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Background: One-week triple therapy with vonoprazan is endorsed by Japanese guidelines as an alternative to proton pump inhibitor (PPI)-based triple therapy for first-line Helicobacter pylori eradication. This contrasts with Western guidelines recommending 2-week PPI-based triple therapy., Aim: To verify the non-inferiority of 1-week vonoprazan-based triple therapy versus 2-week PPI-based triple therapy as first-line H. pylori eradication in a multiracial Asian cohort., Methods: Randomised controlled trial of treatment-naïve patients with H. pylori infection assigned 1:1 to either 7 days amoxicillin 1 g + clarithromycin 500 mg + vonoprazan 20 mg twice per day or 14 days amoxicillin 1 g + clarithromycin 500 mg + omeprazole OR esomeprazole OR rabeprazole 20 mg twice/day. Subjects were randomly assigned to each PPI 1:1:1 Demographics, H. pylori resistance, CYP 2C19 genotype, eradication success and safety profiles were compared between groups., Results: Between June 2019 and June 2021, 252 of 1097 subjects screened were randomised. 244 (age [SD] 51.7 [14.6]) received vonoprazan- (n = 119) or PPI-based (n = 125) triple therapy. Eradication rates by intention-to-treat analysis were 87.4% (vonoprazan-based triple therapy) versus 88.0% (PPI-based triple therapy. By per protocol analysis: 96.3% (vonoprazan-based triple therapy) versus 94.0% (PPI-based triple therapy). Clarithromycin resistance predicted treatment failure on multivariate analysis: RR 11.4; 95% CI [1.4-96.3], p = 0.025. No significant differences in CYP 2C19 genotypes or adverse events occurred between groups., Conclusion: One-week vonoprazan-based triple therapy achieved comparable efficacy to 2-week PPI-based triple therapy and was well tolerated., (© 2022 John Wiley & Sons Ltd.)

The primary objectives of this study were to investigate the suitability of a 6-probe cocktail (caffeine, tolbutamide, omeprazole, dextromethorphan, midazolam, and digoxin) to be used as a tool for assessing the activity of drug metabolizing enzymes and transporters, and examine differences in the way drugs are handled among groups with different genetic regulation of these processes. This was a single-center, open-label, phase I clinical study involving 20 young, healthy Chinese volunteers (equal gender distribution). The subjects were administered a single, oral dose of the 6-probe cocktail and serum samples were collected to assess the disposition of the different probe substrates and produced metabolites. The serum samples were analyzed using ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry technology. The DNA samples were subjected to whole exome sequencing. Nineteen healthy volunteers completed the study. The 6-probe cocktail was safe and well-tolerated by all the subjects. The parent substrates and metabolites-caffeine (paraxanthine), dextromethorphan (dextrorphan), digoxin, midazolam (1-hydroxy-midazolam), omeprazole (5-hydroxy-omeprazole), and tolbutamide (4-hydroxy-tolbutamide)-were within the detectable window. Genetic variations known to alter drug metabolism (CYP2D6*10, CYP2C19*2, CYP2C19*3, and CYP2C9*3) were identified and generally correlated with phenotypic status. The 6-probe cocktail appeared to be suitable for assessing drug metabolizing activities. This, in conjunction with individual genetics, will pave the way for the implementation of personalized medicine in clinical practice. This will hopefully improve efficacy and reduce the incidence of adverse drug reactions., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Myelodysplastic syndromes (MDS) are heterogeneous groups of clonal myeloid disorders characterized by unexplained persistent peripheral blood (PB) cytopenia(s) of one or more of the hematopoietic lineages, or bone marrow (BM) morphologic dysplasia in hematopoietic cells, recurrent genetic abnormalities, and an increased risk of progression to acute myeloid leukemia (AML). In the past several years, diagnostic, prognostic, and therapeutic approaches have substantially improved with the development of Next Generation Sequencing (NGS) diagnostic testing and new medications. However, there is no single diagnostic parameter specific for MDS, and correlations with clinical information, and laboratory test findings are needed to reach the diagnosis.

