Peritoneal Biopsies Research Articles

Annexin A1, FPR2/ALX, and inflammatory cytokine expression in peritoneal endometriosis

To characterize Annexin A1 (ANXA1), FPR2/ALX and cytokines expression in peritoneal endometriosis and to clarify their role in its etiology, a cross-sectional study was performed with forty women in reproductive age (22 patients with endometriosis and 18 control women) that had undergone laparoscopic surgery. Peritoneal biopsy and fluid aspirations from endometriosis and control samples were analyzed for the expression of ANXA1, FPR2/ALX and cytokines. ANXA1 and FPR2 / ALX levels were measured by Western blotting and interleukin 1ß (IL-1β), interleukin 4 (IL-4), interleukin 6 (IL-6), and interleukin 10 (IL-10) levels were quantified by enzyme-linked immunosorbent assay (ELISA). The present study identified the presence in human peritoneal tissue of ANXA1 and FPR2 / ALX both in healthy condition and in women with peritoneal endometriosis, however, was lower in endometriosis samples than in control samples. By quantifying the IL-6 and IL-1β cytokines in the peritoneal fluid by ELISA, this study identified a higher IL-6 concentration in endometriosis group, but no significative difference in IL-1ß levels. The IL-4 and IL-10 levels could not be detected.These results indicate that the reduction of the inflammatory resolution mediators could be responsible for the inflammatory process perpetuation, maintenance and worsening of endometriosis.

Benign Multicystic Peritoneal Mesothelioma in a Male Patient with Previous Wilms’ Tumor: A Case Report and Review of the Literature

Benign multicystic peritoneal mesothelioma (BMPM) is a rare condition, more common in females of reproductive age, which arises from the peritoneal mesothelium. A 33-year-old male presented to our unit with abdominal pain and constipation. His past medical history included a previous unilateral nephrectomy for Wilms' tumor and the previous incidental finding of some intra-abdominal cystic formations at the level of the mesentery. After performing a CT scan, an exploratory laparotomy was done and a voluminous cystic mesenteric mass, composed of 3 confluent formations, was observed. Some other similar but significantly smaller lesions were found. An en bloc resection of the mesenteric mass together with the corresponding intestinal loops, an appendicectomy, and some peritoneal biopsies were performed. The postoperative period was complicated by a peritonitis due to dehiscence of the intestinal anastomosis, which required another operation, and a delayed return of normal bowel function, which was resolved through prokinetic therapy. Through histological examination, a BMPM was diagnosed. At 8 months of follow-up, the patient is free of symptoms. BMPM exact etiopathogenesis still remains uncertain. Given his high recurrence rate, a long-term follow-up is recommended.

Is the peritoneal dialysis biocompatibility hypothesis dead?

The peritoneal dialysis (PD) biocompatibility hypothesis is that conventional PD solutions with high levels of glucose degradation products (GDPs), glucose and lactate, and low pH cause morphological and functional damage to the peritoneal membrane and that this damage may be attenuated by biocompatible solutions. Functional findings from randomized trials have not supported this hypothesis, and now new data from a large European pediatric peritoneal biopsy study provide a morphologic correlate for this. The implications are discussed.

Peritoneal liquid biopsy used to predict cancer recurrence after gastrointestinal cancer surgery.

e16191Background: To elucidate recurrence pathophysiology after gastrointestinal cancer surgery, the prognostic effect of peritoneal cancer cell spillage during surgery was evaluated in patients (p...

Diaphragmatic smears are not of additional benefit in the detection of peritoneal spread in gynecological cancers.

Peritoneal biopsies (PB) and peritoneal washing (PW) are routine measures in abdominal staging of gynecological malignancies and are used particularly for the assessment of occult microscopic tumor spread to the peritoneal surface including the diaphragm. Cytological diaphragmatic smears (DS) have been suggested as a supplemental tool; however, they are not routinely taken and their usefulness is still unclear. The present study retrospectively evaluated whether DS provide an additional benefit over PB and PW for the detection of peritoneal malignancies in patients with gynecological cancer. The data from patients who underwent laparotomy for suspected gynecological cancer and had DS and either PB, PW or ascites were reviewed. Sensitivity and specificity, and the number upstaged patients were determined. A total of 43 patients were excluded due to benign diagnosis (those with negative DS or PW) and 2 out of the remaining had 2 carcinomas simultaneously. Among these 41 malignancies, DS were positive in 12, PW in 18 and PB in 19 cases. No case was DS-positive while negative for both PB and PW. Four cases were missed when only PB and 5 when only PW was performed. Notably, no case of peritoneal disease was identified solely on positive DS, indicating that all 23 positive cases (presence of occult peritoneal disease in 56.1%) were identified by PB and PW together (100% sensitivity; 62% specificity). In addition, none of the cases was upstaged solely on positive DS results. Taken together, these data demonstrated that DS do not present an additional benefit to PW and PB in the detection of peritoneal gynecological disease.

