DNA flow cytometry for detection of genomic instability as a cancer precursor in the gastrointestinal tract.

One of the earliest applications of flow cytometry was the measurement of DNA content in cells. This method is based on the ability to stain DNA in a stoichiometric manner (i.e., the amount of stain is directly proportional to the amount of DNA within the cell). For more than 40years, a number of studies have consistently demonstrated the utility of DNA flow cytometry as a potential diagnostic and/or prognostic tool in patients with most epithelial tumors, including pre-invasive lesions (such as dysplasia) in the gastrointestinal tract. However, its availability as a clinical test has been limited to few medical centers due to the requirement for fresh tissue in earlier studies and perceived technical demands. However, more recent studies have successfully utilized formalin-fixed paraffin-embedded (FFPE) tissue to generate high-quality DNA content histograms, demonstrating the feasibility of this methodology. This review summarizes step-by-step methods on how to perform DNA flow cytometry using FFPE tissue and analyze DNA content histograms based on the published consensus guidelines in order to assist in the diagnosis and/or risk stratification of many different epithelial tumors, with particular emphasis on dysplasia associated with Barrett's esophagus and inflammatory bowel disease.

DNA content abnormality frequently develops in the right/proximal colon in patients with primary sclerosing cholangitis and inflammatory bowel disease and is highly predictive of subsequent detection of dysplasia.

AIMS: Patients with primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD, termed PSC-IBD) have a higher risk of harbouring nonconventional and/or invisible dysplasias, especially in the right/proximal colon, than those with IBD alone. We postulated that DNA content abnormality may be frequently detected in the right/proximal colon in PSC-IBD patients, even in the absence of dysplasia, and that this may predispose to progression to nonconventional and/or invisible dysplasias that are often associated with increased rates of aneuploidy and advanced neoplasia. METHODS AND RESULTS: DNA flow cytometry was performed on 96 morphologically benign colon biopsies taken throughout the colon from 25 PSC-IBD patients during the surveillance colonoscopy that preceded the next procedure that detected dysplasia. Thirty (31%) of the 96 benign colon biopsies in this dysplasia group demonstrated abnormal DNA content, with a propensity for the right/proximal colon (70%) (P < 0.001). In contrast, only one (1%) of 87 benign colon biopsies from 20 IBD patients without neoplasia (control group) demonstrated DNA content abnormality, and it was from the left colon. For analysis per patient, 48% (12 of 25) of the patients in the dysplasia group had abnormal DNA content compared with 5% (1 of 20) of the control group (P = 0.002). Of the 12 PSC-IBD patients with DNA content abnormality, invisible dysplasia was detected in 10 (83%) patients on follow-up, nine (75%) of whom had nonconventional dysplasia. CONCLUSION: PSC-IBD patients have an increased risk of developing abnormal DNA content in the right/proximal colon, predating the detection of dysplasia.

Mitochondrial proton leak in cardiac aging.

Age-associated diseases are becoming progressively more prevalent, reflecting the increased lifespan of the world's population. However, the fundamental mechanisms of physiologic aging are poorly understood, and in particular, the molecular pathways that mediate cardiac aging and its associated dysfunction are unclear. Here, we focus on certain ion flux abnormalities of the mitochondria that may contribute to cardiac aging and age-related heart failure. Using oxidative phosphorylation, mitochondria pump protons from the matrix to the intermembrane space to generate a proton gradient across the inner membrane. The protons are returned to the matrix by the ATPase complex within the membrane to generate ATP. However, a portion of protons leak back to the matrix and do not drive ATP production, and this event is called proton leak or uncoupling. Accumulating evidence suggests that mitochondrial proton leak is increased in the cardiac myocytes of aged hearts. In this mini-review, we discuss the measurement methods and major sites of mitochondrial proton leak with an emphasis on the adenine nucleotide transporter 1 (ANT1), and explore the possibility of inhibiting augmented mitochondrial proton leak as a therapeutic intervention to mitigate cardiac aging.

Fundic Gland Polyps Lack DNA Content Abnormality Characteristic of Other Adenomatous Precursor Lesions in the Gastrointestinal Tract.