Objectives: Antibiotic resistance is a key reason for Helicobacter pylori (H. pylori) treatment failure and updated local data are important to guide the choice of empiric first-line therapy. Clarithromycin-based triple therapy (TT) is currently the first-line treatment in Singapore. Our primary aim was to determine the change in pattern of primary antibiotic resistance of H. pylori in Singapore as well as TT success rate., Methods: A registry of H. pylori isolates was tested for primary antibiotic resistance and were analyzed from year 2001 to 2020. Treatment outcome data based on 13 C-urea breath test ( 13 C-UBT) results of empiric first-line 2-week TT from 2019 to 2020 were also analyzed retrospectively., Results: A total of 387 H. pylori isolates were included. Rates of resistance to clarithromycin as well as tetracycline showed a downtrend in the last 5 years (clarithromycin: 2001-2002: 8.57%; 2007-2008: 14.14%; 2013-2014: 18.09%; 2019-2020: 13.71%; tetracycline: 2001-2002: 5.71%; 2007-2008: 7.07%; 2013-2014: 7.45%; 2019-2020: 0%). The resistance rate for amoxicillin remained low (2001-2002: 2.86%; 2007-2008: 5.05%; 2013-2014: 4.26%; 2019-2020: 7.26%). There was a steady increase in the rate of levofloxacin resistance (2001-2002: 5.71%; 2007-2008: 11.11%; 2013-2014: 15.96%; 2019-2020: 16.94%). From 2019 to 2020, the rate of successful TT was 91.67% (891/972)., Conclusion: The latest rate of clarithromycin resistance in 2019 to 2020 remained below the threshold of 15% and eradication rates remain high; thus, TT remains appropriate as an empiric first-line treatment in Singapore., (© 2022 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

GASTROSWOT is a strategic analysis of the current and projected states of the different subspecialties in gastroenterology that aims to provide guidance for research, clinical, and financial planning in gastroenterology. We executed a consensus-based international strengths, weaknesses, opportunities, and threats (SWOT) analysis. Four general coordinators, six field coordinators, and 12 experts participated in the study. SWOTs were provided for the following fields: neurogastroenterology, functional gastrointestinal disorders, and upper gastrointestinal diseases; inflammatory bowel disease; pancreatology and biliary diseases; endoscopy; gastrointestinal oncology; and hepatology. The GASTROSWOT analysis highlights the following in the current state of the field of gastroenterology: the incidence and complexity of several gastrointestinal diseases, including malignancies, are increasing; the COVID-19 pandemic has affected patient care on several levels; and with the advent of technical innovations in gastroenterology, a well trained workforce and strategic planning are required to optimise health-care utilisation. The analysis calls attention to the following in the future of gastroenterology: artificial intelligence and the use of big data will speed up discovery and smarter health-care provision in the field; the growth and diversification of gastroenterological specialties will improve specialised care for patients, but could promote fragmentation of care and health system inefficiencies; and furthermore, thoughtful planning is needed to reach an effective balance between the need for subspecialists and the value of general gastroenterology services., Competing Interests: Declaration of interests EdM is a consultant for Takeda Pharmaceutical Company, Abbott, and Mylan. WA received consultancy fees from AbbVie, Amgen, Arena Pharmaceuticals, Dynacare, Janssen, Merck, Novartis, Pfizer, Sandoz, and Takeda. SL received speaker and advisor fees from Gilead and AbbVie, and grants from Gilead. RP is a consultant to HCL Technologies, and on the scientific advisory board at Nestlé. VKS is consultant to AbbVie and Nestlé Health Science and has received grant funding from Orgenesis and Theraly. JV is a consultant to Olympus America, and on the scientific advisory board at Aspero Medical. AC consults for, is on the advisory board of, and has received education grants from from AbbVie, Takeda, Janssen, and Pfizer. The Radboud University Medical Center, on behalf of JPHD, received honoraria or research grants from Gilead and AbbVie. All other authors declare no competing interests., (Crown Copyright © 2022 Published by Elsevier Ltd. All rights reserved.)

