AMACR Research Articles

Redefining the phenotype of alpha-methylacyl-CoA racemase (AMACR) deficiency

BackgroundAlpha-methylacyl-CoA racemase (AMACR) deficiency is a rare peroxisomal enzyme deficiency caused by biallelic variants in the AMACR gene. This deficiency leads to the accumulation of toxic bile acid intermediates (R)-trihydroxycholestenoic acid (THCA) and (R)-dihydroxycholestenoic acid (DHCA) and pristanic acid. With less than 20 patients described in literature, the phenotype of AMACR deficiency is poorly defined and no data on the natural history are available.ResultsHere we describe a cohort of 12 patients (9 adults and 3 children) with genetically confirmed AMACR deficiency (median age at diagnosis 56 years, range 3–69), followed for an average of 6 years (between 2015 and 2023). Five novel pathogenic variants are described. In 5/9 adult patients, retinitis pigmentosa was detected at a median age of 45 years (range 30–61). The median delay to diagnosis of AMACR deficiency after the diagnosis of retinitis pigmentosa was 24 years (range 0–33). All adult patients subsequently developed neurological signs and symptoms after the age of 40 years; most frequently neuropathy, ataxia and cognitive decline with prior normal cognitive functioning. One patient presented with a stroke-like episode. All adult patients showed a typical MRI pattern involving the thalami and gray matter structures of the pons and midbrain. One patient had a hepatocellular carcinoma at the time of the AMACR deficiency diagnosis and two patients suffered from gallstones. All three included children had elevated liver transaminases as single presenting sign and showed no brain MRI abnormalities.ConclusionAMACR deficiency can be considered as an adult slowly progressive disease with a predominant neurological phenotype. The main signs comprise retinitis pigmentosa, neuropathy, ataxia and cognitive decline; stroke-like episodes may occur. Recognition of typical MRI abnormalities may facilitate prompt diagnosis. In addition, there is a risk of liver fibrosis/cirrhosis and hepatocellular carcinoma in these patients, requiring active monitoring.

Variable clinical phenotypes of alpha-methylacyl-CoA racemase deficiency: Report of four cases and review of the literature.

Alpha-methylacyl-CoA-racemase (AMACR) deficiency (MIM#604489) is a peroxisomal disorder resulting in the accumulation of pristanic acid, dihydroxycholestanoic acid (DHCA), and trihydroxycholestanoic acid (THCA), with variable clinical features and age of onset from infancy to late adulthood. The purpose of this report is to define clinical variations and follow-up data in AMACR deficiency emphasizing treatment with a review of cases reported in the literature. Here, four patients, from two families, diagnosed with AMACR deficiency and showing phenotypic heterogeneity are presented. A 10-month-old-female presented with coagulopathy, hepatic dysfunction, and elevated pristanic acid, DHCA, and THCA levels. Genetic testing confirmed a homozygous variant c.596G>A in the AMACR gene. Her brother who had macrovesicular hepatosteatosis and elevated pristanic acid levels was diagnosed with family screening. The third patient presented with rhabdomyolysis following a strenuous exercise without any other complaint. Homozygous novel c.1006G>A variant was found on the AMACR gene. His asymptomatic sister carrying the same variant also had elevated pristanic acid levels. They had normal neuropsychologic evaluation. Dietary treatment with low phytanic and pristanic acid content was recommended to the patients but all showed poor compliance. The sibling pairs were followed for periods of 11 and 7 years, respectively. AMACR deficiency is usually described as an adult-onset disorder with neuropsychological problems. The characterization of natural history and new clinical phenotypes may support earlier diagnosis and treatment.

