoid carcinoma (MEC) and surrounding conventional lepidic adenocarcinoma, respectively. Both components had the same TP53 somatic mutation (p.V157F) but not Mastermind-like 2 (MAML2) gene rearrangement. The two components may have developed from an identical origin. The tumor could be trans-differentiating from lepidic adenocarcinoma to MEC, possibly promoted by IPF-induced tissue damage. The final diagnosis was "adenosquamous carcinoma with mucoepidermoid-like features (that may originate from lepidic adenocarcinoma)." This case has implications for the potential histogenesis of peripheral lung MEC. Over time, the MEC would expand and outgrow the lepidic adenocarcinoma, making it impossible to distinguish between fake and true MEC. The present case suggests that peripheral MEC could differ from proximal MEC in its histogenesis and molecular genetics. Thus, careful examination is necessary to diagnose peripheral lung MEC, particularly in patients with interstitial lung diseases.

Mortality associated with the development of acute liver failure after a single dose of nivolumab.

AIM: Immune-related adverse events (irAEs) caused by immune checkpoint inhibitors are reported in all organs; however, the frequency of liver injury is low compared to irAEs in other organs. We describe a case of fulminant hepatitis after administration of the first dose of nivolumab for the management of esophageal cancer. METHODS: A man in his 80s was treated with nivolumab as a second-line therapy after his overall health worsened during preoperative chemotherapy for esophageal cancer. He was admitted to the hospital as an emergency case 30 days later with complaints of vomiting, following which acute liver failure was diagnosed. RESULTS: The patient developed hepatic encephalopathy on the third day after admission and died on the seventh day. The pathological results showed sub-extensive spread hepatocellular necrosis throughout the liver, and immunostaining confirmed the presence of CD8-positive cells, which is consistent with irAEs. CONCLUSIONS: Immune checkpoint inhibitors have proven to be effective for the treatment of malignant tumors, and although fatalities due to acute liver failure are extremely rare, such cases have been reported previously. Among the immune checkpoint inhibitors, anti-programmed death-1 receptor is associated with less hepatotoxicity. However, even a single dose of this treatment can cause acute liver failure, which could be fatal.

Biphenotypic Sinonasal Sarcoma: A Genetically Confirmed Case Showing Bone Invasion Accompanying a Non-neoplastic Respiratory Epithelium.

Biphenotypic sinonasal sarcoma is a newly established tumor entity that is associated with distinct clinicopathological findings. Biphenotypic sinonasal sarcoma is a rare, low-grade spindle cell sarcoma that arises in middle-aged females, exclusively in the sinonasal tract. A fusion gene involving PAX3 is detected in most biphenotypic sinonasal sarcomas, which aids in its diagnosis. Here, we report a case of biphenotypic sinonasal sarcoma with its cytological findings. The patient was a 73-year-old woman who presented with purulent nasal discharge and dull pain in the left cheek area. Computed tomography showed a mass extending from the left nasal cavity to the left ethmoid sinus, the left frontal sinus, and the frontal skull base. She underwent a combined transcranial and endoscopic approach for en bloc resection with a safety margin. Histologically, spindle-shaped tumor cells have been thought to proliferate mainly in the subepithelial stroma. Here, nasal mucosal epithelial hyperplasia was noted, and the tumor had invaded the bone tissue accompanying the epithelial cells. Fluorescence in situ hybridization (FISH) analysis showed a PAX3 rearrangement, and next-generation sequencing identified a PAX3::MAML3 fusion. Based on FISH, split signals were observed not in respiratory cells but in stromal cells. This indicated that respiratory cells were non-neoplastic. In the diagnosis of biphenotypic sinonasal sarcoma, the inverted growth of the respiratory epithelium can be a diagnostic pitfall. FISH analysis using a PAX3 break-apart probe is helpful not only for an accurate diagnosis but also for detecting the true neoplastic cells.

A distinctive cytomorphological feature of interstitial pneumonia-related lung adenocarcinoma: The potential issues and solutions in practical diagnosis.