Background: Anorectal manometry (ARM) is essential for identifying sphincteric dysfunction. The International Anorectal Physiology Working Group (IAPWG) protocol and London Classification provide a standardized format for performing and interpreting ARM. However, there is scant evidence to support timing and number of constituent maneuvers., Aims: To assess the impact of protocol modification on diagnostic accuracy in patients with fecal incontinence., Methods: Retrospective analysis of high-resolution ARM recordings from consecutive patients based on the current IAPWG protocol and modifications thereof: (1) baseline rest period (60 vs. 30 vs. 10 s); (2) number of abnormal short squeezes (SS) out of 3 (SS1/SS2/SS3) based on maximal incremental squeeze pressures over 5 s; (3) resting anal pressures (reflecting recovery) at 25-30 versus 15-20 s after SS1., Results: One hundred patients (86 F, median age 55 [IQR: 39-65]; median St. Mark's incontinence score 14 [10-17]) were studied. 26% and 8% had anal hypotonia and hypertonia, respectively. Compared with 60-s resting pressure, measurements had perfect correlation (κ = 1.0) over 30 s, and substantial correlation (κ = 0.85) over 10 s. After SS1, SS2, and SS3, 43%, 49%, and 46% had anal hypocontractility, respectively. Correlation was substantial between SS1 and SS2 (κ = 0.799) and almost perfect between SS2 and SS3 (κ = 0.9). Compared to resting pressure of 5 s before SS1, pressure recordings at 25-30 and 15-20 s after SS1 were significantly correlated., Conclusions: A 30-s resting anal pressure, analysis of 2 short-squeezes with a 20-s between-maneuver recovery optimizes study duration without compromising diagnostic accuracy. These findings indicate the IAPWG protocol has redundancy., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: We aimed to provide a practical and evidence-based guide on the indications, performance and reporting of high-resolution oesophageal manometry (HRM) and ambulatory pH monitoring (PHM) in adult patients in Singapore., Methods: The guideline committee comprised local gastroenterologists from public and private sectors with particular expertise in aspects of HRM and PHM, and it was tasked to produce evidence-based statements on the indications, performance and reporting of these tests. Each committee member performed literature searches to retrieve relevant articles within the context of domains to which they were assigned., Results: Twelve recommendation statements were created and summarised., Conclusion: Standardising key aspects of HRM and PHM is imperative to ensure the delivery of high-quality care. We reported the development of recommendations for the performance and interpretation of HRM and ambulatory reflux monitoring in Singapore.

Introduction: The objectives of this study were to examine the effects of ethnicity, gender and a proton pump inhibitor (PPI), omeprazole, on the human gut microbiome. PPIs are commonly used for the treatment of acid-related disorders. We hypothesised that PPI therapy might perturb microbial communities and alter the gut microbiome., Methods: Healthy subjects of Chinese (n = 12), Malay (n = 12) and Indian (n = 10) ancestry, aged 21-37 years, were enrolled. They provided a baseline stool sample (Day 1) and were then given a course of omeprazole at therapeutic dose (20 mg daily) for seven days. Stool samples were collected again on Day 7 and 14 (one week after stopping omeprazole). Microbial DNA was extracted from the stool samples, followed by polymerase chain reaction, library construction, 16S rRNA sequencing using Illumina MiSeq, and statistical and bioinformatics analyses., Results: The findings showed an increase in species richness (p = 0.018) after omeprazole consumption on Day 7, which reverted to baseline on Day 14. There were significant increases in the relative abundance of Streptococcus vestibularis (p = 0.0001) and Veillonella dispar (p = 0.0001) on Day 7, which diminished on Day 14. Faecalibacterium prausnitzii, Sutterella stercoricanis and Bacteroides denticanum were characteristic of Chinese, Malays and Indians, respectively. Lactobacillaceae and Bacteroides xylanisolvens were the signature taxa of male and female subjects, respectively., Conclusion: The study demonstrated alterations in the gut microbiome following omeprazole treatment. This may explain the underlying pathology of increased risk of Clostridium difficile infections associated with omeprazole therapy., (Copyright: © Singapore Medical Association.)