Fundic gland polyps (FGPs) develop sporadically (frequently after proton pump inhibitor therapy) or in the setting of a hereditary polyposis syndrome, such as familial adenomatous polyposis (FAP). FAP-related FGPs often demonstrate low-grade dysplasia (LGD) and are frequently associated with APC mutations, even in the absence of dysplasia. Sporadic FGPs with dysplasia are molecularly similar to FAP-related FGPs and demonstrate frequent mutations in APC gene. Despite having similar molecular alterations with colorectal and other adenomatous precursor lesions in the gastrointestinal (GI) tract, FGPs rarely progress to advanced gastric neoplasia (high-grade dysplasia [HGD] or adenocarcinoma), and their role in gastric tumorigenesis remains unclear but likely limited. The clinicopathologic features of 192 patients diagnosed with FGPs, including 86 with FAP-related FGPs (33 with dysplastic FGPs and 53 with nondysplastic FGPs) and 106 with sporadic FGPs (12 with dysplastic FGPs and 94 with nondysplastic FGPs), were analyzed. DNA flow cytometry was performed on 111 FAP-related FGP biopsies, including 32 FGPs with LGD and 79 nondysplastic FGPs, to assess the presence of abnormal DNA content (ie, aneuploidy or elevated 4N fraction). Moreover, 40 sporadic FGP biopsies, including 14 dysplastic (13 LGD and 1 HGD) and 26 nondysplastic FGPs, were examined for DNA content abnormality. Patients with FAP and nondysplastic FGPs were more likely to be younger (mean age, 32 years) and present with multiple FGPs (92%, defined as having ≥2 FGPs) than those with sporadic nondysplastic FGPs (61 years and 65%, respectively; P < .001). They also recorded higher rates of previous or concurrent gastric epithelial dysplasia not occurring in a FGP (8%, P = .016), nongastric GI dysplasia (96%, P < .001), and nongastric GI malignancy (17%, P = .001) compared with those with sporadic nondysplastic FGPs (0%, 52%, and 2%, respectively). The sporadic group was more frequently associated with proton pump inhibitor therapy (78%, P < .001), gastric intestinal metaplasia (24%, P = .004), and a family history of gastric cancer (10%, P = .027) than the FAP group (19%, 6%, and 0%, respectively). Almost all FAP-related FGPs had a polypoid endoscopic appearance (98% vs 84% for sporadic FGPs; P = .009). The mean size of the largest FAP-related FGPs (0.5 cm) was similar to that of sporadic FGPs (0.7 cm) (P = .069). None of the 147 patients with FAP-related or sporadic nondysplastic FGPs were associated with subsequent detection of advanced gastric neoplasia within a mean follow-up time of 54 months (range, <1 to 277 months). However, 2 (4%) of the 45 patients with FAP-related or sporadic dysplastic FGPs developed advanced gastric neoplasia within a mean follow-up time of 59 months (range, <1 to 236 months). One (3%) of the 33 patients with FAP and dysplastic FGPs developed signet ring cell adenocarcinoma, whereas 1 (8%) of the 12 patients with sporadic dysplastic FGPs developed HGD (P = .445). However, none of the FAP-related and sporadic FGP biopsies, regardless of the presence or absence of dysplasia, demonstrated DNA content abnormality. In conclusion, FGPs lack large-scale chromosomal changes that are characteristic of the typical adenoma-carcinoma sequence involved in the development of other GI malignancies. Progression to advanced gastric neoplasia is rare in FGPs, which may be partly explained by the apparent lack of the chromosomal instability phenotype in these lesions.

Role of DNA Flow Cytometry in the Diagnosis of Malignancy in Bile Duct Biopsies Using Paraffin-Embedded Tissue.

OBJECTIVES: Histopathologic evaluation of bile biopsies for biliary strictures is frequently challenging and is affected by interobserver disagreement. Reliable ancillary tests that can help differentiate benign from malignant are not available. This study aimed to evaluate whether DNA content abnormalities detected by flow cytometry on formalin-fixed, paraffin-embedded (FFPE) tissue can help differentiate benign/reactive, dysplastic from malignant cell populations in bile duct biopsies. METHODS: We performed DNA flow cytometry on 30 FFPE bile duct biopsies in 5 well-defined diagnostic categories: (1) negative for dysplasia (NED), (2) low-grade dysplasia (LGD), (3) high-grade dysplasia (HGD), (4) carcinoma (CA), and (5) indefinite for dysplasia (IND). RESULTS: Abnormal DNA content was detected in 0 NED, 5 LGD (62.5%), 2 HGD (33.3%), 3 CA (60%), and 4 IND (80%) samples. As a diagnostic marker, the estimated sensitivity, specificity, positive predictive value, and negative predictive value were 63%, 100%, 100%, and 50%, respectively, for diagnosing HGD or CA. CONCLUSIONS: DNA flow cytometry analysis is a useful ancillary test for the interpretation of bile duct biopsies. DNA content abnormalities, when correlated with histologic findings, will not only help confirm the morphologic impression but also identify patients who are at a higher risk of developing malignancy.

An Analysis of Metabolic Changes in the Retina and Retinal Pigment Epithelium of Aging Mice.

Purpose: The purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside retinal responses to light. Methods: Scotopic and photopic electroretinogram (ERG) responses in young (3-6 months) and aged (23-26 months) C57Bl/6J mice were recorded. Metabolic flux in retina and eyecup explants was quantified using U-13C-glucose or U-13C-glutamine with gas chromatography-mass spectrometry (GC-MS), O2 consumption rate (OCR) in a perifusion apparatus, and quantifying adenosine triphosphatase (ATP) with a bioluminescence assay. Results: Scotopic and photopic ERG responses were reduced in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected in aged mice. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable. Conclusions: Our examination of metabolism showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The metabolic stability of these tissues ex vivo suggests age-related metabolic alterations may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or structural changes influence ocular metabolism in vivo.