Features and Treatment of Peritoneal Metastases from Solid Pseudopapillary Neoplasms of the Pancreas.

BackgroundSolid pseudopapillary neoplasms (SPNs) of the pancreas may present widespread peritoneal metastases, but the treatment of this malignancy has not been well described and requires further investigation.Material/MethodsFour cases of SPN with significant peritoneal metastases in our department were operated and retrospectively summarized after long-term follow-up. Eight more cases of peritoneal metastatic SPN from the PubMed database were also included in the analysis.ResultsPeritoneal metastases of SPNs have different gross features. The benign nodules were tenacious and well encapsulated, while the malignant nodules were soft and prone to slow bleeding. However, neither of these nodules invaded the small intestines or mesentery. Of the 12 disseminated cases, 7 had history of primary tumor rupture, whereas the others had tumors malignant in nature. A total of 14 surgical events were documented, including 3 complete cytoreductive surgeries (CCRS), 9 cytoreductive surgeries (CRS), and 2 debulking surgeries. After follow-up ranging from 0.3 to 6.1 years, the results of the Fisher’s exact test showed no difference between CCRS and CRS in treating either low-grade or high-grade malignant SPNs (P=0.257 and P=0.203, respectively). For all cases of SPN with peritoneal metastases, the CCRS procedure could significantly improve tumor-free survival (TFS) compared to the CRS procedure (P=0.046).ConclusionsSPN rupture could cause significant peritoneal metastases, and either disruption or biopsy of these lesions should be avoided. Peritoneal metastases from SPNs vary both in gross features and biological mechanisms. CCRS may offer optimal therapeutic outcomes and longer TFS for individuals with significant peritoneal metastases of SPNs.

Sclerosing encapsulating peritonitis and intra-abdominal fistulas secondary to Mycobacterium bovis from remote consumption of unpasteurized cow’s milk

Mycobacterium bovis tuberculosis acquired in Canada is extremely rare, thanks to eradication and surveillance programs as well as to milk pasteurization practices initiated in the early twentieth century. This case report presents a case of abdominal tuberculosis in a 69-year-old Canadian-born white man presumed to be secondary to consumption of unpasteurized cow’s milk in childhood. He initially presented with non-specific abdominal symptoms and underwent extensive work-up, including CT-guided biopsy, which failed to elucidate the diagnosis. After multiple hospitalizations, he underwent exploratory laparotomy with peritoneal biopsy, from which M. bovis was cultured. Diagnosis of abdominal tuberculosis is notoriously challenging because of its long latency period and the non-specific nature of presenting symptoms. In addition, imaging similarities between abdominal tuberculosis and neoplastic processes can cause further confusion. This case highlights the diagnostic challenges of this disease while illustrating the importance of a thorough history, particularly in the context of a difficult diagnosis.

242 A Rare Case of Ovarian Serous Borderline Tumor With Brain Metastasis

Serous borderline tumor (SBT) represents a group of noninvasive tumor of ovary bridging between benign adenoma and invasive carcinoma. They are commonly seen in younger women and have excellent outcome, except occasional local recurrence. Metastasis to the lymph nodes has rarely occurred. So far, the only distant metastases reported were to the supraclavicular and internal mammary lymph node. Here, we present a 33-year-old nulliparous woman diagnosed with Stage IV-B serous borderline tumor to the brain. Her serous borderline tumor with 1 mm focus of microstromal invasion of left ovary eight years ago, with a recurrent tumor of the right ovary occurred 4 years ago, for which she underwent two unilateral salpingo-oophorectomies with omentectomy on separate occasions with extensive abdominal, peritoneal, and pelvic lymph node biopsies. No evidence of extraovarian spread on both times was identified. However, 4 years after her last surgery, the patient presented to our institution with a migraine. MRI revealed four supratentorial lesions with two dominant proteinaceous mass lesions (6.0 x 4.5 x 3.5 cm and 4.5 x 3.5 x 2.8 cm) having irregular lobulations in the bilateral temporal lobes consistent with metastatic disease. Biopsy of the dominant lesion shows clusters of broad papillae with hierarchical branching lined by polygonal to columnar serous epithelium with mild to moderate atypia. Findings are consistent with metastatic serous borderline tumor. To our best knowledge, this is the first case reported of a serous borderline tumor with metastasis to the brain. No current review or literature supports this distant metastasis.

