Unbiased screen for pathogens in human paraffin-embedded tissue samples by whole genome sequencing and metagenomics.

Identification of bacterial pathogens in formalin fixed, paraffin embedded (FFPE) tissue samples is limited to targeted and resource-intensive methods such as sequential PCR analyses. To enable unbiased screening for pathogens in FFPE tissue samples, we established a whole genome sequencing (WGS) method that combines shotgun sequencing and metagenomics for taxonomic identification of bacterial pathogens after subtraction of human genomic reads. To validate the assay, we analyzed more than 100 samples of known composition as well as FFPE lung autopsy tissues with and without histological signs of infections. Metagenomics analysis confirmed the pathogenic species that were previously identified by species-specific PCR in 62% of samples, showing that metagenomics is less sensitive than species-specific PCR. On the other hand, metagenomics analysis identified pathogens in samples, which had been tested negative for multiple common microorganisms and showed histological signs of infection. This highlights the ability of this assay to screen for unknown pathogens and detect multi-microbial infections which is not possible by histomorphology and species-specific PCR alone.

COVID-19 Autopsies Reveal Underreporting of SARS-CoV-2 Infection and Scarcity of Co-infections.

Coronavirus disease 2019 (COVID-19) mortality can be estimated based on reliable mortality data. Variable testing procedures and heterogeneous disease course suggest that a substantial number of COVID-19 deaths is undetected. To address this question, we screened an unselected autopsy cohort for the presence of SARS-CoV-2 and a panel of common respiratory pathogens. Lung tissues from 62 consecutive autopsies, conducted during the first and second COVID-19 pandemic waves in Switzerland, were analyzed for bacterial, viral and fungal respiratory pathogens including SARS-CoV-2. SARS-CoV-2 was detected in 28 lungs of 62 deceased patients (45%), although only 18 patients (29%) were reported to have COVID-19 at the time of death. In 23 patients (37% of all), the clinical cause of death and/or autopsy findings together with the presence of SARS-CoV-2 suggested death due to COVID-19. Our autopsy results reveal a 16% higher SARS-CoV-2 infection rate and an 8% higher SARS-CoV-2 related mortality rate than reported by clinicians before death. The majority of SARS-CoV-2 infected patients (75%) did not suffer from respiratory co-infections, as long as they were treated with antibiotics. In the lungs of 5 patients (8% of all), SARS-CoV-2 was found, yet without typical clinical and/or autopsy findings. Our findings suggest that underreporting of COVID-19 contributes substantially to excess mortality. The small percentage of co-infections in SARS-CoV-2 positive patients who died with typical COVID-19 symptoms strongly suggests that the majority of SARS-CoV-2 infected patients died from and not with the virus.

A Rare Case of Cutaneous Trichophyton verrucosum of the Forearm in a 51-Year-Old Cattle Farmer.

Trichophyton verrucosum is a pathogen causing superficial mycoses in cattle worldwide and is one of the few zoophilic dermatophytes. Farmers and veterinarians are at a higher risk for infection owing to frequent direct animal contact. An increase in cases among humans has been observed in the past few years. We report a rare case of T verrucosum of the forearm in a 51-year-old cattle farmer, who after initial treatment with antibiotics and surgery, and in whom diagnosis was delayed, was finally successfully treated with terbinafine and itraconazole.

Two distinct immunopathological profiles in autopsy lungs of COVID-19.

Coronavirus Disease 19 (COVID-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has grown to a worldwide pandemic with substantial mortality. Immune mediated damage has been proposed as a pathogenic factor, but immune responses in lungs of COVID-19 patients remain poorly characterized. Here we show transcriptomic, histologic and cellular profiles of post mortem COVID-19 (n = 34 tissues from 16 patients) and normal lung tissues (n = 9 tissues from 6 patients). Two distinct immunopathological reaction patterns of lethal COVID-19 are identified. One pattern shows high local expression of interferon stimulated genes (ISGhigh) and cytokines, high viral loads and limited pulmonary damage, the other pattern shows severely damaged lungs, low ISGs (ISGlow), low viral loads and abundant infiltrating activated CD8+ T cells and macrophages. ISGhigh patients die significantly earlier after hospitalization than ISGlow patients. Our study may point to distinct stages of progression of COVID-19 lung disease and highlights the need for peripheral blood biomarkers that inform about patient lung status and guide treatment.

Postmortem examination of COVID-19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction.