Background: The use of high-resolution manometry (HRM) to diagnose oesophageal motility disorders is based on ten single water swallows (SWS); however, this approach might not be representative of oesophageal function during the ingestion of normal food. We tested whether inclusion of a standardised solid test meal (STM) to HRM studies increases test sensitivity for major motility disorders. Additionally, we assessed the frequency and cause of patient symptoms during STM., Methods: Consecutive patients who were referred for investigation of oesophageal symptoms were recruited at Nottingham University Hospitals (Nottingham, UK) in the development study and at University Hospital Zürich (Zürich, Switzerland) in the validation study. HRM was done in the upright, seated position with a solid-state assembly. During HRM, patients ingested ten SWS, followed by a standardised 200 g STM. Diagnosis of oesophageal motility disorders was based on the Chicago Classification validated for SWS (CCv3) and with STM (CC-S), respectively. These studies are registered with ClinicalTrials.gov, numbers NCT02407938 and NCT02397616., Findings: The development cohort included 750 patients of whom 360 (48%) had dysphagia and 390 (52%) had reflux or other symptoms. The validation cohort consisted of 221 patients, including 98 (44%) with dysphagia and 123 (56%) with reflux symptoms. More patients were diagnosed with a major motility disorder by use of an STM than with SWS in the development set (321 [43%] patients diagnosed via STM vs 163 [22%] via SWS; p<0·0001) and validation set (73 [33%] vs 49 [22%]; p=0·014). The increase was most evident in patients with dysphagia (241 [67%] of 360 patients on STM vs 125 [35%] patients on SWS in the development set, p<0·0001), but was also present in those referred with reflux symptoms (64 [19%] of 329 patients vs 32 [10%] patients in the development set, p=0·00060). Reproduction of symptoms was reported by nine (1%) of 750 patients during SWS and 461 (61%) during STM (p<0·0001). 265 (83%) of 321 patients with major motility disorders and 107 (70%) of 152 patients with minor motility disorders reported symptoms during the STM (p=0·0038), compared with 89 (32%) of 277 patients with normal motility as defined with CC-S (p<0·0001)., Interpretation: The diagnostic sensitivity of HRM for major motility disorders is increased with use of the STM compared with SWS, especially in patients with dysphagia. Observations made during STM can establish motility disorders as the cause of oesophageal symptoms., Funding: None., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: The factors that determine how people eat when they are healthy or have disease have not been defined. We used high resolution manometry (HRM) to assess pharyngeal swallowing and oesophageal motility during ingestion of a solid test meal (STM) in healthy volunteers and patients with motility disorders., Methods: This study was based at University Hospital Zurich (Zürich, Switzerland). Healthy volunteers who responded to an advertisement completed HRM with ten single water swallows (SWS) in recumbent and upright positions followed by a 200 g rice STM in the upright position. Healthy volunteers were stratified for age and sex to ensure a representative population. For comparison, consecutive patients with major motility disorders on SWS and patients with dysphagia but no major motility disorders on SWS (disease controls) were selected from a database that was assembled prospectively; the rice meal data were analysed retrospectively. During STM, pharyngeal swallows were timed and oesophageal contractions were classified as representing normal motility or different types of abnormal motility in accordance with established metrics. Factors that could potentially be associated with eating speed were investigated, including age, sex, body-mass index, and presence of motility disorder. We compared diagnoses based on SWS findings, assessed with the Chicago Classification v3.0, with those based on STM findings, assessed with the Chicago Classification adapted for solids. These studies are registered with ClinicalTrials.gov, numbers NCT02407938 and NCT02397616., Findings: Between April 2, 2014, and May 13, 2015, 72 healthy volunteers were recruited and underwent HRM. Additionally, we analysed data from 54 consecutive patients with major motility disorders and 53 with dysphagia but no major motility disorders recruited between April 2, 2013, and Dec 18, 2014. We found important variations in oesophageal motility and eating speed during meal ingestion in healthy volunteers and patients. Increased time between swallows was accompanied by more effective oesophageal contractions (in healthy volunteers, 20/389 [5%] effective swallows at <4 s between swallows vs 586/900 [65%] effective swallows at >11 s between swallows, p<0·0001). Obstructive, spastic, or hypercontractile swallows were rare in healthy volunteers (total <1%). Patients with motility disorders ate slower than healthy volunteers (14·95 g [IQR 11-25] per min vs 32·9 g [25-40] per min, p<0·0001) and pathological oesophageal motility were reproduced when patients consumed the STM. In healthy volunteers, eating speed was associated only with frequency of swallows (slope 2·5 g per min per pharyngeal swallow per min [95% CI 1·1-4·0], p=0·0009), whereas in patients with dysphagia, it was correlated with frequency of effective oesophageal contractions (6·4 g per min per effective contraction per min [4·3-8·5], p<0·0001). Diagnostic agreement was good between the HRM with SWS and rice STM (intra-class correlation coefficient r=0·81, 95% CI 0·74-0·87, p<0·0001)., Interpretation: Our results show normative values for pharyngeal swallowing and oesophageal motility in healthy volunteers. Detailed analysis of HRM data acquired during an STM shows that the rate-limiting factor for intake of solids in health is the frequency of pharyngeal swallowing and not oesophageal contractility. The reverse is true in patients with oesophageal motility disorders, in whom the frequency of effective oesophageal contractions determines eating speed., Funding: University Hospital Zurich., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

About one-third of patients with suspected gastro-oesophageal reflux disease (GERD) do not respond symptomatically to proton pump inhibitors (PPIs). Many of these patients do not suffer from GERD, but may have underlying functional heartburn or atypical chest pain. Other causes of failure to respond to PPIs include inadequate acid suppression, non-acid reflux, oesophageal hypersensitivity, oesophageal dysmotility and psychological comorbidities. Functional oesophageal tests can exclude cardiac and structural causes, as well as help to confi rm or exclude GERD. The use of PPIs should only be continued in the presence of acid reflux or oesophageal hypersensitivity for acid reflux-related events that is proven on functional oesophageal tests., (Copyright: © Singapore Medical Association.)