Utility of p63 and Alpha Methyl Acyl CoA Racemase in the Lesions of Prostatic Mimickers: An Observational Study

Introduction: Prostate adenocarcinoma is the most commonly occurring cancer in men. Differentiation of prostatic adenocarcinoma from prostatic mimickers sometimes difficult on the sole basis of histologic findings. Diagnosis of carcinoma is based on Immunohistochemistry (IHC) by negative immune reaction with p63 and positive staining with Alpha Methyl Acyl CoA Racemase (AMACR). Aim: To evaluate the utility of p63 and AMACR in the prostatic mimickers. Materials and Methods: In this analytical observational study, all prospective prostatic mimickers with transrectal ultrasound (TRUS) guided biopsies received in the pathology department of Bharati Vidyapeeth Medical College, Pune, India in a tertiary care hospital from July 2020 to July 2022 were studied. 70 cases were collected, studied and analysed which included 38 Transurethral Resection of the Prostate (TURP) specimens and 32 TRUS guided tissue biopsies. The Haematoxylin & Eosin (H&E) stained slides of all the cases were examined and divided into three categories-malignant (08 cases), prostatic mimickers (53 cases) and suspicious for malignancy (09 cases). Furthur, these cases were subjected to IHC for p63 and AMACR. Statistical Package for Social Sciences (SPSS) version 25.0 software was used for data analysis using chi square test along with p-value. Results: Out of the 53 cases of prostatic mimickers and 09 cases were suspicious for malignancy, 56 were positive for p63 and 06 were negative for p63. There was a statistically significant association between p63 IHC staining and the differentiation of benign cases from malignant prostatic lesions (p<0.001). The sensitivity and specificity of p63 were 93.3% and 100% respectively. There were 08 cases of prostate adenocarcinoma stained with AMACR, all were positive. The sensitivity and specificity being 100%. Results revealed that there was a significant statistical association between the positive IHC staining of AMACR and prostatic adenocarcinoma (p<0.001). Conclusion: The diagnostic performance of p63 and AMACR proves to be significant in differentiating between the mimickers of prostatic lesions and adenocarcinoma. IHC is recommended to reduce diagnostic error in suspected cases.

Evaluation of TURP specimen with special reference to prostatic intraepithelial neoplasia and prostatic cancer using light microscopy and immunohistochemical markers of P63, CK5/6, AMACR (p504s), and PSA

Background: Prostate cancer is the world’s leading cause of cancer and the second cause of cancer-related death in men after lung cancer. The incidence of prostatic carcinoma is increasing with age. Prostate intraepithelial neoplasia can only be diagnosed by histopathological examination of prostate tissue. In practice, small foci of cancer and several morphological mimickers of different Gleason Grades were encountered leading to diagnosing difficulty. To overcome this pitfall, the use of HMW Cytokeratin’s 5 and 6 (CK5/6) or p63 or combination of these two with alpha-methyl acyl- COA-racemase (AMACR) is triple cocktail which is recommended. Aims and Objectives: The objectives of this study were to analyze the histomorphology of various non-neoplastic and neoplastic lesions of the prostate and to know the incidence of prostatic intraepithelial neoplasia in transurethral resection of prostate (TURP) specimen, to assess the usefulness of immunohistochemical analysis with a panel of markers (p63, CK5/6, AMACR [p504s], and prostate-specific antigen [PSA]) to confirm the diagnosis of prostatic intraepithelial neoplasia and prostate cancer, and to differentiate several benign mimickers of prostatic adenocarcinoma. Materials and Methods: It is a prospective study performed for 18 month from January 2020 to June 2021. Thirty TURP specimens were collected in 10% formalin and processed in the department of pathology, MCH, Kolkata. Results: In the present study, benign prostatic hyperplasia formed majority of cases (80%). The presence of AMACR and absence of basal cell specific marker p63 and CK5/6 confirm the presence of prostate cancer. Conclusion: High degree of association of HGPIN with prostatic Carcinoma, it is suggested that these HGPIN patients need close follow-up, observation, and investigation to rule out existence of carcinoma, especially in the peripheral zone.