BACKGROUND: The cytological features of interstitial pneumonia (IP)-related lung adenocarcinoma (LADC) have not been clearly described. This study aimed to describe its cytomorphological features, uncover potential problems in practical cytological diagnosis, and provide possible solutions. METHODS: Bronchial brushing cytology samples from 40 IP-related LADC cases (the IP group) and 110 control cases (LADC unrelated to IP; the non-IP group) were analyzed. All patients underwent surgery after brushing cytology, and their histopathological subtypes were determined. The authors reviewed the cytological features and focused particularly on cytoplasmic mucin production. RESULTS: In the IP group, neoplastic cells with cytoplasmic mucin were detected at a significantly higher frequency (44.4% [8 of 18] vs. 6.3% [4 of 64]), and most of them were invasive mucinous adenocarcinomas (IMAs). Twenty-two of the 40 LADC cases in the IP group failed to be judged as "malignant/positive" (thus, they were judged to be "equivocal and/or negative"). The frequency of equivocal and/or negative judgments was 55.0% (22 of 40) in the IP group and 41.8% (46 of 110) in the non-IP group. The cytological diagnosis of IMA was difficult because it showed only slight nuclear atypia. Therefore, the authors examined the immunocytochemical expression of hepatocyte nuclear factor 4α (HNF4α), a diagnostic marker for IMA. As a result, four of the six cases that were judged to be equivocal in the IP group showed positive signals and could be retrospectively judged as malignant/positive. CONCLUSIONS: The cytological diagnosis of IP-related LADC may be more difficult because of the larger proportion of IMA. Immunocytochemistry for HNF4α can be used to improve diagnostic confidence in IP-related LADC.

Rhabdomyosarcoma With FUS::TFCP2 Fusion in the Scalp: A Rare Case Report Depicting Round and Spindle cell Morphology.

Rhabdomyosarcoma (RMS) is a nonepithelial malignant tumor that differentiates into immature skeletal muscle. It is currently classified into 4 main subtypes according to the WHO classification. However, based on clinicopathological and molecular findings, there has been an increasing number of cases that do not fit into any of these subtypes. TFCP2-rearranged RMS is a rare tumor with characteristic clinicopathological findings including a preference for the craniofacial bones, a spindle and epithelioid histomorphology, and positive immunohistochemistry for epithelial markers, ALK, and myogenic markers. In this report, we describe a rare case of RMS with FUS::TFCP2 fusion in the scalp of a 58-year-old man. Histologically, the tumor showed a biphasic pattern, with solid proliferation of round cells in the superficial areas and of spindle cells in the deep areas. Immunohistochemically, tumor cells were positive for pan keratin, myogenic markers (desmin, MYOD1, and myogenin), and ALK. Additionally, fluorescence in situ hybridization using a break-apart FUS probe revealed FUS rearrangement. RMS with FUS::TFCP2 fusion was suspected, and the fusion gene was finally confirmed by target fusion sequencing. We believe that detailed histological, immunohistochemical, and genetic findings were important for the diagnosis. The unique traits of this tumor were the biphasic histological appearance consisting of round and spindle cells and development in the skin and soft tissue.

Earliest histopathological changes in COVID-19 pneumonia with comprehensive gene expression analyses: A case series study.

AIMS: In COVID-19 pneumonia, early detection and appropriate treatment are essential to prevent severe exacerbation. Therefore, it is important to understand the initiating events of COVID-19 pneumonia. However, at present, the literature about early stage disease has been very limited. Here, we investigated the earliest histopathological changes and gene expression profiles associated with COVID-19 pneumonia. METHODS AND RESULTS: We carefully examined 25 autopsied cases with different clinical courses. Dilation of capillaries and edematous thickening of the alveolar septa were found even in areas that macroscopically looked almost normal. Pneumocytes, histocytes/macrophages, and vascular endothelial cells were immunohistochemically positive for tissue factor, which is an important early responder to tissue injuries. Comprehensive gene expression analyses revealed that those lesions presented differential profiles compared to those of control lungs and were associated with a significant upregulation of the lysosomal pathway. CONCLUSIONS: Alveolar capillary dilation and edematous thickening may be the earliest histopathological change detected in COVID-19 pneumonia. Intensive investigations of such lesions may lead to an understanding of the initiating event of not only COVID-19 pneumonia but also of general diffuse alveolar damage.

Post-COVID-19 Granulomatous Inflammation in the Lung, Distinct From Sarcoidosis: A Report of Two Cases.

This report describes two cases of granulomatous lung inflammation following coronavirus disease 2019 (COVID-19), presenting as a sarcoidosis-like reaction with granuloma formation in airspaces and interstitium. Clinical and pathological findings in both cases were similar to but still distinct from sarcoidosis. In the first case, the chest CT of a 55-year-old male with a history of polymerase chain reaction (PCR)-confirmed COVID-19 showed well-defined multiple nodules in the bilateral lung fields. He underwent video-assisted thoracic surgery for diagnostic purposes. The pathological specimen showed loose non-caseous granulomas with mild lymphoplasmacytic infiltration and early fibroblastic proliferation in alveolar spaces. In the second case, a 68-year-old male, who presented with consolidation in the anterior segment of the right upper lobe, underwent bronchoscopy and transbronchial lung biopsy showed non-caseous granulomas with mild lymphoplasmacytic infiltration in the peribronchiolar interstitium. The opacities improved spontaneously in both cases. Further studies are needed to determine whether COVID-19 could cause granulomatous lung inflammation distinct from sarcoidosis.