The management of patients with refractory GERD (rGERD) is a major clinical challenge for gastroenterologists. In up to 30% of patients with typical GERD symptoms (heartburn and/or regurgitation), acid-suppressive therapy does not provide clinical benefit. In this Review, we discuss the current management algorithm for GERD and the features and management of patients who do not respond to treatment (such as those individuals with an incorrect diagnosis of GERD, inadequate PPI intake, persisting acid reflux and persisting weakly acidic reflux). Symptom response to existing surgical techniques, novel antireflux procedures, and the value of add-on medical therapies (including prokinetics and reflux inhibitors) for rGERD symptoms are discussed. Pharmaceutical agents targeting oesophageal sensitivity, a condition that can contribute to symptom generation in rGERD, are also discussed. Finally, on the basis of available published data and our expert opinion, we present an outline of a current, usable algorithm for management of patients with rGERD that considers the timing and diagnostic use of pH-impedance monitoring on or off PPI, additional diagnostic tests, the clinical use of baclofen and the use of add-on neuromodulators (tricyclic agents and selective serotonin reuptake inhibitors).

The phosphatidylinositol-3-kinase pathway is one of the most commonly altered molecular pathways in invasive breast carcinoma, with phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) mutations in 25% of invasive carcinomas. Ductal carcinoma in situ (DCIS), benign papillomas, and small numbers of columnar cell lesions harbor an analogous spectrum of PIK3CA and AKT1 mutations, yet there is little data on usual ductal hyperplasia and atypical ductal and lobular neoplasias. We screened 192 formalin-fixed paraffin-embedded breast lesions from 75 patients for point mutations using a multiplexed panel encompassing 643 point mutations across 53 genes, including 58 PIK3CA substitutions. PIK3CA point mutations were identified in 31/62 (50%) proliferative lesions (usual ductal hyperplasia and columnar cell change), 10/14 (71%) atypical hyperplasias (atypical ductal hyperplasia and flat epithelial atypia), 7/16 (44%) lobular neoplasias (atypical lobular hyperplasia and lobular carcinoma in situ), 10/21 (48%) DCIS, and 13/37 (35%) invasive carcinomas. In genotyping multiple lesions of different stage from the same patient/specimen, we found considerable heterogeneity; most notably, in 12 specimens the proliferative lesion was PIK3CA mutant but the concurrent carcinoma was wild type. In 11 additional specimens, proliferative epithelium and cancer contained different point mutations. The frequently discordant genotypes of usual ductal hyperplasia/columnar cell change and concurrent carcinoma support a role for PIK3CA-activating point mutations in breast epithelial proliferation, perhaps more so than transformation. Further, these data suggest that proliferative breast lesions are heterogeneous and may represent non-obligate precursors of invasive carcinoma.