AIMS: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly evolved into a sweeping pandemic. Its major manifestation is in the respiratory tract, and the general extent of organ involvement and the microscopic changes in the lungs remain insufficiently characterised. Autopsies are essential to elucidate COVID-19-associated organ alterations. METHODS AND RESULTS: This article reports the autopsy findings of 21 COVID-19 patients hospitalised at the University Hospital Basel and at the Cantonal Hospital Baselland, Switzerland. An in-corpore technique was performed to ensure optimal staff safety. The primary cause of death was respiratory failure with exudative diffuse alveolar damage and massive capillary congestion, often accompanied by microthrombi despite anticoagulation. Ten cases showed superimposed bronchopneumonia. Further findings included pulmonary embolism (n = 4), alveolar haemorrhage (n = 3), and vasculitis (n = 1). Pathologies in other organ systems were predominantly attributable to shock; three patients showed signs of generalised and five of pulmonary thrombotic microangiopathy. Six patients were diagnosed with senile cardiac amyloidosis upon autopsy. Most patients suffered from one or more comorbidities (hypertension, obesity, cardiovascular diseases, and diabetes mellitus). Additionally, there was an overall predominance of males and individuals with blood group A (81% and 65%, respectively). All relevant histological slides are linked as open-source scans in supplementary files. CONCLUSIONS: This study provides an overview of postmortem findings in COVID-19 cases, implying that hypertensive, elderly, obese, male individuals with severe cardiovascular comorbidities as well as those with blood group A may have a lower threshold of tolerance for COVID-19. This provides a pathophysiological explanation for higher mortality rates among these patients.

Digital image analysis improves precision of PD-L1 scoring in cutaneous melanoma.

AIMS: Immune checkpoint inhibitors have become a successful treatment in metastatic melanoma. The high response rates in a subset of patients suggest that a sensitive companion diagnostic test is required. The predictive value of programmed death ligand 1 (PD-L1) staining in melanoma has been questioned due to inconsistent correlation with clinical outcome. Whether this is due to predictive irrelevance of PD-L1 expression or inaccurate assessment techniques remains unclear. The aim of this study was to develop a standardised digital protocol for the assessment of PD-L1 staining in melanoma and to compare the output data and reproducibility to conventional assessment by expert pathologists. METHODS AND RESULTS: In two cohorts with a total of 69 cutaneous melanomas, a highly significant correlation was found between pathologist-based consensus reading and automated PD-L1 analysis (r = 0.97, P < 0.0001). Digital scoring captured the full diagnostic spectrum of PD-L1 expression at single cell resolution. An average of 150 472 melanoma cells (median 38 668 cells; range = 733-1 078 965) were scored per lesion. Machine learning was used to control for heterogeneity introduced by PD-L1-positive inflammatory cells in the tumour microenvironment. The PD-L1 image analysis protocol showed excellent reproducibility (r = 1.0, P < 0.0001) when carried out on independent workstations and reduced variability in PD-L1 scoring of human observers. When melanomas were grouped by PD-L1 expression status, we found a clear correlation of PD-L1 positivity with CD8-positive T cell infiltration, but not with tumour stage, metastasis or driver mutation status. CONCLUSION: Digital evaluation of PD-L1 reduces scoring variability and may facilitate patient stratification in clinical practice.

Grover's-like drug eruption in a patient with metastatic melanoma under ipilimumab therapy.

BACKGROUND: Dermatologic toxicity is an important adverse effect of immune checkpoint inhibitors targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death 1 receptor (PD-1) or PD ligand 1 (PD-L1). Skin toxicity most commonly includes a maculopapular erythematous rash and pruritus. Rarely life threatening complications such as Steven's Johnson syndrome or toxic epidermal necrolysis may occur. CASE PRESENTATION: Here we report the uncommon event of a drug-induced transient acantholytic dermatosis (Grover's disease) in a 73-year-old Caucasian male treated with ipilimumab for metastatic melanoma. Five weeks after initiation of therapy, the patient developed a widespread polymorphic papulovesicular dermatosis on the trunk and proximal extremities with intense pruritus. Skin biopsy showed acantholytic dyskeratosis with interface dermatitis consistent with a Grover's-like drug eruption. CONCLUSIONS: These findings should raise awareness for uncommon immune-related dermatological toxicities of immunomodulatory antibodies targeting the CTLA-4 signaling axis. We recommend biopsies of unexpected skin lesions to rapidly identify dermatological adverse events of immune checkpoint inhibitors.