Pathological criteria for multiplex gene-panel testing using next-generation sequencing in non-small cell lung cancer.

BACKGROUND: Multiplex gene-panel tests have recently been developed, including the Oncomine Dx Target Test multi-CDx system (ODxTT), and are commonly used to determine the adaptation of molecular-targeting drugs in non-small cell lung cancer. However, in actual clinical settings, we obtain false results owing to the small biopsy samples. We aimed to optimize tissue preparation methods to improve the success rate. PATIENTS AND METHODS: We investigated 88 biopsy samples. The area and nucleated cell count in the first cut section were quantified using a morphometric software. Pathological parameters, including "total tissue area" and "total nucleated cell count," were calculated by multiplying the total number of slides submitted to ODxTT. Optimal cutoff values to obtain the best success rate were also determined. Additionally, we morphometrically measured actual tumor cell proportions and attempted to determine the lower limit possible to detect mutations. RESULTS: Optimal cutoff values for "total nucleated cell count" and "total tissue area" were 132,885 and 32.94 mm2, respectively. The actual tumor cell proportions ranged from 4.6 to 97.7%. Even in cases with actual tumor cell proportions of less than 20% (ranging from 4.6 to 19.7%), there was no false negative. CONCLUSION: Thus, we proposed the pathological criteria for accurate ODxTT. Our result suggested that tumor cell proportions of less than 20% (around 5%) could be applicable for ODxTT. We hope that our results will help pathologists to choose between the multi-plex test (ODxTT) or single-plex test in routine diagnostics.

Rapid anticalcification treatment for glutaraldehyde-fixed autologous tissue in cardiovascular surgery.

BACKGROUND: Glutaraldehyde (GA)-fixed autologous tissues, including the pericardium, are widely used as patches and valve substitutes in cardiovascular surgery. However, GA treatment causes tissue calcification. No rapid anticalcification method has been established for use during surgery. Here, we aimed to establish a rapid anticalcification method using ethanol, as has already been demonstrated for bioprosthetic valves. METHODS: Thoracic aorta tissues were first fixed with GA for 3 min and then treated with ethanol for 0 (group 2), 10 (group 3), 20 (group 4), and 30 (group 5) min; untreated tissues (group 1) served as the control. The treated tissues were subdermally implanted into 3-week-old male Wistar rats and kept in place for 28 days. The calcification in each explant was semiquantitatively evaluated by annotating and measuring the area using virtual slides, and the data obtained were statistically analyzed. RESULTS: Semiquantitative analysis revealed that calcification of the implants from the untreated group (group 1; P = 0.0014) and groups 4 (P = 0.0014) and 5 (P = 0.0031) was significantly lower than that of implants from group 2. Moreover, implants from group 3 showed a tendency toward decreased calcification, although it was not significant (P = 0.0503). CONCLUSIONS: A rapid ethanol treatment prevents calcification of GA-fixed tissues in a rat model of subdermal implantation. This method may facilitate effective and rapid anticalcification of autologous tissues for use during cardiovascular surgery.

Significant accumulation of KRAS mutations in bronchiolar metaplasia-associated honeycomb lesions of interstitial pneumonia.

Interstitial pneumonia (IP) is a major risk factor for lung adenocarcinoma (LADC). IP-related LADC predominantly develops in the bronchiolar metaplasia lining in honeycomb lesions. Kirsten rat sarcoma virus (KRAS) is the most common oncogene mutated in IP-related LADC. The present study examined the metaplastic epithelia in honeycomb lesions for KRAS mutations using digital droplet polymerase chain reaction (ddPCR), a sensitive method used to detect infrequent mutations. Significantly higher KRAS mutation variant allele frequencies (VAFs) were detected in the metaplastic lung epithelia from 13 patients with IP compared with those in 46 non-lesioned lung samples from patients without IP (G12V, P=0.0004, G12C, P=0.0181, and G12A, P=0.0234; Mann Whitney U test). Multivariate analyses revealed that higher KRAS G12V (logistic regression model; P=0.0133, odds ratio=7.11) and G12C (P=0.0191, odds ratio=5.81) VAFs in patients with IP were independent of confounding variables, such as smoking and age. In patients with IP, metaplastic epithelia exhibited significantly higher KRAS G12V and G12C VAFs compared with the non-lesioned counterparts (paired t-test; G12V, P=0.0158, G12C, P=0.0465). These results suggested that IP could increase KRAS mutations and supported the hypothesis that bronchiolar metaplasia could be a precursor for IP-related LADC.