Role of Two Antibodies Panel High Molecular Weight Cytokeratin and Alpha-MethylacylCoA Racemase in Diagnosing Prostatic Lesions: A Cross-sectional Study

Introduction: Prostatic diseases cause significant morbidity and mortality. Although histopathological examination is the gold standard for diagnosing prostatic lesions but diagnosis may be challenging in the presence of benign mimickers or a very small focus of malignancy. Immunohistochemical aid to morphology helps in making a timely and accurate diagnosis. Aim: This study was done to evaluate the role of two antibodies panel High Molecular Weight Cytokeratin (HMWCK) and AlphaMethylacyl-CoA Racemase (AMACR) in improving the diagnostic accuracy of prostatic lesions. Materials and Methods: This was an observational cross-sectional study conducted in the Department of Pathology, Shri Guru Ram Rai Institute of Medical and Health Sciences (SGRRIM and HS), Dehradun, Uttarakhand, India, from May 2019 to October 2020. Haemotoxylin and Eosin (H&E) stained sections of prostatic biopsies were classified into benign and malignant. Amongst malignant lesions, prostatic adenocarcinomas were graded according to Gleason’s grading system and Gleason’s scores were noted. One section from each was subjected to AMACR and HMWCK antibody tests. HMWCK was interpreted as negative/positive and continuous/ discontinuous. For AMACR, both location and intensity of stain was observed. The parameters studied were Gleason’s score, group grade, expression of HMWCK and AMACR. Categorical data was presented in form of frequency and percentage. Independent t-test, Yates Chi-square test were used. Data was entered in Microsoft (MS) Excel sheet and analysis was done using CRAN R 2.1. Results: Total of 80 prostatic biopsies were taken, 24 were malignant and 55 were benign and one was Benign Prostatic Hyperplasia (BPH) with a focus suspicious for malignancy showing atypical small acinar proliferation on histopathological examination. The mean age of non neoplastic cases was 67.68±8.56 years, while that of neoplastic lesions was 75.41±9.34 years. Amongst benign, 56.3% (31/55) cases were BPH, 43.6% (24/55) cases were BPH with associated lesions which included 62.5% (15/24) cases of BPH with non specific prostatitis; 29.2% (7/24) cases of BPH with adenosis and 8.3% (02/24) cases of BPH with basal cell hyperplasia. Of malignant cases, 24 cases were of adenocarcinoma with maximum cases having Gleason’s score 9 (11/24;45.8%) and Group grade V (18/24;75%). The sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) of HMWCK and AMACR were calculated using histopathology as the gold standard. Conclusion: Although histopathology is the gold standard in prostatic biopsies but immunohistochemistry is additional diagnostic aid in confirmation of diagnosis. Immunohistochemistry not only confirms the histological diagnosis but is of great help in challenging cases. It has markedly increased the diagnostic accuracy

Neonatal cholestasis – A case report on congenital bile acid synthetic defect type 4 and severe anemia

Congenital defects of bile acid synthesis are rare disorders that cause progressive liver dysfunction. We present a case of alpha methylacyl-CoA racemase (AMACR) deficiency with non-spherocytic hemolytic anemia who presented with rapidly progressive severe cholestasis and liver failure with normal gamma-glutamyl transferase levels. After extensive investigation, he was found to have AMACR deficiency with HBB gene mutation associated with non-spherocytic hemolytic anemia possibly explaining the severity of the disease. To the best of our knowledge, a similar association has not been reported so far.

IMMUNOHISTOCHEMICAL EVALUATION OF ALPHA-METHYL ACYL CO-A RACEMASE EXPRESSION IN GASTRIC BIOPSIES AND RESECTED SPECIMEN WITH HISTOPATHOLOGICAL CORRELATION

The third most common cause of cancer associated deaths worldwide is due to gastric cancer. Finding specic targets for neoadjuvant therapy is essential which will lead to better survival of the patients. Various molecular pathways are involved in gastric cancer pathogenesis. Of these pathways , deregulation of PI3K/AKT/mTOR pathway plays an important role in cell growth, cell proliferation and metabolism. The mTOR is activated by multiple growth factors including IGF-1R, EGFR and HER2. mTOR is regulated by activation of various molecules one of which isalpha methyl acyl coA racemase (AMACR). This alpha methyl acyl coA racemase can be tried as a molecular target as it is involved in one of the molecular pathways needed for gastric carcinoma development.