Tumor-associated B cells in cutaneous primary melanoma and improved clinical outcome.

B cells often infiltrate the microenvironment of human tumors. B cells can both positively and negatively regulate antitumor immune responses. In several human cancers, higher numbers of CD20(+) TAB are associated with a favorable prognosis, whereas in human primary melanomas, this association is contentious. In this study, we determined the association of TAB numbers in cutaneous primary melanoma tissue samples and patients' overall survival. The CD20 immunohistochemistry on archival nonmetastasized and metastasized cutaneous primary melanoma tissues from 2 independent patient cohorts was performed. One cohort was used in class comparison for metastasis, the most important prognostic factor for overall survival, and the other cohort for a subsequent survival analysis. Survival association was further validated with RNA data from a third independent cohort. Whole tissue sections were read automatically via quantitative digital imaging and analysis. Survival data were analyzed by Cox proportional hazard modeling. We discovered that cutaneous primary melanomas without metastasis contain significantly more TAB than primary melanomas that had metastasized. At time of first diagnosis, a higher number of TAB is associated with a significantly better overall survival in patients with cutaneous primary melanomas of >1 mm Breslow depth. Also, higher CD20/CD19 tumor mRNA levels are correlated with a significantly better overall survival. Thus, our data support TAB numbers as a prognostic biomarker in cutaneous primary melanoma patients with a tumor of >1 mm Breslow depth. For a survey in larger studies, whole tissue section analysis seems to be key to accurate assessment of TAB numbers.

Systemic inflammation in a melanoma patient treated with immune checkpoint inhibitors-an autopsy study.

BACKGROUND: Immune checkpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) have been recently approved for treatment of patients with metastatic melanoma and non-small cell lung cancer (NSCLC). Despite important clinical benefits, these therapies are associated with a diverse spectrum of immune-related adverse events (irAEs) that are typically transient, but occasionally severe or even fatal. CASE PRESENTATION: This autopsy case illustrates that clinically overt irAEs may represent only a fraction of the total spectrum of immune-related organ pathology in patients treated with immune checkpoint inhibitors. We report a comprehensive analysis of systemic irAE pathology based on the autopsy of a 35-year-old female patient with metastatic melanoma treated first with ipilimumab and then nivolumab. The clinical course was characterized by a mixed tumor response with regression of skin and lung metastases and fatal progression of metastatic disease in the small bowel, peritoneum and brain. During therapy with ipilimumab, radiographic features of immune-related pneumonitis were noted. The autopsy examination established a sarcoid-like granulomatous reaction of the lung, pulmonary fibrosis and diffuse alveolar damage. Importantly, a clinically unapparent but histologically striking systemic inflammation involving the heart, central nervous system, liver and bone marrow was identified. Severe immune-related end-organ damage due to lymphocytic myocarditis was found. CONCLUSIONS: Autopsy studies are an important measure of quality control and may identify clinically unapparent irAEs in patients treated with immunotherapy. Pathologists and clinicians need to be aware of the broad spectrum of irAEs for timely management of treatment-related morbidity.

Liver Cirrhosis/Severe Fibrosis Is a Risk Factor for Anastomotic Leakage after Colorectal Surgery.

Purpose. Liver cirrhosis associated with high perioperative morbidity/mortality. This retrospective study determines whether liver cirrhosis represents a risk factor for anastomotic leakage after colonic anastomosis or not. Methods. Based on a prospective database with all consecutive colorectal resections performed at the authors' institution from 07/2002 to 07/2012 (n = 2104) all colonic and rectal anastomoses were identified (n = 1875). A temporary loop ileostomy was constructed in 257 cases (13.7%) either due to Mannheimer Peritonitis-Index > 29 or rectal anastomosis below 6 cm from the anal verge. More than one-third of the patients (n = 691) had postoperative contrast enema, either at the occasion of another study or prior to closure of ileostomy. The presence of liver cirrhosis and the development of anastomotic leakage were assessed by chart review. Results. The overall anastomotic leakage rate was 2.7% (50/1875). In patients with cirrhosis/severe fibrosis, the anastomotic leakage rate was 12.5% (3/24), while it was only 2.5% (47/1851) in those without (p = 0.024). The difference remained statistically significant after correction for confounding factors by multivariate analysis. Conclusion. Patients with liver cirrhosis/severe fibrosis have an increased risk of leakage after colonic anastomosis.