Implications of thyroid transcription factor-1 gene methylation in carcinogenesis of interstitial pneumonia-related non-terminal respiratory unit lung adenocarcinoma.

The present study aimed to elucidate the mechanisms underlying the histogenesis of interstitial pneumonia (IP)-related lung adenocarcinoma (LADC). We focused on the methylation of thyroid transcription factor 1 (TTF-1). The TTF-1 locus was highly methylated in IP-LADCs compared to non-IP-LADCs. Among the IP-LADCs, the non-terminal respiratory unit (TRU) LADCs showed marked hypermethylation in CpG sites in a particular intragenic region. This region was also found to be highly methylated in the IP lungs. The hierarchical dendrogram based on methylation levels divided the IP lungs into three different clusters. One of them showed a methylation profile similar to that of non-TRU LADCs. The non-TRU LADCs developed from this cluster with a significantly higher frequency. Moreover, bronchiolar metaplasia lining honeycomb/cystic lesions in IP lungs, IP-related non-TRU LADCs, and bronchiolar epithelia in healthy lungs were separately collected by microdissection and examined for methylation. Bronchiolar metaplasia showed hypermethylation, but bronchiolar epithelia did not. The methylation patterns in bronchiolar metaplasia were similar to those in non-TRU LADCs. In summary, a particular region of TTF-1 was highly methylated in IP-related non-TRU LADCs and bronchiolar metaplasia, supporting the theory that IP-related non-TRU LADCs may develop from bronchiolar metaplasia lining honeycomb/cystic lesions.

Case of inverted papillary urothelial carcinoma in the ureter.

INTRODUCTION: Urothelial neoplasms with a varus growth pattern are rare, and few urologists have encountered inverted urothelial carcinoma of the ureter. CASE PRESENTATION: An 82-year-old man was referred to our hospital for investigation of gross hematuria. Magnetic resonance imaging revealed nodules measuring 1-2 mm in diameter in the left upper ureter with slight reduction in signal intensity on diffusion-weighted imaging. Ureteroscopy showed a pedunculated smooth tumor that had the appearance of an inverted papilloma rather than the papillary shape typical of urothelial carcinoma. The tumor was biopsied and histopathological examination revealed a noninvasive, low-grade urothelial carcinoma with inverted multiple layers. Laparoscopic radical nephroureterectomy was subsequently performed, and a pedunculated tumor measuring 20 mm in diameter was found in the left upper ureter. The histopathological diagnosis was inverted papillary urothelial carcinoma, low-grade, pTa, pN0. CONCLUSION: This report provides the first clinical description of inverted papillary urothelial carcinoma of the ureter.

Prognostic impact of HNF4α expression in interstitial lung disease.

Pneumocyte injury is a crucial factor influencing the severity of interstitial lung disease (ILD). In this study, we investigated the potential of hepatocyte nuclear factor α (HNF4α) as an immunohistochemical marker to detect pneumocyte injury and as a prognostic marker. Surgical lung biopsy specimens were collected from 309 patients with different types of ILDs (61 idiopathic pulmonary fibrosis (IPF), 173 non-IPF, and 75 unclassifiable ILD). HNF4α expression were examined and the frequency of positive cells (per mm2 ) was calculated. HNF4α was strongly expressed in regenerating pneumocytes present on fibroblastic foci, Masson bodies/organizing alveoli. In the non-IPF and unclassifiable ILD groups, cases with high frequency expression showed significantly poorer outcome. Particularly, in the unclassifiable ILD group, the prognostic impact was more significant (death due to ILD, log-rank test, p < 0.0001), with a 10-year survival rate (hazard ratio 11.1, Wald test, p = 0.0003), as compared to the non-IPF group (log-rank test, p = 0.0269; hazard ratio 2.7, Wald test, p = 0.0334). Multivariable analysis focusing on the unclassifiable ILD group confirmed that the frequent HNF4α expression was an independent prognostic factor (hazard ratio 28.6; Wald test, p = 0.0033). Thus, HNF4α can be utilized as an immunohistochemical marker for pneumocyte injury and have prognostic impact particularly in unclassifiable ILD.

Diagnostic utility of transbronchial biopsy for Hodgkin's lymphoma: A case study.