Tuberculous peritonitis. 'Forgotten' disease

To analyze diagnosis and treatment of patients with tuberculous peritonitis, to develop the algorithms for instrumental examination and differential diagnosis. There were 48 patients with tuberculous peritonitis. The examination included radiography, abdominal and thoracic computed tomography, ultrasound, and laparoscopy. All patients underwent histological, cytological, microbiological and molecular-genetic analysis of abdominal exudate and peritoneal biopsy. Exclusion criterion was signs of secondary peritonitis. Clinical picture of tuberculous peritonitis was accompanied by nonspecific symptoms. Previously identified pulmonary tuberculosis and HIV-infection were present in 93.8 and 70.8% of patients. Diagnostic laparoscopy of abdominal cavity as the main method of instrumental diagnosis together with cytological, molecular-genetic and microbiological research of peritoneal exudate and tissue specimens were useful to determine diagnosis in 87.2-95.8% of cases. Tuberculous peritonitis may be assumed in patients with previous tuberculosis of lungs or other localizations, HIV-infection. Computed tomography is the most informative method to diagnose tuberculous peritonitis. Diagnostic laparoscopy is indicated for suspected tuberculous peritonitis. This procedure is supplemented by peritoneal biopsy, cytological, molecular-genetic and microbiological examination of peritoneal exudate and tissue specimens.

The abdominal cocoon: A management dilemma

Abdominal cocoon is a rare entity with no known aetiology and is a clinical curiosity in the surgical field, and a management dilemma for surgeons. Treatment may include excision of the accessory peritoneal sac with lysis of the inter-loop adhesions. Bowel resection is unnecessary unless a nonviable segment is found. However, if there are no signs of acute abdominal symptoms after diagnosing abdominal cocoon syndrome pre-operatively, surgery is unnecessary. We presenting a case of a 47-year-old male who had laparotomy noted an entirely frozen abdomen which was depicted as a cocoon. The stomach was only identified due to feeling the position of the NGT, no other anatomy was delineated. A peritoneal biopsy was taken prior to closure. Post operatively he was given parenteral and trickle enteral nutritional support and was discharged home with PEG gastrostomy for feeding.

Current Status of Peritoneal Dialysis in Japan.

As an effective renal replacement therapy, peritoneal dialysis (PD) is as important as hemodialysis (HD) and renal transplantation. PD is beneficial for social rehabilitation and home medical care. However, in Japan, there are fewer PD patients than HD patients. According to the JSDT 2015 registry, PD patients accounted for only 2.9% of all dialysis patients, and they have been gradually decreasing since 2009. One of the reasons why PD is not widely used in Japan is that there is insufficient evidence supporting its use. In this review, some recent reports about changes and challenges of PD, including survival rates, residual renal function, peritonitis, encapsulating peritoneal sclerosis (EPS), and combination therapy are summarized. These also indicate some problems and strategies related to PD treatment in Japan. According to the PDOPPS study, the incidence of peritonitis and culture-negative peritonitis was higher in Japan than in other countries. Further, the International Society of Peritoneal Dialysis (ISPD) Recommendation about peritonitis and catheter-related infection that topical application of antibiotic cream or ointment to the catheter exit site should be used daily could be a strategy for decreasing the incidence of peritonitis. The prevention and treatment of EPS are other challenges in PD. An EPS recommendation was published by the ISPD in 2017. Although the Next PD study showed that the occurrence of EPS is now decreasing compared to the time when acidic dialysates were used, the predictors for the development of EPS and strategies to reduce EPS have not been established. Peritoneal pathological parameters of groups that did and did not develop EPS were compared using peritoneal biopsy tissues at the time of cessation of PD, and it was found that the L/V ratio could be an independent predictor of EPS development. In Japan, 20% of PD patients are receiving combination therapy, which shows promising results. However, some problems, such as the risk of EPS, still limit long-term PD. Key Messages: PD treatment is changing as patients' situations change and with advances in technology. It is necessary to translate evidence to Japan from overseas reports and ISPD guidelines.