[Small red sports on the feet and diarrhoea with 63]. / Eine 63-jährige Patientin mit Petechien an den Füssen und Diarrhö

We report a case of a 63 year old woman with progressive small red spots that can not be diminished with pressure. They appeared on the feet first and spread out to the legs, trunk and arms. This was accompanied with non-bloody diarrhoea and the laboratory diagnostics showed minor impairment of the kidney. The biopsy of the skin showed deposits of immunoglobulin A and C3 complement in the stratum papillare of the small vessels and a necrosis of the wall, which made the diagnosis of the Schönlein-Henoch purpura clear. It was accompanied by a systemic participation of the gastrointestinal tract. The quick reversible renal insufficiency was rather interpreted as a prerenal cause, than as an origin of the Schönlein-Henoch purpura. We further discussed the diagnostic methods, the aetiology and the possible therapy options.

The impact of pulmonary bone component embolism: an autopsy study.

AIMS: Pulmonary bone marrow embolism (BME) and pulmonary bone fragment embolism (BFE) are two types of non-thrombotic pulmonary embolism (NTPE). While BME can be found consistently in autopsies, BFE is a rarely observed event. Both these conditions have bone lesions as source of embolism and are not considered to be causative for death. METHODS: A retrospective autopsy study was performed and lung whole tissue slides were reviewed for the presence of pulmonary embolism. Clinicopathological data were screened for osseous lesions considered as risk factors for BME and BFE. RESULTS: We reviewed 985 consecutive, unselected autopsies and identified 29 cases of BME (2.9%) and 5 cases of BFE (0.5%). Both conditions were mutually exclusive. While BME showed a significant association with costal fractures, BFE was significantly associated with osteomyelitis and previously performed femur nailing. There were between 1 and 346 bone emboli in BFE with a density ranging from 0.74 to 30.5 emboli/cm(2) with mean embolic diameter of 45.8±37.6 µm. In two patients, BFE contributed significantly to fatal outcome. CONCLUSIONS: BME was associated with costal fractures, while BFE was associated with orthopaedic procedures and osteomyelitis. BFE can result in patient death. Both conditions appeared exclusively, indicating that although they originate from osseous lesions their underlying pathogenesis may likely be different.

Mandatory resection of strangulation marks in small bowel obstruction?

BACKGROUND: No evidence is available on how to treat intraoperatively detected band-shaped strangulation marks of the bowel wall originating from an adhesive band or hernia ring. The authors prefer to resect these hazardous strangulation marks to avoid secondary small bowel perforation. This retrospective study investigated the prevalence of intraoperatively unrecognized ulceration and transmural necrosis at the site of the strangulation marks. METHODS: From July 2003 to July 2011, a total of 31 of 461 patients with acute bowel obstruction underwent small bowel resection due to strangulation marks, exclusively. Seven patients had two strangulation marks, resulting in 38 strangulation marks to be analyzed. RESULTS: From 38 examined strangulation marks, 14 (36.8 %) exhibited deep ulceration or transmural necrosis. Four (10.5 %) necrotic lesions had already been recognized intraoperatively, while 7 (18.4 %) unsuspicious strangulation marks showed deep ulceration and 3 (7.9 %) showed transmural necrosis exclusively at final histopathologic examination. The number of strangulation marks that needed to be resected for prevention of one missed deep ulceration and/or transmural necrosis of the small bowel was 3.4. The presence of deep ulceration or transmural necrosis is associated with an obvious decrease in bowel diameter caudad to the strangulation mark. No anastomotic leak occurred. CONCLUSION: The severity of small bowel damage at the site of band-shaped strangulation marks may be underestimated by surgeons. The present series showed favorable results with a resection-per-principle policy for these strangulation marks. If an obvious decrease of bowel diameter aborally to the strangulation mark is present, resection or seromuscular invagination of the later is particularly recommended.

Prevalence and clinical implications of diverticulosis of the vermiform appendix.