Lung lesions of Hodgkin's lymphoma (HL) are rare and difficult to diagnose by nonsurgical biopsy. We herein present the case of a 72-year-old Japanese male who presented with accumulation of lung infiltrates and masses bilaterally on the lungs for 3 years. Although transbronchial lung biopsy (TBB) and computed tomography-guided biopsy were conducted several times, his diagnosis remained inconclusive. On further deterioration of lung lesions, the patient was transferred to our hospital. Positron emission tomography revealed increased accumulation in the bilateral lungs and right supraclavicular lymph nodes. Surgical biopsy of the lymph node was performed. He was finally diagnosed with HL and underwent chemotherapy with doxorubicin, vinblastine, dacarbazine, and brentuximab vedotin. After chemotherapy, the lung lesion showed significant regression. A literature review indicated that the diagnostic success rate of TBB was low (18.5%) in cases of lung lesions in HL.

The nonsmokers' and smokers' pathways in lung adenocarcinoma: Histological progression and molecular bases.

There could be two carcinogenetic pathways for lung adenocarcinoma (LADC): the nonsmokers' pathway and the smokers' pathway. This review article describes the two pathways with special reference to potential relationships between histological subtypes, malignant grades, and driver mutations. The lung is composed of two different tissue units, the terminal respiratory unit (TRU) and the central airway compartment (CAC). In the nonsmokers' pathway, LADCs develop from the TRU, and their histological appearances change from lepidic to micropapillary during the progression process. In the smokers' pathway, LADCs develop from either the TRU or the CAC, and their histological appearances vary among cases in the middle of the progression process, but they are likely converged to acinar/solid at the end. On a molecular genetic level, the nonsmokers' pathway is mostly driven by EGFR mutations, whereas in the smokers' pathway, approximately one-quarter of LADCs have KRAS mutations, but the other three-quarters have no known driver mutations. p53 mutations are an important factor triggering the progression of both pathways, with unique molecular alterations associated with each, such as MUC21 expression and chromosome 12p13-21 amplification in the nonsmokers' pathway, and HNF4α expression and TTF1 mutations in the smokers' pathway. However, investigation into the relationship between histological progression and genetic alterations is in its infancy. Tight cooperation between traditional histopathological examinations and recent molecular genetics can provide valuable insight to better understand the nature of LADCs.

[Postoperative Relapse of Combined Large‒Cell Neuroendocrine Carcinoma of the Lung with a Remarkable Transient Response to Amrubicin Monotherapy in an Elderly Patient-A Case Report].

An 86‒year‒old man with chronic kidney disease underwent surgical resection for combined large‒cell neuroendocrine carcinoma of the left lower lobe of the lung(pT2aN1M0, stage ⅡB). Five months later, multiple liver and bone metastases and mediastinal lymph node recurrence were detected. After 9 courses of amrubicin monotherapy(32 mg/m2 for 3 consecutive days), his tumor marker levels normalized, and radiological examination revealed a complete tumor response. Adverse events occurred, but they were tolerable except a decrease in the neutrophil count. The patient remained in good condition for several months but died of tumor relapse 22 months after the initial recurrence. Amrubicin monotherapy was considered to be one of the treatment choices for recurrent large‒cell neuroendocrine carcinoma of the lung in elderly patients.

A method to obtain reproducible Ki-67 indices in lung adenocarcinoma.

AIMS: Proliferative activity, evaluated from the Ki-67 index, is a strong prognostic factor in lung adenocarcinoma (LADC). Here, we optimised a procedure to measure the Ki-67 index and establish the best cut-off value. METHODS AND RESULTS: We examined 342 stage I LADCs for the immunohistochemical expression of Ki-67 using different antibodies, MIB1 and SP6. The results revealed the superior specificity of SP6; therefore, SP6 was used in subsequent analyses. Slides were scanned with a virtual slide system. Using software, tumour cells were counted in a whole tumour. Thereafter, the tumour was evenly subdivided into 0.25-mm2 tiles. The frequency of positive cells was counted in each tile of an invasive area or the whole tumour. We calculated the number of tumour cells required to produce a 95% confidence interval (CI) <0.05. Additionally, we calculated coverage probabilities (CP) using two different methods, counting any number or 200 cells per tile. The results showed that we could meet our goal by counting 2000 cells from 10 random tiles (200 cells each) in invasive areas. CONCLUSIONS: We successfully developed an optimal procedure for determination of the Ki-67 labelling index using an SP6 antibody, which provided CP > 70% and CI of <0.05 in more than 90% of cases. Furthermore, we identified an optimal cut-off value of 0.12 with an alternative of 0.15, based on disease recurrence. This procedure and the cut-off values may be used in the routine pathological diagnosis of LADC.