The Diagnostic Usefulness of Ultrasound-Guided Peritoneal Biopsy for the Solitary Peritoneal Thickening of an Unknown Cause Visualized as Only Infiltrated Fat Tissue on a CT Scan

The Diagnostic Usefulness of Ultrasound-Guided Peritoneal Biopsy for the Solitary Peritoneal Thickening of an Unknown Cause Visualized as Only Infiltrated Fat Tissue on a CT Scan

Bicarbonate buffered peritoneal dialysis fluid upregulates angiopoietin-1 and promotes vessel maturation.

BackgroundUltrafiltration decline is a progressive issue for patients on chronic peritoneal dialysis (PD) and can be caused by peritoneal angiogenesis induced by PD fluids. A recent pediatric trial suggests better preservation of ultrafiltration with bicarbonate versus lactate buffered fluid; underlying molecular mechanisms are unknown.MethodsAngiogenic cytokine profile, tube formation capacity and Receptor Tyrosine Kinase translocation were assessed in primary human umbilical vein endothelial cells following incubation with bicarbonate (BPDF) and lactate buffered (LPDF), pH neutral PD fluid with low glucose degradation product content and lactate buffered, acidic PD fluid with high glucose degradation product content (CPDF). Peritoneal biopsies from age-, PD-vintage- and dialytic glucose exposure matched, peritonitis-free children on chronic PD underwent automated histomorphometry and immunohistochemistry.ResultsIn endothelial cells angiopoietin-1 mRNA and protein abundance increased 200% upon incubation with BPDF, but decreased by 70% with LPDF as compared to medium control; angiopoietin-2 remained unchanged. Angiopoietin-1/Angiopoietin-2 protein ratio was 15 and 3-fold increased with BPDF compared to LPDF and medium. Time-lapse microscopy with automated network analysis demonstrated less endothelial cell tube formation with BPDF compared to LPDF and CPDF incubation. Receptor Tyrosine Kinase translocated to the cell membrane in BPDF but not in LPDF or CPDF incubated endothelial cells. In children dialyzed with BPDF peritoneal vessels were larger and angiopoietin-1 abundance in CD31 positive endothelium higher compared to children treated with LPDF.ConclusionBicarbonate buffered PD fluid promotes vessel maturation via upregulation of angiopoietin-1 in vitro and in children on dialysis. Our findings suggest a molecular mechanism for the observed superior preservation of ultrafiltration capacity with bicarbonate buffered PD fluid with low glucose degradation product content.

Results of Fertility-Sparing Surgery for Expansile and Infiltrative Mucinous Ovarian Cancers.

No series had been reported focusing on the results of fertility-sparing surgery in stage I mucinous ovarian cancers according to histotype (infiltrative vs. expansile). Investigating such outcomes was the aim of the present study. The present study was a retrospective analysis of patients treated conservatively with preservation of the uterus and contralateral ovary from 1976 to 2016. The pathology of the tumors was reviewed by two expert pathologists according to the 2014 World Health Organization (WHO) classification criteria. Oncologic and fertility results were analyzed. Twenty-one patients fulfilled the inclusion criteria, twelve with expansile and nine with infiltrative cancer. All patients had a unilateral tumor and underwent unilateral salpingo-oophorectomy in one-step (n = 6) or two-step (n = 15) surgeries. All but one had complete peritoneal staging surgery based on cytology, omentectomy, and random peritoneal biopsies. Ten had nodal staging surgery. The International Federation of Gynecology and Obstetrics stages were IA (n = 9), IC1 (n = 6), and IC2 (n = 6); the nuclear grades were grade 1 (n = 9), grade 2 (n = 5), and grade 3 (n = 1). Two patients recurred (one expansile and one infiltrative type) 19 and 160 months after surgery, respectively. One stage IA, nuclear grade 2 expansile tumor recurred on the spared ovary; the patient remains alive. The other stage IA infiltrative tumor recurred as peritoneal spread; the patient is alive with disease. Six patients became pregnant; four with expansile tumors and two with infiltrative tumors. The type of mucinous cancer has no impact on the oncologic outcome in this series of patients treated conservatively. Fertility-sparing surgery should be considered for early-stage infiltrative-type tumors. According to the most recently updated World Health Organization classification guidelines, mucinous cancers should be classified as either expansile or infiltrative. The infiltrative type has a poorer prognosis, but there are no data about the safety of fertility-sparing surgery (FSS) in this context. A collection of 21 cases reviewed by two expert pathologists this study is the first devoted to the conservative treatment of mucinous tumors according to both subtypes. The key result was that the type of mucinous cancer has no impact on the oncologic outcome; thus, FSS may be considered in both subtypes.