Expression of Alpha Methyl Acyl-CoaRacemase (AMACR) Immunohistochemistry in Carcinoma Prostate in Tertiary Care Hospital

Objective: Prostate needle biopsy samples from cancer patients at a tertiary care hospital were analysed for the presence of the potential screening indicator alpha-methylacyl-COA RACEMES. Material and Methods: This was a Cross-sectional study carried out at Department of Histopathology, Lady Reading Hospital Peshawar from June, 2021 to December, 2021.Regardless of the patient's age, tumour grade, or histological type, all adenocarcinoma prostate specimens diagnosed at the Lady Reading Hospital Peshawar on the basis of immunohistochemistry and routine histology were examined. Standard and mean deviation were computed for quantitative factors of patient like age and frequencies, percentages were calculated for qualitative factors like AMACR expressions. Results: Outof 90 cases, AMACR positivity was found in 68 (85%), while negativity was seen in 22 (15%). Nine (11.3%) of the negative cases had one staining (weak, non-circumferential) and three cases (3%) had no stainings (No cytoplasmic staining). Conclusion: Positive AMACR staining on prostate needle core necropsies may be utilised to support a cancer diagnosis when the focus at issue is 1 mm in highest diameter. Basic hematoxylin and eosin standards for malice require analysis of cell-specific markers like P63 or 34E12. AMACR labelling must be interpreted in the context of other labels that can be used to find out more. Keywords: Adenocarcinoma of the prostate, Alphamethylacyl-CoA racemase (AMACR), Immunohistochemistry.

Diagnosis and treatment of an inborn error of bile acid synthesis type 4: A case report

BACKGROUNDInborn error of bile acid synthesis type 4 is a peroxisomal disease with impaired bile acid synthesis caused by a-methylacyl-CoA racemase (AMACR) gene mutation. The disease is usually found in children with mild to severe liver disease, cholestasis and poor fat-soluble vitamin absorption. At present, there is no report of inborn errors of bile acid synthesis type 4 in adults with liver disease and poor fat-soluble vitamin absorption.CASE SUMMARYA 71-year-old man was hospitalized in our department for recurrent liver dysfunction. The clinical manifestations were chronic liver disease and yellow skin and sclera. Serum transaminase, bilirubin and bile acid were abnormally increased; and fat-soluble vitamins decreased. Liver cirrhosis and ascites were diagnosed by computed tomography. The patient had poor coagulation function and ascites and did not undergo liver puncture. Genetic testing showed AMACR gene missense mutation. The patient was diagnosed with inborn error of bile acid synthesis type 4. He was treated with ursodeoxycholic acid, liver protection and vitamin supplementation, and jaundice of the skin and sclera was reduced. The indicators of liver function and the quality of life were significantly improved.CONCLUSIONWhen adults have recurrent liver function abnormalities, physicians should be alert to genetic diseases and provide timely treatment.

An Example to a Rare Multisystemic and Challenging Metabolic Disorder: Peroxisomal Disease