Elamipretide (SS-31) treatment attenuates age-associated post-translational modifications of heart proteins.

It has been demonstrated that elamipretide (SS-31) rescues age-related functional deficits in the heart but the full set of mechanisms behind this have yet to be determined. We investigated the hypothesis that elamipretide influences post-translational modifications to heart proteins. The S-glutathionylation and phosphorylation proteomes of mouse hearts were analyzed using shotgun proteomics to assess the effects of aging on these post-translational modifications and the ability of the mitochondria-targeted drug elamipretide to reverse age-related changes. Aging led to an increase in oxidation of protein thiols demonstrated by increased S-glutathionylation of cysteine residues on proteins from Old (24 months old at the start of the study) mouse hearts compared to Young (5-6 months old). This shift in the oxidation state of the proteome was almost completely reversed by 8 weeks of treatment with elamipretide. Many of the significant changes that occurred were in proteins involved in mitochondrial or cardiac function. We also found changes in the mouse heart phosphoproteome that were associated with age, some of which were partially restored with elamipretide treatment. Parallel reaction monitoring of a subset of phosphorylation sites revealed that the unmodified peptide reporting for Myot S231 increased with age, but not its phosphorylated form and that both phosphorylated and unphosphorylated forms of the peptide covering cMyBP-C S307 increased, but that elamipretide treatment did not affect these changes. These results suggest that changes to thiol redox state and phosphorylation status are two ways in which age may affect mouse heart function, which can be restored by treatment with elamipretide.

Nonampullary Duodenal Adenomas in Familial Adenomatous Polyposis and Sporadic Patients Lack the DNA Content Abnormality That Is Characteristic of the Adenoma-Carcinoma Sequence Involved in the Development of Other Gastrointestinal Malignancies.

Nonampullary duodenal adenomas (NADAs) develop sporadically or in the setting of a hereditary syndrome such as familial adenomatous polyposis (FAP). Although they are thought to progress into duodenal adenocarcinomas via an adenoma to carcinoma sequence similar to colorectal cancer, limited data suggested that they may be biologically dissimilar to colorectal adenomas. The clinicopathologic features of 71 patients diagnosed with NADAs (37 FAP and 34 sporadic) were analyzed. From the 71 patients, 89 NADA biopsies (42 FAP and 47 sporadic) were evaluated by DNA flow cytometry. Eighty-two samples showed low-grade dysplasia, and 7 demonstrated high-grade dysplasia (HGD). Twenty-one low-grade adenomas of the ileal pouch (n=19) and jejunum (n=2) from 15 FAP patients who underwent total proctocolectomy were also analyzed by DNA flow cytometry. The FAP patients were more likely to be younger (mean: 28 y) and have multifocal disease (92%) than the sporadic patients (66 y and 24%, respectively) (P<0.001). Most NADAs presented as polypoid lesions (87%) in the duodenal bulb and/or second portion of the duodenum (94%). Sporadic NADAs (mean: 2.4 cm) were significantly larger than FAP-related NADAs (1.3 cm) (P=0.005). Three (4%) patients (2 sporadic and 1 FAP) had high-grade NADAs at the first endoscopy, while the remaining 68 (96%) patients had low-grade dysplasia. Two additional sporadic and 1 FAP patients developed HGD on follow-up. Although the overall detection rate of advanced neoplasia (either HGD or adenocarcinoma) was similar between the FAP (n=5; 14%) and sporadic groups (n=4; 12%) (P=1.000), 3 FAP patients (all with Spigelman stage III to IV) developed adenocarcinoma in the duodenum (n=2) or in the ileal pouch (n=1) within a mean follow-up time of 76 months, while no adenocarcinoma was found in the sporadic group. Of the 37 FAP patients, 29 (78%) had a history of total proctocolectomy, and 15 (52%) developed low-grade adenomas in the ileal pouch with (n=2) or without (n=13) jejunal involvement (vs. 0% in the sporadic patients, P<0.001). All 15 patients had ≥Spigelman stage II. Aneuploidy was detected in only 1 (1%) sporadic NADA with HGD, whereas the remaining 109 duodenal, ileal pouch, and jejunal adenomas showed normal DNA content. The overall 3-, 9-, and 15-year detection rates of adenocarcinoma (in the duodenum and ileal pouch) in all NADA patients were 1.4%, 7.2%, and 18.8%, respectively. Three-, 9-, and 15-year detection rates of adenocarcinoma in the FAP patients were 2.7%, 9.7%, and 22.6%, respectively, while these rates remained at 0% in the sporadic patients. In conclusion, FAP-related NADAs have distinct clinicopathologic features compared with their sporadic counterpart. However, the vast majority of both FAP-related and sporadic NADAs (99%) lack the DNA content abnormality that is characteristic of the typical adenoma-carcinoma sequence involved in other gastrointestinal carcinogenesis. Although adenocarcinoma is more likely to develop in FAP patients with a high adenoma burden, probably due to the higher likelihood that some advanced lesions are missed endoscopically, FAP-related and sporadic NADAs may have a comparable risk of developing advanced neoplasia on a per-adenoma basis.