A case of peritoneal malignant mesothelioma following radiation therapy for cervical cancer.

The present study presents a case of peritoneal malignant mesothelioma (PMM) following radiation therapy for cervical cancer. A 34-year-old Japanese woman, without asbestos exposure, was referred to the Department of Gynecologic Oncology, Saitama Medical University International Medical Center due to a cervical mass, and was diagnosed with cervical squamous cell carcinoma (SCC). The serum levels of tumor markers, including SCC antigen and cancer antigen 125 (CA125) were 229.0 ng/ml and 54.4 U/ml, respectively. The patient underwent concurrent chemoradiotherapy (CCRT), and a complete response was achieved. After 54 months, ascites was found at the rectouterine pouch, but peritoneal cytology suggested reactive mesothelial cell. After 62 months of CCRT, magnetic resonance imaging revealed masses in both the salpinges. The serum levels of SCC and CA125 were 0.9 ng/ml and 506.1 U/ml, respectively. Following this, left salpingectomy and peritoneal biopsy were performed laparoscopically. Histologic examination revealed atypical mesothelial cells with no continuity of background tubal epithelium. Immunohistochemistry showed positive staining for calretinin, thrombomodulin, mesothelin and glucose transporter 1. Based on these findings, the patient was diagnosed with PMM epithelioid type and underwent systemic chemotherapy; stable disease status has been obtained for 3 months. This case demonstrates the possibility of PMM occurrence within 10 years after radiotherapy, and indicates the importance of histological and immunohistochemical examination, particularly in cases of an atypical tumorigenesis pattern from the primary cancer.

Challenging and uncommon diagnosis of long-evolution ascites

This is a case report of a 45-year-old Caucasian man with chronic alcoholism. No history of liver disease or asbestos exposure. He complained of ascites during the last 3 years...

Extra-pulmonary tuberculosis in immunocompetent patients: Case series and mini review of the literature

Introduction: Tuberculosis (TB) is still one of the most frequent infectious diseases, causing 2 million deaths a year worldwide. The prevalence of TB has increased in both immunocompetent and immunocompromised and virtually, it can affect any organ. Up to 25% of tuberculosis cases present extra-pulmonary involvement. The diagnosis of extra-pulmonary TB (EPTB) is difficult, often requiring biopsy. Material and methods: We present two cases of EPTB in immunocompetent young women. The first case is about a 15-years old female who presented with persistent high fever, ascites and an increased CA-125 serum level. Biochemical analysis of ascites fluid revealed a high value of adenosine deaminase (ADA), negative Ziehl-Neelsen stain and undetectable Mycobacterium tuberculosis at PCR assay. The peritoneal biopsy confirmed the diagnosis of peritoneal TB. The second case, a 29-years old female with a seven-month history of cervical lymph nodes swelling, with drainage and excision biopsy performed, misdiagnosed as cats-scratch disease based on the histological description as granulomatous lymphadenitis, which turned out to be tuberculous lymphadenitis at the second histopathological opinion. In conclusion, these cases highlight the need for tuberculosis consideration in the differential diagnosis of extra-pulmonary involvement, even in immunocompetent individuals, in order to decrease the misdiagnoses and complications rates.