Objective: Peroxisomal diseases (PDs) are autosomal recessively inherited inborn errors of metabolism (IEMs) that have a wide range of clinical presentations, with variable severity. The aim of this study was to describe cases with various subtypes of PDs with different clinical presentations and to increase the awareness of pediatricians to this metabolic disorder. Material and Methods: The study was conducted in Dr. Sami Ulus Maternity and Child Health Treatment and Research Hospital, division of pediatric metabolism, between December 2020 and January 2021. Patients of whom very long chain fatty acids (VLCFAs) were analysed due to various conditions between January 2020 and December 2020 were retrospectively evaluated. Among patients that VLCFA analyses were performed, 71 tests were found to have abnormal results and 20 of these patients had undergone molecular genetic analysis with the suspicion of PDs. An underlying genetic pathology was detected in 6 patients. Data of patients were obtained from patients files. Results: Six patients were evaluated. Diagnoses of patients were as following: Zellweger spectrum disorders (n=4), rhisomelic chondrodysplasia punctata type 1 (n=1), and AMACR deficiency (n=1). Age of onset of clinical symptoms ranged from the neonatal period to 6 years. Main clinical symptoms were hypotonia, developmental delay and dysmorphic findings. Three patients had cataracts and 5 had sensorineural deafness. Elevated transaminases were detected in 5/6 patients, of whom 4 had abnormal liver function tests. VLCFAs were found to be elevated in all patients. Variants in PEX1, PEX5, PEX6, PEX7, PEX12, PEX 26 and AMACR genes were detected. Patients with liver insufficiency received cholic acid treatment. Conclusion: PDs are very rare IEMs that comprise various subtypes with range of clinical phenotypes with different clinical presentations. Although genetic panel analyses is required for the diagnosis of, VLCFA analysis is a very simple and quick tool for the detection of PDs, and should be included in the screening of patients with visual and hearing impairments, liver disease and dysmorphic findings.

Utility of Alpha-Methyl Acyl-CoA Racemase Marker in Prostatic Adenocarcinomas

Introduction: Prostatic adenocarcinoma is the second leading cause of cancer related death in men in the western world and its incidence is increasing in Asian countries. Hence, it is of diagnostic challenge for pathologists to report in tissue biopsies especially in small focus of suspicious glands in radical prostatectomies and needle biopsies. Aim: To evaluate the role of Alpha-Methyl Acyl CoA Racemase (AMACR) immunohistochemical marker in diagnosing prostatic adenocarcinomas and in benign conditions of prostate. Materials and Methods: This was a cross-sectional study of 26 cases of prostatic adenocarcinomas, two cases of High Grade Prostatic Intraepithelial Neoplasias (HGPIN), 139 cases of Benign Prostatic Hyperplasia (BPH) and two cases of atypical adenomatous diagnosed on routine Haemotoxylin and Eosin (H&E) stained sections during a period of four years from January 2016 to December 2019 at ASRAM Medical College, Eluru. Immunohistochemistry with AMACR marker was done in all cases. Membranous staining pattern was accepted for prostatic adenocarcinomas. Semiquantitative scoring method was employed and tissue sections were examined at high power magnification (X400) by Olympus light microscope to evaluate AMACR expression. Results: Out of the 26 cases of prostatic adenocarcinomas in the study, majority were Grade group IV according to Gleason scoring system. Majority of the prostatic carcinomas showed strong and diffuse AMACR positivity. All BPH and Atypical Adenomatous Hyperplasia (AAH) were negative to AMACR immunohistochemical marker. Conclusion: The AMACR was found to be important diagnostic immune marker in prostatic adenocarcinomas especially in problematic situations where quantity and quality of tissue is limited.

Utility of Alpha-Methyl Acyl-CoA Racemase Marker in Prostatic Adenocarcinomas

Introduction: Prostatic adenocarcinoma is the second leading cause of cancer related death in men in the western world and its incidence is increasing in Asian countries. Hence, it is of diagnostic challenge for pathologists to report in tissue biopsies especially in small focus of suspicious glands in radical prostatectomies and needle biopsies. Aim: To evaluate the role of Alpha-Methyl Acyl CoA Racemase (AMACR) immunohistochemical marker in diagnosing prostatic adenocarcinomas and in benign conditions of prostate. Materials and Methods: This was a cross-sectional study of 26 cases of prostatic adenocarcinomas, two cases of High Grade Prostatic Intraepithelial Neoplasias (HGPIN), 139 cases of Benign Prostatic Hyperplasia (BPH) and two cases of atypical adenomatous diagnosed on routine Haemotoxylin and Eosin (H&E) stained sections during a period of four years from January 2016 to December 2019 at ASRAM Medical College, Eluru. Immunohistochemistry with AMACR marker was done in all cases. Membranous staining pattern was accepted for prostatic adenocarcinomas. Semiquantitative scoring method was employed and tissue sections were examined at high power magnification (X400) by Olympus light microscope to evaluate AMACR expression. Results: Out of the 26 cases of prostatic adenocarcinomas in the study, majority were Grade group IV according to Gleason scoring system. Majority of the prostatic carcinomas showed strong and diffuse AMACR positivity. All BPH and Atypical Adenomatous Hyperplasia (AAH) were negative to AMACR immunohistochemical marker. Conclusion: The AMACR was found to be important diagnostic immune marker in prostatic adenocarcinomas especially in problematic situations where quantity and quality of tissue is limited.