Persistent or recurrent Barrett's neoplasia after an endoscopic therapy session is associated with DNA content abnormality and can be detected by DNA flow cytometric analysis of paraffin-embedded tissue.

Endoscopic therapy is currently the standard of care for the treatment of high-grade dysplasia (HGD) or intramucosal adenocarcinoma (IMC) in patients with Barrett's esophagus (BE). Visible lesions are treated with endoscopic mucosal resection (EMR), which is often coupled with radiofrequency ablation (RFA). However, endoscopic therapy may require multiple sessions (one session every 2-3 months) and does not always assure complete eradication of neoplasia. Furthermore, despite complete eradication, recurrences are not uncommon. This study assesses which potential risk factors can predict a poor response after endoscopic sessions. Forty-five BE patients who underwent at least one endoscopic session (EMR alone or ablation with or without preceding EMR) for the treatment of HGD/IMC, low-grade dysplasia (LGD), or indefinite for dysplasia (IND) were analyzed. DNA flow cytometry was performed on 82 formalin-fixed paraffin-embedded samples from the 45 patients, including 78 HGD/IMC, 2 LGD, and 2 IND. Eight non-dysplastic BE samples were used as controls. Three to four 60-micron thick sections were cut from each tissue block, and the area of HGD/IMC, LGD, or IND was manually dissected. Potential associations between clinicopathologic risk factors and persistent/recurrent HGD/IMC following each endoscopic session were examined using univariate and multivariate Cox models with frailty terms. Sixty (73%) of the 82 specimens showed abnormal DNA content (aneuploidy or elevated 4N fraction). These were all specimens with HGD/IMC (representing 77% of that group). Of these 60 HGD/IMC samples with abnormal DNA content, 42 (70%) were associated with subsequent development of persistent/recurrent HGD/IMC (n = 41) or esophageal adenocarcinoma (EAC; n = 1) within a mean follow-up time of 16 months (range: 1 month to 9.4 years). In contrast, only 6 (27%, all HGD/IMC) of the 22 remaining samples (all with normal DNA content) were associated with persistent/recurrent HGD/IMC. For outcome analysis per patient, 11 (24%) of the 45 patients developed persistent/recurrent HGD/IMC or EAC, despite multiple endoscopic sessions (mean: 3.6, range: 1-11). In a univariate Cox model, the presence of abnormal DNA content (hazard ratio [HR] = 3.8, p = 0.007), long BE segment ≥ 3 cm (HR = 3.4, p = 0.002), endoscopic nodularity (HR = 2.5, p = 0.042), and treatment with EMR alone (HR = 2.9, p = 0.006) were significantly associated with an increased risk for persistent/recurrent HGD/IMC or EAC. However, only abnormal DNA content (HR = 6.0, p = 0.003) and treatment with EMR alone (HR = 2.7, p = 0.047) remained as significant risk factors in a multivariate analysis. Age ≥ 60 years, gender, ethnicity, body mass index (BMI) ≥ 30 kg/m2, presence of hiatal hernia, and positive EMR lateral margin for neoplasia were not significant risk factors for persistent/recurrent HGD/IMC or EAC (p > 0.05). Three-month, 6-month, 1-year, 3-year, and 6-year adjusted probabilities of persistent/recurrent HGD/IMC or EAC in the setting of abnormal DNA content were 31%, 56%, 67%, 79%, and 83%, respectively. The corresponding probabilities in the setting of normal DNA content were 10%, 21%, 28%, 38%, and 43%, respectively. In conclusion, in BE patients with baseline HGD/IMC, both DNA content abnormality and treatment with EMR alone were significantly associated with persistent/recurrent HGD/IMC or EAC following each endoscopic session. DNA content abnormality as detected by DNA flow cytometry identifies HGD/IMC patients at highest risk for persistent/recurrent HGD/IMC or EAC, and it also serves as a diagnostic marker of HGD/IMC with an estimated sensitivity of 77%. The diagnosis of HGD/IMC in the setting of abnormal DNA content may warrant alternative treatment strategies as well as long-term follow-up with shorter surveillance intervals.

Gastric Intestinal Metaplasia in Mucosa Adjacent to Gastric Cancers Is Rarely Associated With the Aneuploidy That Is Characteristic of Gastric Dysplasia or Cancer.