KRAS mutations define a clinically distinct subgroup of lung adenocarcinoma patients, characterized by smoking history, resistance to EGFR-targeted therapies, and adverse prognosis. Whether KRAS-mutated lung adenocarcinomas also have distinct histopathological features is not well established. We tested 180 resected lung adenocarcinomas for KRAS and EGFR mutations by high-sensitivity mass spectrometry-based genotyping (Sequenom) and PCR-based sizing assays. All tumors were assessed for the proportion of standard histological patterns (lepidic, acinar, papillary, micropapillary, solid, and mucinous), several other histological and clinical parameters, and TTF-1 expression by immunohistochemistry. Among 180 carcinomas, 63 (35%) had KRAS mutations (KRAS+), 35 (19%) had EGFR mutations (EGFR+), and 82 (46%) had neither mutation (KRAS-/EGFR-). Solid growth pattern was significantly over-represented in KRAS+ carcinomas: the mean±s.d. for the amount of solid pattern in KRAS+ carcinomas was 27±34% compared with 3±10% in EGFR+ (P<0.001) and 15±27% in KRAS-/EGFR- (P=0.033) tumors. Furthermore, at least focal (≥20%) solid component was more common in KRAS+ (28/63; 44%) compared with EGFR+ (2/35; 6%; P<0.001) and KRAS-/EGFR- (21/82; 26%; P=0.022) carcinomas. KRAS mutations were also over-represented in mucinous carcinomas and were significantly associated with the presence of tumor-infiltrating leukocytes and heavier smoking history. EGFR mutations were associated with non-mucinous non-solid patterns, particularly lepidic and papillary, lack of necrosis, lack of cytological atypia, hobnail cytology, TTF-1 expression, and never/light smoking history. In conclusion, extended molecular and clinicopathological analysis of lung adenocarcinomas reveals a novel association of KRAS mutations with solid histology and tumor-infiltrating inflammatory cells and expands on several previously recognized morphological and clinical associations of KRAS and EGFR mutations. Solid growth pattern was recently shown to be a strong predictor of aggressive behavior in lung adenocarcinomas, which may underlie the unfavorable prognosis associated with KRAS mutations in these tumors.

The pathognomonic genetic alteration in chronic myeloid leukemia is the formation of the BCR-ABL1 fusion gene, which produces a constitutively active tyrosine kinase that drives leukemic transformation. Targeted tyrosine kinase inhibitor treatment with imatinib, nilotinib, dasatinib, bosutinib, and ponatinib is the cornerstone of modern therapy for this hematologic malignancy. Real-time quantitative RT-PCR (RT-qPCR, also RQ-PCR) of BCR-ABL1 RNA is a necessary laboratory technique for monitoring the efficacy of tyrosine kinase inhibitor therapy and quantitatively assessing minimal residual disease. The molecular response measured by BCR-ABL1 RT-qPCR assists in identifying suboptimal responses and can help inform the decision to switch to alternative therapies that may be more efficacious (or to pursue more stringent monitoring). Furthermore, the tyrosine kinase inhibitor-mediated molecular response provides valuable risk stratification and prognostic information on long-term outcomes. Despite these attributes, informed, universal, practical utilization of this well-established monitoring test will require heightened efforts by the molecular diagnostics laboratory community to adopt the standardized reporting units of the International Scale. Without widespread adoption of the International Scale, the consensus major molecular response and early molecular response treatment thresholds will not be definable, and optimal clinical outcomes for patients with chronic myeloid leukemia may not be achieved., (Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Micropapillary carcinoma of the breast is associated with increased rates of lymph node metastasis and lymphovascular invasion. While activating point mutations in PIK3CA (encoding phosphatidylinositol-3-kinase catalytic subunit) or AKT1 are found in 25% to 30% of invasive ductal carcinomas, the mutational profile of invasive micropapillary carcinomas has not been characterized in detail. Micropapillary carcinomas, concurrent metastatic and precursor breast lesions from 19 patients were identified. Lesional tissue was punched from paraffin-tissue blocks, and genomic DNA was extracted and screened for a large panel of known hotspot mutations using multiplex polymerase chain reaction and mass-spectroscopy analysis (643 mutations in 53 genes). Hotspot point mutations were identified in 35% (7/20) of micropapillary breast carcinomas, including PIK3CA exons 7, 9 and 20 hotspots, as well as the AKT1 plekstrin homology domain mutation (E17K); mutations in TP53 and KRAS were each found in a single patient. In 6 patients, micropapillary and