Evaluation of the diagnostic value of immunohistochemistry staining for P63, Alpha-methyl Acyl-CoA Racemase, CK5/6, and 34βE12 in prostate carcinoma

Evaluation of the diagnostic value of immunohistochemistry staining for P63, Alpha-methyl Acyl-CoA Racemase, CK5/6, and 34βE12 in prostate carcinoma

Partial Nephrectomy For a Presumed Single Renal Mass Revealing Multiple Tumor Histologies: A Series of 4 Patients

Abstract Introduction/Objective Renal mass biopsy is known to have a low but unavoidable diagnostic error rate. However, the occurrence of multiple adjacent masses mimicking one mass clinically has been minimally studied. Methods We report a series of four patients who were radiologically presumed to have a single renal mass and treated with partial nephrectomy, yet who were found to have multiple demarcated renal cell carcinoma histologies at pathologic evaluation. Results All were men aged 63–70 years. Grossly, tumors were red brown with scant, bright yellow foci in one of them. Dominant tumors followed by smaller tumors were: patient 1 - clear cell renal cell carcinoma (5.0 cm), clear cell papillary renal cell carcinoma (0.5 cm), and papillary adenoma (0.6 cm); patient 2 - clear cell renal cell carcinoma (1.5 cm) and clear cell papillary renal cell carcinoma (0.5 cm); patient 3 - papillary renal cell carcinoma (5.0 cm) and eosinophilic variant of chromophobe renal cell carcinoma (1.0 cm); patient 4 - chromophobe renal cell carcinoma (4.0 cm) and clear cell papillary renal cell carcinoma (0.6 cm). Immunohistochemical studies for cytokeratin 7, carbonic anhydrase IX, high molecular weight cytokeratin, CD10, and alpha-methyl acyl-CoA racemase (AMACR) confirmed the separate components in all. Conclusion This series adds to the spectrum of causes that may contribute to discordant results of renal mass biopsy and resection specimens. Secondary smaller tumors appear to be predominantly nonaggressive histologies, enriched for clear cell papillary renal cell carcinoma. Pathologists and urologists should be aware of this occurrence when considering the role of renal mass biopsy and interpreting the results.

Serum total prostate-specific antigen (tPSA): correlation with diagnosis and grading of prostate cancer in core needle biopsy.

Aim To investigate the impact of pre-treatment serum total prostate-specific antigen (PSA) level on prevalence of prostate carcinoma detection in prostate core needle biopsy, and its correlation with established prognostic factors. Methods Prostate needle biopsy samples of 115 patients with available pre-treatment serum total PSA (tPSA) level were analysed. For all cases where morphology alone was insufficient, immunohistochemistry was performed using p63, CKHMW and AMACR antibody panel in order to confirm or exclude the existence of prostate carcinoma. Results Statistically significant positive correlation between serum total PSA values and prevalence of finding prostate carcinoma in needle biopsy specimens was found (p=0.011), as well as in the case when the patients were classified into groups according to tPSA levels (p=0.028). Serum total PSA values and levels (level groups) showed significant positive correlation with Gleason score (p=0.029 and p=0.036, respectively) and Grade Group of prostate carcinomas (p=0.044 and p=0.046, respectively). Sensitivity of the screening test by using 4 ng/mL as cut off value for tPSA was 94.12% (CI: 80.32-99.28%), specificity 8.64% (CI: 3.55-17.00%), positive predictive value 30.19% (CI: 21.65-39.87%) and negative predictive value 77.78% (CI: 39.99-97.19%). Conclusion The increase of serum tPSA value increases the likelihood of finding prostate cancer on needle biopsy specimens. Due to such findings and its positive correlation with a grade of prostate cancer, our study indicates that tPSA can still be considered as a useful tool both in detecting and predicting aggressiveness of prostate cancer.