Most gastric cancers (GCs) are thought to develop via gastric intestinal metaplasia (GIM)-dysplasia-carcinoma pathway. Patients with extensive and/or incomplete GIM have been reported to have a higher risk of GC. GIM can also display dysplasia-like cytoarchitectural atypia limited to the bases of gastric pits without surface involvement. However, only a small proportion of GIM patients will develop gastric neoplasia, and it remains questionable if GIM is a direct precursor. A cohort of 82 GC patients with GIM who underwent gastrectomy were analyzed. DNA flow cytometry was performed on 109 GIM samples (including 88 predominantly complete GIM and 21 predominantly incomplete GIM subclassified based on morphology) obtained from adjacent mucosa of the 82 GCs. Only 2 (2%) of the 109 GIM samples demonstrated aneuploidy, both from 2 minority patients (Asian and Hispanic) with limited and complete GIM and no cytoarchitectural atypia. The remaining 107 GIM samples showed mild to focally moderate basal gland (metaplastic) atypia limited to the bases of gastric pits, but they all demonstrated normal DNA content regardless of anatomic location, histologic GIM subtype, or varying degrees of basal gland atypia. In conclusion, the vast majority of the GIM samples (98%) lack the aneuploidy that is characteristic of gastric dysplasia or cancer. This indicates that aneuploidy usually occurs after the development of gastric dysplasia rather than at the stage of GIM. The finding also suggests that the presence of GIM alone may not be sufficient to suggest an increased risk for GC and that the inclusion of other high-risk features (ie, extensive GIM, dysplasia, racial minorities, and/or family history of GC in a first-degree relative) and/or aneuploidy ought to play a role in the selection of GIM patients who may warrant endoscopic surveillance. Finally, GIM with mild to focally moderate basal gland atypia is likely to represent metaplastic atypia in most cases.

Non-conventional dysplasia in inflammatory bowel disease is more frequently associated with advanced neoplasia and aneuploidy than conventional dysplasia.

AIMS: Several different non-conventional morphological patterns of epithelial dysplasia have been recently described in inflammatory bowel disease (IBD), but there is limited information regarding their clinicopathological and molecular features, as well as potential risk for high-grade dysplasia (HGD) or colorectal cancer (CRC) compared with conventional dysplasia developing in IBD. METHODS AND RESULTS: A total of 317 dysplastic lesions from 168 IBD patients were analysed. All lesions were re-reviewed and subtyped as either conventional [including tubular adenoma-like (n = 183) and tubulovillous/villous adenoma-like (n = 56)] or non-conventional dysplasia [including dysplasia with increased Paneth cell differentiation (DPD, n = 40), crypt cell dysplasia (CCD, n = 14), goblet cell deficient (GCD, n = 10), hypermucinous (n = 7), sessile serrated lesion (SSL)-like (n = 4) and traditional serrated adenoma (TSA)-like (n = 3)]. DNA flow cytometry was performed on 70 low-grade conventional (n = 24) and non-conventional (n = 46) dysplastic biopsies to determine their malignant potential and molecular pathways to HGD or CRC. Eleven sporadic tubular adenomas with low-grade dysplasia (LGD) were utilised as controls. Seventy-eight non-conventional dysplastic lesions were identified in 56 (33%) of the 168 patients, whereas 239 conventional dysplastic lesions were identified in 149 (89%) patients. Although both non-conventional and conventional dysplasias were most often graded as LGD at diagnosis (83% and 84%, respectively), non-conventional dysplasia (38%) was more likely to develop HGD or CRC in the same colonic segment than conventional dysplasia (19%) on follow-up (P < 0.001). Almost half (46%) of non-conventional dysplastic samples showed aneuploidy, whereas only 8% of conventional dysplasia (P = 0.002) and 9% of sporadic tubular adenomas (P = 0.037) did. Also, non-conventional dysplasia more frequently presented as a flat/invisible lesion (41%) compared with conventional dysplasia (18%) (P < 0.001). Among the non-conventional subtypes (n = 78), DPD was the most common (n = 40; 51%), followed by CCD (n = 14; 18%), GCD (n = 10; 13%), hypermucinous (n = 7; 9%), SSL-like (n = 4; 5%) and TSA-like (n = 3; 4%) variants. Hypermucinous dysplasia (mean = 2.1 cm) was significantly larger than DPD, SSL-like, TSA-like and GCD variants (mean = 1.0, 1.2, 1.2 and 1.9 cm, respectively) (P = 0.037). HGD or CRC was more likely to be associated with CCD (n = 13; 93%), hypermucinous (n = 4; 57%), GCD (n = 4; 40%) and SSL-like (n = 3; 75%) variants than DPD (n = 6; 15%) and TSA-like dysplasia (n = 0; 0%) on follow-up (P < 0.001). Furthermore, the rate of aneuploidy was significantly higher in CCD (100%), hypermucinous (80%) and GCD (25%) variants than in DPD (12%), SSL-like dysplasia (0%) and TSA-like dysplasia (0%) (P < 0.001). CONCLUSIONS: Non-conventional morphological patterns of dysplasia are not uncommon in IBD, detected in 33% of the patients. The higher frequencies of advanced neoplasia (HGD or CRC) and aneuploidy in non-conventional dysplasia, in particular CCD, hypermucinous and GCD variants, suggest that they may have a higher malignant potential than conventional dysplasia or sporadic tubular adenomas, and thus need complete removal and/or careful follow-up. Greater than 40% of non-conventional dysplasia presented as a flat/invisible lesion, suggesting that IBD patients may benefit from random biopsy sampling in addition to targeted biopsies. The majority of non-conventional subtypes appear to develop via the chromosomal instability pathway, whereas an alternative serrated pathway may be responsible for the development of at least a subset of SSL-like and TSA-like dysplasias.