Endoscopic Ultrasound (EUS) Guided Fine Needle Aspiration: A New Modality to Diagnose Peritoneal Tuberculosis in Presence of Decompensated Cirrhosis—A Case Series and Review of Literature

The gold-standard for diagnosis is growth of Mycobacterium tuberculosis on ascitic fluid or peritoneal culture. Due to the non-specific signs and symptoms of disease, its early diagnosis is difficult, especially in patients with decompensated cirrhosis. The reported sensitivity of ascitic fluid is low and to obtain tissue for peritoneal biopsy in patients with cirrhosis is difficult. Endoscopic ultrasound (EUS) guided fine needle aspiration (FNA) is a good alternative to obtain peritoneal tissue for establishing the diagnosis of peritoneal TB. To assess the role of EUS-FNA in the diagnosis of peritoneal tuberculosis in patients with decompensated cirrhosis. Consecutive patients with peritoneal thickening, ascites and decompensated cirrhosis underwent EUS-FNA from the thickened omentum. Presence of granuloma or demonstration of acid fast bacilli was diagnostic of peritoneal tuberculosis. A total of 5 patients with CLD underwent EUS-FNA from omentum. FNA cytology revealed granuloma with multinucleated cells in all patients (100%) and AFB stain was positive in 2 of them (40%). We hereby report the use of EUS guided fine needle aspiration (FNA) of peritoneum as a newer, safe and unexplored technique for diagnosis of peritoneal TB.

Systematic para-aortic and pelvic lymphadenectomy in ovarian cancer. A literature review

Ovarian cancer represents one of the most frequent gynecological malignancies. The risk of developing this malignancy increases with age, especially after menopause. Surgery remains the only potentially curative treatment for early stage ovarian cancer, and it usually consists in salpingo-oophorectomy, omentectomy, peritoneal biopsies, and lymphadenectomy. Some studies revealed that systematic lymphadenectomy in early stage ovarian cancer leads to higher survival rates, while others showed the opposite. Identification of the sentinel ganglion using indocyanine-green, through open or laparoscopic approach, can decrease postoperative morbidity associated with systematic para-aortic lymphadenectomy. In advanced ovarian cancer, the goal is to obtain the absence of residual disease, which increases the overall survival. In this setting, there is an ongoing debate regarding the best method, various authors recommending either systematic para-aortic and pelviclymphadenectomy, or just the resection of bulky masses. After reviewing the available evidence, we may conclude that in patients with ovarian cancer the goal should be complete resection of the malignant disease, including grossly invaded or micro-metastatic lymph nodes. Extensive lymphadenectomy contributes to a more precise staging, but more studies are needed to reveal the subgroup of patients who benefit most regarding overall and progression-free survival.

Significance of new membrane formation in peritoneal biopsies of peritoneal dialysis patients: a case\u2013control study

BackgroundNewly formed membrane (NFM) on the peritoneal membrane proper is a unique pathological hallmark of encapsulating peritoneal sclerosis (EPS), but its definition and diagnostic significance have not been well described. This study investigated the pathological features of NFM in EPS and prevalence of NFM in peritoneal biopsy at catheter removal.MethodsThis multicenter retrospective, observational study was conducted by the Japanese Society of Peritoneal Dialysis and enrolled ten patients with and 52 without EPS at peritoneal biopsy during enterolysis surgery or catheter removal. All patients were treated using conventional, acidic peritoneal dialysis (PD) solutions. Thirty of the 52 non-EPS patients perform peritoneal lavage once daily after completing PD to prevent the development of EPS and 22 discontinued PD without lavage. NFM was defined as additional membrane structure on the peritoneum that is properly characterized by exudative or fibrous matrices and fibroblast-like cells. Immunostaining of fibrin and podoplanin was performed for the evaluation of NFM.ResultsNFM was confirmed histologically in eight of the ten patients with EPS. It was also detected in 13 of the 30 patients (43.3%) in the post-PD lavage group and in one of the 22 patients (4.5%) in the non-lavage group. The NFM histology showed various stages of EPS pathology, from early exudative changes with fibrin deposition (stage I; n = 5), progressing to proliferative and fibrosing changes with podoplanin-positive fibroblast-like cells (stage II; n = 9), and finally resulting in adhesive and fibrous scar formation (stage III; n = 8). Immunostaining of fibrin and podoplanin was helpful for evaluating the stage of the NFM.ConclusionsNFM contributes the encapsulation of intestines and is a pathological hallmark of EPS, but can be detected microscopically in patients without EPS, especially those with peritoneal lavage after PD. New membrane formation starts insidiously in peritoneal membrane damaged by PD treatment, but does not necessarily lead to the development of EPS.