Association of polymorphisms of PTEN, AKT1, PI3K, AR, and AMACR genes in patients with prostate cancer.

Polymorphic variants in the PTEN (rs2735343), PI3K (rs2699887), AKT1 (rs2494750), AR (rs17302090), and AMACR (rs3195676) genes were evaluated as possible molecular markers of susceptibility, prognosis, and progression of prostate cancer (PCa), in a case-control study. Samples consisted of 277 patients with PCa and 277 controls from Londrina, PR, Brazil. SNPs were analyzed by real-time PCR. A family history of cancer, including PCa, as well as level of schooling were risk factors for PCa. The data were obtained via logistic regression, using odds ratios with a CI 95%. The genotypes of AKT1 and AKT1+AR demonstrated an association with protection for the disease. The combination of SNPs with the histopathological tumor data between allele variants of AMACR, AKT1+AR, and AKT1+AMACR indicated an association with protection against seminal vesicle invasion. The polymorphisms AKT1+AR and PI3K+AR were associated with protection against tumor bilaterality. The genotype combinations PTEN+AMACR and PTEN+AR were associated with the risk of extracapsular extension. Of the five genes studied, two were associated with protection for PCa, four were associated with protection for some prognostic variables, and only one was associated with risk. Thus, these SNPs are candidates for markers to discriminate men with better or worse prognosis for PCa.

Diagnostic utility of Alpha Methylacyl CoA Racemase in prostatic adenocarcinoma: An institutional experience

P504S is a prostate cancer specific gene that encodes for ?-methylacyl CoA racemase (AMACR). AMACR has been shown to be selectively over expressed in prostatic adenocarcinoma with minimal to undetectable expression in benign prostatic tissue. We studied the expression of AMACR in 30 cases of prostatic adenocarcinoma using polyclonal anti-AMACR antibody and correlated it with Gleason score. Extent of staining/proportion score (0: no positive cells; 1+:1-10% positive cells; 2+11-50% positive cells; 3+>50% positive cells) was also recorded and correlated with Gleason score. All the 30 cases showed strong, cytoplasmic granular AMACR staining irrespective of their Gleason score with 26 (86.7%) cases showing immunostaining in more than 50% tumor cells (3+ proportion score). Benign prostate tissue adjacent to adenocarcinoma showed negative AMACR staining. No correlation was seen between Gleason score and AMACR proportion score. We concluded that AMACR is a highly sensitive positive marker of prostatic adenocarcinoma. Keywords: Prostate adenocarcinoma, Gleason score, AMACR.

Circulating mRNA signature as a marker for high-risk prostate cancer

Prostate cancer (PCa) is the second most common cancer in men. The indolent course of the disease makes the treatment choice a challenge for physicians and patients. In this study, a minimally invasive method was used to evaluate the potential of molecular markers in identifying patients with aggressive disease. Cell-free plasma samples from 60 PCa patients collected before radical prostatectomy were used to evaluate the levels of expression of eight genes (AMACR, BCL2, NKX3-1, GOLM1, OR51E2, PCA3, SIM2 and TRPM8) by quantitative real-time PCR. Overexpression of AMACR, GOLM1, TRPM8 and NKX3-1 genes was significantly associated with aggressive disease characteristics, including extracapsular extension, tumor stage and vesicular seminal invasion. A trio of genes (GOLM1, NKX3-1 and TRPM8) was able to identify high-risk PCa cases (85% of sensitivity and 58% of specificity), yielding a better overall performance compared with the biopsy Gleason score and prostate-specific antigen, routinely used in the clinical practice. Although more studies are required, these circulating markers have the potential to be used as an additional test to improve the diagnosis and treatment decision of high-risk PCa patients.