Reduction of elevated proton leak rejuvenates mitochondria in the aged cardiomyocyte.

Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. Age-related heart impairment is lacking a clinically effective treatment. Using the model of naturally aging mice and rats, we show direct evidence of increased proton leak in the aged heart mitochondria. Moreover, our data suggested ANT1 as the most likely site of mediating increased mitochondrial proton permeability in old cardiomyocytes. Most importantly, the tetra-peptide SS-31 prevents age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore opening, rejuvenates mitochondrial function by direct association with ANT1 and the mitochondrial ATP synthasome, and leads to substantial reversal of diastolic dysfunction. Our results uncover the excessive proton leak as a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 can reverse this clinically important complication of cardiac aging.

Mitochondrial protein interaction landscape of SS-31.

Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.

Utility of DNA flow cytometry in distinguishing between malignant and benign intrahepatic biliary lesions.

The distinction between well-differentiated intrahepatic cholangiocarcinoma (iCCA) from its morphological mimics such as bile duct adenoma (BDA) and hamartoma (BDH) can be challenging, particularly in small biopsies. Although a few cases of BDA and BDH have been reported to undergo malignant transformation into iCCA, their neoplastic versus benign nature remains debated. DNA flow cytometry was performed on 47 formalin-fixed paraffin-embedded samples of iCCA, 14 BDA, and 18 BDH. Aneuploidy was detected in 22 iCCA (47%) but in none of the 32 BDA and BDH samples. Among the 34 iCCA patients who underwent complete resection and were followed up to tumor recurrence, tumor-related death, or at least for 1 year, the overall recurrence or death rates (regardless of flow cytometric results) were 18, 56, and 71% within 1, 3, and 5 years, respectively. The 1-, 3-, and 5-year recurrence or death rates in 18 iCCA patients with aneuploidy were 28, 66, and 66%, respectively, whereas 16 iCCA patients in the setting of normal DNA content had 1-, 3-, and 5-year rates of 6, 44, and 72%, respectively. Although aneuploid tumors were associated with worse outcomes during the first 3 years, this difference was not statistically significant (hazard ratio = 1.4, p = 0.473) in the present sample size. In conclusion, the frequency of aneuploidy was significantly higher in iCCA (47%) than in its benign morphological mimics (0%), suggesting that it may potentially serve as a diagnostic marker of malignancy in challenging situations. Our findings also suggest that most BDAs and BDHs, if not all, are benign entities and may not represent precursor lesions to iCCAs that often harbor aneuploidy. Although a larger cohort will be necessary to further determine the prognostic significance of aneuploidy in iCCA patients after resection, the patients with aneuploid tumors may have a higher risk for tumor progression, especially during the first 3 years.

DNA flow cytometric analysis of paraffin-embedded tissue for the diagnosis of malignancy in bile duct biopsies.

Differentiation of reactive versus neoplastic epithelial changes can be challenging in bile duct biopsies. The samples are often scant, distorted, and mixed with significant inflammation, ulceration, and/or debris. Histological confirmation of malignancy is often required before the initiation of surgical therapy, and an erroneous diagnosis of malignancy can lead to unnecessary clinical management. Aneuploidy assessment by DNA flow cytometry was performed on formalin-fixed paraffin-embedded (FFPE) tissue from 63 bile duct biopsies: 10 with a malignant diagnosis (7 with adenocarcinoma and 3 with at least high-grade dysplasia [HGD]); 3 with an atypical diagnosis showing rare atypical glands/cells, concerning but not definite for malignancy; 28 likely reactive biopsies with acute/chronic inflammation, ulceration, and/or mild nuclear atypia; and 22 additional benign biopsies without significant inflammation, ulceration, or nuclear atypia. Aneuploidy was detected in 7 (70%) of the 10 biopsies with definite neoplasia (5 of 7 adenocarcinoma cases and 2 of 3 at least HGD cases), all 3 (100%) atypical biopsies, and none of the 50 benign biopsies. All 3 atypical cases with aneuploidy were subsequently found to have adenocarcinoma (n = 2) or HGD (n = 1). Among the 2 cases of at least HGD with aneuploidy, 1 case developed adenocarcinoma, but no follow-up information was available in the other case. The remaining 1 case of at least HGD, despite having normal DNA content, was found to have adenocarcinoma on follow-up. None of the 50 benign cases (further supported by normal DNA content) developed adenocarcinoma within a mean follow-up time of 37 months (range: 0-282 months). The estimated sensitivity of aneuploidy as a diagnostic marker of malignancy (adenocarcinoma and HGD) was 70%, with the specificity of 100%, positive predictive value of 100%, and negative predictive value of 94%. In conclusion, DNA flow cytometry using FFPE tissue from bile duct biopsies demonstrates a high rate of aneuploidy (70%) in malignant cases and normal DNA content in all benign biopsies. Although the sample size is small, the results indicate that this assay can be potentially useful in challenging atypical cases, where morphological evaluation is limited by scarcity of atypical glands/cells, inflammation, and/or ulceration.