OBJECTIVE: The epidemiology and the aetiology of inflammatory diseases of the vermiform appendix remain poorly understood. The prevalence of appendiceal diverticulosis and diverticulitis in patients undergoing appendectomy for suspected acute appendicitis was investigated. METHODS: A retrospective study was completed on patients who underwent appendectomy for suspected acute appendicitis. Pathology reports of all patients were screened for diverticula of the vermiform appendix. Patients with either diverticulitis of the vermiform appendix or normal appendicitis were compared. RESULTS: Out of two sets of consecutive patients (n = 1073), nine (0.8%) were identified with diverticulosis of the vermiform appendix. Two of these patients had diverticulitis of the vermiform appendix without appendicitis, three had diverticulitis with consecutive localized appendicitis, and four had proper acute appendicitis with a noninflamed diverticulum of the vermiform appendix. One patient had perforated appendicitis. Two patients had an obstructing neuroendocrine carcinoid which may have caused diverticular formation. CONCLUSIONS: Diverticula of the vermiform appendix are rare. If inflamed, they mimic acute appendicitis and are treated by appendectomy. If not inflamed, and diagnosed intraoperatively, incidental appendectomy is recommended.

Merkel cell polyomavirus large T antigen is detected in rare cases of nonmelanoma skin cancer.

BACKGROUND: Studies of Merkel cell polyomavirus (MCPyV) in nonmelanoma skin cancers (NMSC) other than Merkel cell carcinoma (MCC) produced controversial results. Therefore, we studied the prevalence of MCPyV in basal cell carcinoma (BCC) and in squamous cell carcinoma (SCC). METHODS: Tissue specimens were analyzed for the presence of MCPyV DNA by conventional polymerase chain reaction (PCR). Expression of MCPyV large T protein was determined by immunohistochemistry. RESULTS: MCPyV DNA was frequently detected in skin cancers by PCR, in 36 of 88 BCCs, in 21 of 75 SCCs and in 10 of 47 normal skin samples. In BCC, a significant difference in the detection rate compared to normal skin was observed. In contrast, weak reactivity for MCPyV large T antigen was detected only sporadically in immunosuppressed patients (2 of 88 BCCs, 1 of 75 SCCs). Mutations of the large T antigen of MCPyV were more frequently observed in MCC than in BCC/SCC. CONCLUSIONS: Our results suggest that the frequent detection of the MCPyV genome in NMSC by PCR reflects ubiquitous spread of the virus. However, the low immunohistochemical detection rate of MCPyV and the lack of MCC-specific MCPyV mutations argue against an essential role of MCPyV in the development of skin cancers other than MCC.

Rhabdoid large cell carcinoma of lung, with illustrative immunohistochemical and molecular findings.

Large cell carcinomas with rhabdoid phenotype (LCC-RP) account for <1% of pulmonary large cell carcinomas and are associated with extremely poor prognosis. We report a case of a 64-year-old male patient who presented at an advanced stage with a LCC-RP, arising from a poorly differentiated adenocarcinoma of the lung. Ninety percent of the tumor consisted of large pleomorphic rhabdoid tumor cells that showed eosinophilic cytoplasmic inclusions and the remaining 10% showed evidence of adenomatous differentiation. Rhabdoid areas were immunohistochemically positive for pan-cytokeratin AE1/3, epithelial membrane antigen, vimentin, thyroid transcription factor-1, integrase interactor-1, and negative for desmin. Nuclear positivity was absent for Myo-D1. The parent adenocarcinoma was positive for thyroid transcription factor-1 and cytokeratins 7. Epidermal growth factor receptor mutation analysis revealed the same mutation (p.delL747-T751) in both areas, suggesting that the malignant rhabdoid phenotype represents a dedifferentiation phenomenon of the adenocarcinoma. This is the first reported case of an Exon 19 deletion in epidermal growth factor receptor of the activating type detected in a LCC-RP associated with a poorly differentiated pulmonary adenocarcinoma.

Giant ectopic liver, hepatocellular carcinoma and pachydermia-a rare genetic syndrome?

Ectopic liver is a very uncommon developmental anomaly that predisposes to the development of hepatocellular carcinoma. We describe the second documented case of a hepatocellular carcinoma developing in the primary liver of a patient with a rare and uncharacterized genetic symptom complex. Also present was the largest ectopic liver ever reported, measuring 12 cm in diameter which contained a solitary focus of metastatic hepatocellular carcinoma. The primary hepatocellular carcinoma is believed to have arisen in the native liver from a hepatic adenoma that was diagnosed 15 years earlier. The patient's uncharacterised condition featured prominent thick, yellow skin over the dorsum of the fingers, and was associated with follicular hyperkeratosis, abnormal plantar creases, digital clubbing, misshaped ears, a lingua plicata and an angioleiomyolipoma of the right kidney. This unique case of hepatocellular carcinoma arising from liver cell adenoma in a patient with an uncharacterised condition featuring a large ectopic liver invites discussion of the role of local factors in carcinogenesis in the parent liver but not the ectopic liver. It also underlines the imperative ongoing need for clinical autopsies.