COMBO-FISH: A Versatile Tool Beyond Standard FISH to Study Chromatin Organization by Fluorescence Light Microscopy

Background: Fluorescence In Situ Hybridization (FISH) has become routine for bio-medical research and medical diagnosis, thereby offering a variety of probes and ready-to-use kits that fulfil requirements for many applications. However, conventional FISH relies on chemo- and/or thermal denaturation to improve target accessibility and use huge amounts of DNAs that needs to be bonded to the target site. COMBinatorial Oligo-nucleotide FISH (COMBO-FISH) offers possibilities to circumvent these shortcomings. Methods: COMBO-FISH uses either a set of oligo-nucleotide probes (15 – 25 mers) uniquely co-localizing at the target site or single oligo-stretches repetitively but exclusively binding to the target. These are designed by systematic sequence data base searches. COMBO-FISH probes form Hoogsteen or Watson-Crick bonds and protocols with or without thermal denaturation can be realized. The latter allows the combination of COMBO-FISH with immunostaining. Low amounts of probe DNA allow the best maintenance of native chromatin organization – a prerequisite for applying super-resolution single molecule localization microscopy (SMLM). Results: Specific labelling of the AMACR gene with an oligo-nucleotide probe set was applied in three different cell types. Hybridization efficiencies were determined by counting spots of high visibility and their radial positions were measured by confocal microscopy. The nuclear architecture revealed a non-random organization. Using uniquely, repetitively binding COMBO-FISH probes for centromere 9, Alu consensus and L1 sequences revealed a characteristic probe distribution in cell nuclei as being measured by SMLM. Comparison to theoretical data allowed determination of chromosome 9 radial positions without painting whole chromosomes. Three-colour staining by COMBO-FISH for Alu and L1 with immunostaining for heterochromatin is successfully demonstrated. Conclusions: COMBO-FISH is a powerful tool that circumvents shortcomings of standard FISH procedures using probes derived e.g., from BAC clones. The application for measurements of nuclear architecture by 3D confocal microscopy and of chromatin nano-architecture by SMLM using COMBO-FISH and immunostaining simultaneously has been demonstrated.

Histological assessment & use of immunohistochemical markers for detection of dysplasia in Barrett’s esophageal mucosa

BackgroundHistological assessment of dysplasia in Barrett’s esophagus (BE) has high inter-observer variability. Hence, use of ancillary markers for early detection of dysplasia in BE is an important clinical question. MethodsIn this retrospective study consecutive cases of BE (n = 59), over a period of 4 years were included. Hematoxylin and eosin stained sections were reviewed independently by 3 senior qualified pathologists, who graded the dysplasia according to the Vienna Classification system and inter-observer agreement was analysed using the Kappa statistics. Subsequently Alpha-Methyl Acyl-CoA Racemase (AMACR), p53, CyclinD1, β-catenin, H2AX and M30 immunohistochemical (IHC) stains were examined on the following disease categories: BE with no dysplasia [NFD] (45), BE with indefinite for dysplasia (IFD) (4), low grade dysplasia (LGD) (3), high grade dysplasia (HGD) (2) and in adenocarcinomas (5). H score was calculated by adding up products of different grades of stain distribution and stain intensities (range of scores 0–300). ResultsAmong the 3 pathologists, overall agreement was poor (k 0.06; 95% CI −0.089 to 0.145), with highest disagreement noted for differentiating the LGD and IFDs (k = 0.21). After revising the histological criteria, the kappa improved to 0.53. Among the IHC stains performed, p53, β-catenin, H2AX and M30 stains were significantly useful to differentiate between IFD and LGD (P values: 0.04, 0.004, 0.05 & 0.04, respectively). AMACR and β-catenin stains though were up-regulated in HGD/adenocarcinomas than in other categories, their expression were not statistically different between the IFD and LGDs. ConclusionsA detail histological scoring system may bring uniformity in histological interpretation of dysplasia in BE. Using a combined panel of IHC stains seems helpful in detection of dysplasia in BE, especially to differentiate the IFD and LGD changes in BE.