DNA flow cytometric and interobserver study of crypt cell atypia in inflammatory bowel disease.

AIMS: The pathological features and diagnostic reliability of crypt cell atypia (CCA) arising in inflammatory bowel disease (IBD) and its clinical significance are unknown. METHODS AND RESULTS: DNA flow cytometry (FCM) was performed on 14 colon biopsies of CCA from seven IBD patients (male-to-female ratio, 5:2; mean age, 53 years; mean IBD duration, 15 years) using paraffin-embedded tissue. Seven gastrointestinal pathologists were asked to diagnose each biopsy as negative for dysplasia (NEG), indefinite for dysplasia (IND), low-grade dysplasia (LGD) or high-grade dysplasia (HGD) by morphology alone, then again with knowledge of FCM results. Aneuploidy was detected in all 14 biopsies, and five of eight biopsies (63%) also showed strong and diffuse nuclear staining for p53 in the areas of CCA. Six (86%) patients developed HGD (n = 5) or adenocarcinoma (n = 1) in the same colonic segment where CCA had been diagnosed within a mean follow-up time of 27 months. No follow-up information was available in the remaining one patient. When diagnoses were grouped as NEG or 'atypical' (including IND, LGD or HGD), the overall agreement rate of 76% (kappa = 0.51) based on morphology alone improved to 90% (kappa = 0.81) with knowledge of FCM results. Even when categorised as NEG or dysplasia (LGD or HGD) with each of the IND diagnoses reclassified into three categories (NEG, LGD or HGD) based on the degree of suspicion for dysplasia, the overall agreement rate of 63% (kappa = 0.25) based on morphology alone improved to 73% (kappa = 0.46) with knowledge of FCM results. However, when grouped as NEG, LGD or HGD, the overall agreement rate was less than 40% (kappa < 0.09) regardless of knowledge of FCM results. CONCLUSIONS: The presence of aneuploidy, p53 positivity and development of HGD or adenocarcinoma on follow-up indicate that CCA likely represents a dysplastic lesion (at least LGD) and is a histological marker of neoplastic progression. Although the grading of CCA, largely based on cytological abnormalities, is subject to significant interobserver variability, CCA can be histologically identified and should lead to a recommendation of increased endoscopic surveillance, especially if aneuploidy is detected.

Diagnosis, risk stratification, and management of ampullary dysplasia by DNA flow cytometric analysis of paraffin-embedded tissue.

The limited accuracy of endoscopic biopsy in detecting high-grade dysplasia or adenocarcinoma within ampullary adenoma or dysplasia has been reported. The natural history of ampullary dysplasia is also unclear, and there are no established guidelines to determine which patients with ampullary dysplasia require resection versus surveillance endoscopy. DNA flow cytometry was performed on 47 ampullary biopsies with low-grade dysplasia, 18 high-grade dysplasia, and 23 negative for dysplasia, as well as 11 cases of ampullary adenocarcinoma. Abnormal DNA content (aneuploidy or elevated 4N fraction > 6%) was identified in 9 (82%) of adenocarcinoma, 13 (72%) of high-grade dysplasia, 7 (15%) of low-grade dysplasia, and none (0%) of non-dysplastic mucosa. One-, 2-, and 7-year detection rates of high-grade dysplasia or adenocarcinoma in low-grade dysplasia patients with abnormal DNA content were 57%, 86%, and 88%, respectively, whereas low-grade dysplasia patients in the setting of normal DNA content had 1-, 2-, and 7-year detection rates of 10%, 10%, and 10%, respectively. The univariate and multivariate hazard ratios (HRs) for subsequent detection of high-grade dysplasia or adenocarcinoma in low-grade dysplasia patients with DNA content abnormality were 16.8 (p = <0.01) and 9.8 (p = <0.01), respectively. Among the 13 high-grade dysplasia patients with DNA content abnormality, 5 patients (38%) were subsequently found to have adenocarcinoma within a mean follow-up time of 3 months, whereas only 1 (20%) of the remaining 5 patients in the setting of normal DNA content developed adenocarcinoma in a month (HR = 2.6, p = 0.39). The overall 1- and 2-year detection rates of adenocarcinoma in all high-grade dysplasia patients (regardless of flow cytometric results) were 34% (95% confidence interval = 16-63%) and 47% (95% confidence interval = 23-79%), respectively. In conclusion, the majority of low-grade dysplasia patients (86%) in the setting of abnormal DNA content developed high-grade dysplasia or adenocarcinoma within 2 years and thus may benefit from resection, whereas those with normal DNA content may be followed with surveillance endoscopy. The presence of DNA content abnormality can also confirm a morphologic suspicion of high-grade dysplasia, which should be managed with resection, as nearly 50% of the high-grade dysplasia patients were found to have adenocarcinoma within 2 years.