non-micropapillary components of the same tumor were separately tested, yielding concordant results in five; one had a wild type micropapillary component, but a PIK3CA mutation in the invasive ductal component. Concurrent lymph node metastases were mostly wild type (2/8 mutant). Accompanying ductal carcinoma in situ had point mutations in 45% (5/11), mostly concordant with invasive carcinoma; however, mutational status of other breast proliferative lesions was generally discordant with accompanying carcinoma. The rate of PIK3CA mutations in this series of micropapillary carcinomas is similar to invasive ductal carcinomas; however, there may be an enrichment of AKT1 mutations (10%). The non-micropapillary components and precursor lesions occasionally had different mutations., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Somatic mutations in PIK3CA are commonly seen in invasive breast cancer and several other carcinomas, occurring in three hotspots: codons 542 and 545 of exon 9 and in codon 1047 of exon 20. We designed a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant PIK3CA DNA with attention to avoiding amplification of a pseudogene on chromosome 22 that has >95% homology to exon 9 of PIK3CA. We tested 60 FFPE breast DNA samples with known PIK3CA mutation status (48 cases had one or more PIK3CA mutations, and 12 were wild type) as identified by PCR-mass spectrometry. PIK3CA exons 9 and 20 were amplified in the presence or absence of LNA-oligonucleotides designed to bind to the wild-type sequences for codons 542, 545, and 1047, and partially suppress their amplification. LNA-PCR sequencing confirmed all 51 PIK3CA mutations; however, the mutation detection rate by standard Sanger sequencing was only 69% (35 of 51). Of the 12 PIK3CA wild-type cases, LNA-PCR sequencing detected three additional H1047R mutations in "normal" breast tissue and one E545K in usual ductal hyperplasia. Histopathological review of these three normal breast specimens showed columnar cell change in two (both with known H1047R mutations) and apocrine metaplasia in one. The novel LNA-PCR shows higher sensitivity than standard Sanger sequencing and did not amplify the known pseudogene., (Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Wireless oesophageal (Bravo) readings in healthy European and American subjects show varied results. Values in Asians remain unstudied., Aims: We performed Bravo studies in healthy Asians to determine if values differed from previously published and identified reflux parameters to differentiate healthy volunteers from patients., Methods: Fifty healthy volunteers were recruited between August 2009 and August 2011 to undergo 48 h wireless pH monitoring. Bravo capsule was introduced transorally and placed 6 cm above the squamocolumnar junction. Acid reflux parameters were compared against 41 patients previously evaluated for non-erosive reflux disease., Results: Five volunteers were excluded. Capsule dislodgement occurred in four and three volunteers on study days 1 and 2 respectively. Forty and 37 volunteers (73% male, mean age 33.0 ± 7.6 years) had interpretable readings at 24 and 48 h, respectively. Percentage of time oesophageal pH<4 in 37 volunteers who completed 48 h recordings was 1.6% (7.5%), 1.5% (6.3%) and 1.9% (5.8%) on days 1, 2 and overall study duration, respectively., Conclusion: Bravo readings in Asians differed from previously published. Percentage of time oesophageal pH<4 on day 2 and DeMeester score on day 2 (95th percentile 22.9) best discriminated healthy volunteers from patients., (Copyright © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Activating point mutations in the phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) are among the most common molecular defects in invasive breast cancer. Point mutations in the downstream kinase AKT1 are seen in a minority of carcinomas. These mutations are found preferentially in estrogen receptor-positive and Her2-positive breast carcinomas; however, special morphologic types of breast cancer have not been well studied. Twenty-nine cases of pure invasive mucinous carcinoma and 9 cases of ductal carcinoma with mucinous differentiation were screened for a panel of point mutations (>321 mutations in 30 genes) using a multiplex polymerase chain reaction panel with mass spectroscopy readout. In addition, associated ductal carcinoma in situ, hyperplasia, or columnar cell lesions were separately tested where available (25 lesions). In 3 invasive cases and 15 ductal carcinoma in situ/proliferative lesions, PIK3CA hotspot mutations were, instead, tested by direct sequencing. No point mutations were identified in invasive mucinous breast carcinoma. This contrasts with the 35% frequency of PIK3CA mutations in a comparative group of invasive ductal carcinomas of no