Alpha 1-antitrypsin retention in an ectopic liver.

Ectopic livers are infrequently reported in the literature. The reported size for ectopic livers range from a few millimeters up to several centimeters. They are often clinically silent and incidentally discovered during imaging of the hepatobiliary tract, regional surgical procedures or autopsy. They are predestined for benign liver diseases otherwise observed in normal livers like fatty change or develop malignancies such as hepatocellular carcinoma, in a manner analogous to the parent orthotopic liver. The presence of abnormal alpha 1-antitrypsin retention in an ectopic liver has, to our knowledge, not been reported in the literature. Hereby, we present the first reported case featuring alpha 1-antitrypsin retention in an ectopic liver attached to the fundus of the gallbladder and present the clinical, radiological and pathological findings in a caucasian woman undergoing cholecystectomy for acute cholecystitis. Special liver stains showed an alpha 1-antitrypsin retention which was confirmed immunohistochemically. Although ectopic livers are rare and usually an incidental finding, the radiologist and the surgeon should take this into the differential diagnosis of a mass attached to the gall bladder. A secondary disease should be considered by the pathologist in such a specimen and alpha 1-antitrypsin retention should be ruled out by special liver stains. Finally, such a finding should prompt the managing clinician to exclude systemic alpha-1 antitrypsin deficiency in the patient through further appropriate tests.

C-reactive protein is superior to bilirubin for anticipation of perforation in acute appendicitis.

OBJECTIVE: Very recently it has been shown that hyperbilirubinemia is a specific predictor of perforation in acute appendicitis. We compared the diagnostic importance of bilirubin, C-reactive protein (CRP), leukocyte count and age as markers of perforation in acute appendicitis. MATERIAL AND METHODS: A two-center retrospective cohort study was completed. Patients with acute appendicitis (n = 725) were divided into two groups, group A with perforation (n = 155) and group B without (n = 570). RESULTS: In group A an elevated CRP (> 5 mg/l) was measured in 98% of cases versus 72.5% in group B. Hyperbilirubinemia (> 20 micromol/l) was measured in 38% of cases in group A versus 22.3% in group B. Leukocytosis (> 10 x 10(9)/l) was measured in 85% of cases in group A versus 79.3% in group B. Analysis of qualitative and quantitative data showed every marker to be significantly correlated with perforation except elevated white cell blood count. However CRP showed the strongest correlation. The logistic regression model showed CRP to be by far the most significant marker of perforation. CONCLUSIONS: Our results confirm hyperbilirubinemia to be a statistically significant marker of perforation in acute appendicitis. However, CRP is superior to bilirubin for anticipation of perforation in acute appendicitis.

Patients with low prostate-specific antigen levels (< or =4 ng/ml) would benefit from a twelve-core biopsy protocol for prostate cancer detection.

INTRODUCTION: Prostate biopsy protocols using twelve cores rather than the standard six cores have consistently shown improved prostate cancer detection rates. The aim of our study was to evaluate whether the improved rate of prostate cancer detection in patients with low prostate-specific antigen levels warrants the standardization of a twelve-core biopsy protocol in this group. PATIENTS AND METHODS: The clinical and pathological records from 241 patients treated between 2000 and 2003 were evaluated, and the impact of a twelve-core biopsy protocol on the prostate cancer detection rate relative to prostate-specific antigen levels compared to the standardized six-core biopsies was analyzed. RESULTS: Prostate cancer was detected in 34% (81/241) of the patients who underwent transrectal ultrasound-guided biopsy. An additional 23.5% (19/81) of the carcinomas were diagnosed using the twelve-core biopsy protocol, and 84.2% (16/19) of these fulfilled the clinical significance criterion developed by Epstein and coworkers (see text). Interestingly, the greatest increase was found in the patient group with prostate-specific antigen levels < or =4 ng/ml. CONCLUSIONS: Patients with low prostate-specific antigen levels (< or =4 ng/ml) would benefit from the standardized use of a twelve-core biopsy protocol using peripheral cores.