Improving mitochondrial function with SS-31 reverses age-related redox stress and improves exercise tolerance in aged mice.

Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day SS-31. Mitochondrial function was assessed in vivo using 31P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.

Utility of DNA Flow Cytometric Analysis of Paraffin-embedded Tissue in the Risk Stratification and Management of 'Indefinite for dysplasia' in Patients With Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The clinical significance of 'indefinite for dysplasia' [IND] in patients with inflammatory bowel disease remains unclear. Currently, no biomarker can reliably differentiate reactive changes from true dysplasia and/or risk stratify IND. METHODS: A total of 52 IND colon biopsies were analysed by DNA flow cytometry. The follow-up result of each biopsy was determined by reviewing all subsequent biopsies and endoscopic reports for the occurrence of high-grade dysplasia [HGD] or colorectal cancer [CRC] at the site of previous biopsy or in the same segment of colon. RESULTS: The overall 1-, 3-, 5-, and 7-year detection rates of HGD or CRC in all 52 IND cases were 4.6% (95% confidence interval [CI], 0.0%-10.6%), 18.2% [95% CI, 3.5%-30.7%], 26.3% [95% CI, 8.4%-40.7%], and 31.6% [95% CI, 11.2%-47.4%], respectively. More interestingly, 10.6% of IND cases with aneuploidy were subsequently found to have HGD or CRC within 1 year [95% CI, 0.0%-23.7%], with 36.4% [95% CI, 7.1%-56.5%], 51.7% [95% CI, 16.1%-72.2%], and 59.8% [95% CI, 21.4%-79.5%] detected within 3, 5, and 7 years, respectively. By comparison, in the setting of normal DNA content, 1-, 3-, 5-, and 7-year detection rates of HGD or CRC were 0.8% [95% CI, 0.0%-2.7%], 3.3% [95% CI, 0.0%-9.6%], 5.2% [95% CI, 0.0%-14.7%], and 6.5% [95% CI, 0.0%-18.1%], respectively. Only the presence of aneuploidy was found to be a significant predictor of HGD or CRC with the estimated univariate and multivariate hazard ratios of 13.8 [p = 0.016] and 50.3 [p = 0.010], respectively. CONCLUSIONS: IND may not be a low-risk condition for HGD or CRC. In this regard, the presence of aneuploidy can identify a subset of IND cases that are at increased risk for subsequent detection of HGD or CRC.

Heart specific knockout of Ndufs4 ameliorates ischemia reperfusion injury.

RATIONALE: Ischemic heart disease (IHD) is a leading cause of mortality. The most effective intervention for IHD is reperfusion, which ironically causes ischemia reperfusion (I/R) injury mainly due to oxidative stress-induced cardiomyocyte death. The exact mechanism and site of reactive oxygen species (ROS) generation during I/R injury remain elusive. OBJECTIVE: We aim to test the hypothesis that Complex I-mediated forward and reverse electron flows are the major source of ROS in I/R injury of the heart. METHODS AND RESULTS: We used a genetic model of mitochondrial Complex I deficiency, in which a Complex I assembling subunit, Ndufs4 was knocked out in the heart (Ndufs4H-/-). The Langendorff perfused Ndufs4H-/- hearts exhibited significantly reduced infarct size (45.3 ±â€¯5.5% in wild type vs 20.9 ±â€¯8.1% in Ndufs4H-/-), recovered contractile function, and maintained mitochondrial membrane potential after no flow ischemia and subsequent reperfusion. In cultured adult cardiomyocytes from Ndufs4H-/- mice, I/R mimetic treatments caused minimal cell death. Reintroducing Ndufs4 in Ndufs4H-/- cardiomyocytes abolished the protection. Mitochondrial NADH declined much slower in Ndufs4H-/- cardiomyocytes during reperfusion suggesting decreased forward electron flow. Mitochondrial flashes, a marker for mitochondrial respiration, were inhibited in Ndufs4H-/- cardiomyocytes at baseline and during I/R, which was accompanied by preserved aconitase activity suggesting lack of oxidative damage. Finally, pharmacological blockade of forward and reverse electron flow at Complex I inhibited I/R-induced cell death. CONCLUSIONS: These results provide the first genetic evidence supporting the central role of mitochondrial Complex I in I/R injury of mouse heart. The study also suggests that both forward and reverse electron flows underlie oxidative cardiomyocyte death during reperfusion.