special type. Interestingly, PIK3CA hotspot point mutations were identified in associated ductal carcinoma in situ (3/14) and hyperplasia (atypical ductal hyperplasia [2/3], usual ductal hyperplasia [2/3], columnar cell change [1/5]), suggesting that PIK3CA mutations may play a role in breast epithelial proliferation. This series represents the largest study, to date, of PIK3CA genotyping in mucinous carcinoma and supports the unique pathogenetics of invasive mucinous breast carcinoma., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: EGFR mutation status is the best predictor of response to tyrosine kinase inhibitors (TKIS) in primary lung adenocarcinoma. Approximately 70% of lung cancers are diagnosed in advanced stages where small biopsies and cytological specimens are the only source of material for both diagnosis and mutation testing. Specific antibodies that can detect mutant EGFR protein were evaluated for the detection of EGFR mutation by immunohistochemistry (IHC) in cytology and small biopsy specimens., Methods: Assessment of EGFR mutation status was performed by using antibodies specific to the two major forms of mutant EGFR, exon 21 L858R and exon 19 deletion (15bp). The study was performed in 145 lung adenocarcinomas, including cytology material, core biopsy, and decalcified bone biopsy. Stains were scored as negative (0), 1+ (weak and focal), 2+ (moderate intensity and focal), and 3+ (strong and diffuse). The result of the IHC stains was correlated with mutations status determined by standard molecular methods., Results: Validation using clinical material showed deletions in exon 19 were detected in 35% and L858R mutation in 17.6% of all cases by standard molecular methods. A cutoff value of 2+ was used as positive by IHC. No wild type cases were immunoreactive. The positive predictive value (PPV) and specificity for both antibodies was 100%. The antibodies performed well in cytology, core biopsies and decalcified bone biopsies., Conclusion: Immunostaining to detect specific mutant EGFR shows a good correlation with mutation analysis and can be used as a screening method to identify patients for TKI therapy. IHC methodology is potentially useful when molecular analysis is not available and for use in small biopsies when material is too scant for molecular tests. Importantly mutation specific antibodies are useful in determining EGFR status in tissues obtained from bone biopsy as decalcification processes used in molecular based studies often result in DNA degradation hindering mutation detection., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Immunohistochemistry is increasingly utilized to differentiate lung adenocarcinoma and squamous cell carcinoma. However, detailed analysis of coexpression profiles of commonly used markers in large series of whole-tissue sections is lacking. Furthermore, the optimal diagnostic algorithm, particularly the minimal-marker combination, is not firmly established. We therefore studied whole-tissue sections of resected adenocarcinoma and squamous cell carcinoma (n=315) with markers commonly used to identify adenocarcinoma (TTF-1) and squamous cell carcinoma (p63, CK5/6, 34βE12), and prospectively validated the devised algorithm in morphologically unclassifiable small biopsy/cytology specimens (n=38). Analysis of whole-tissue sections showed that squamous cell carcinoma had a highly consistent immunoprofile (TTF-1-negative and p63/CK5/6/34βE12-diffuse) with only rare variation. In contrast, adenocarcinoma showed significant immunoheterogenetity for all 'squamous markers' (p63 (32%), CK5/6 (18%), 34βE12 (82%)) and TTF-1 (89%). As a single marker, only diffuse TTF-1 was specific for adenocarcinoma whereas none of the 'squamous markers,' even if diffuse, were entirely specific for squamous cell carcinoma. In contrast, coexpression profiles of TTF-1/p63 had only minimal overlap between adenocarcinoma and squamous cell carcinoma, and there was no overlap if CK5/6 was added as a third marker. An algorithm was devised in which TTF-1/p63 were used as the first-line panel, and CK5/6 was added for rare indeterminate cases. Prospective validation of this algorithm in small specimens showed 100% accuracy of adenocarcinoma vs squamous cell carcinoma prediction as determined by subsequent resection. In conclusion, although reactivity for 'squamous markers' is common in lung adenocarcinoma, a two-marker panel of TTF-1/p63 is sufficient for subtyping of the majority of tumors as adenocarcinomas vs squamous cell carcinoma, and addition of CK5/6 is needed in only a small subset of cases. This simple algorithm achieves excellent accuracy in small specimens while conserving the tissue for potential predictive marker testing, which is now an essential consideration in advanced lung cancer specimens.