Molecular classification of metastatic and recurrent endometrial endometrioid carcinoma: prognostic relevance among low- and high-stage tumours.

AIMS: Molecular classification according to The Cancer Genome Atlas (TCGA) improves endometrial endometrioid carcinoma (EEC) prognostication and has specific treatment implications; however, original data were skewed towards low-grade and low-stage tumours. Herein, we molecularly classify EECs metastatic at the time of diagnosis or with subsequently documented recurrent/metastatic disease to examine correlation with clinical outcomes. METHODS: TCGA categories include POLE-mutated, microsatellite instability (MSI), p53 abnormal (p53 abnl) and no specific molecular profile (NSMP). POLE targeted sequencing at exons 9, 11, 13 and 14 and immunohistochemistry (IHC) for PMS2, MSH6 and p53 were performed to establish molecular classification. RESULTS: The distribution in our cohort of 141 EECs was similar to that generally reported in EEC, with nine POLE-mutated (6%), 45 MSI (32%), 16 p53 abnl (11%) and 71 NSMP (50%), with similar distributions between low- and high-stage cohorts. We demonstrate that when stratified by molecular subtype, disease-specific survival from the time of high-stage (stages III-IV) presentation or time of recurrence in low-stage (stages I-II) disease among metastatic and/or recurrent EEC is strongly associated with TCGA classification (high-stage P = 0.02, low-stage P = 0.017). Discordant molecular classification between primary and metastatic/recurrent tumours occurred in four of 105 (3.8%) patients, two related to PMS2/MSH6 IHC and two related to p53 IHC. CONCLUSIONS: We demonstrate that molecular classification is prognostically relevant not only at the time of diagnosis, but also at the time of recurrence and in the metastatic setting. Rare subclonal alterations occur and suggest a role for confirming TCGA classification in recurrent/metastatic tumours.

Targetable ERBB2/HER2 Mutations in Gynecologic Malignancies: Clinicopathological, Immunohistochemical, and Molecular Correlations.

Targeted anti-HER2 therapy has been recently added to the standard treatment recommendations in endometrial serous carcinoma. Current eligibility requires testing for HER2 overexpression and/or gene amplification by immunohistochemistry and by fluorescence in situ hybridization. However, clinical trials have also demonstrated the efficacy of anti-HER2 drugs against activating ERBB2/HER2 mutations in a variety of solid tumor types, and fam-trastuzumab deruxtecan is now approved by the US Food and Drug Administration for HER2-mutant non-small cell lung cancer. This study aimed at evaluating the detailed clinical, histomorphological, immunohistochemical, and molecular characteristics of gynecologic malignancies with ERBB2/HER2 mutations. We identified 16 tumors with 19 ERBB2/HER2 mutations in our departmental archives: 11 endometrial primaries, 2 endocervical adenocarcinomas, 1 ovarian mucinous adenocarcinoma, 1 tubo-ovarian undifferentiated carcinoma, and 1 high-grade endometrioid adenocarcinoma of Mullerian origin. ERBB2/HER2 mutations most often involved the tyrosine kinase domain (52.6%), and the most frequent specific mutation was R678Q (31.6%), involving the juxtamembrane domain. More than half (54.5%) of endometrial carcinomas and half of all tumors were MMR-deficient, resulting from MSH6 loss in all but 2 tumors. None of the tumors (0%) were POLE-mutated, while 18.8% were TP53-mutated. HER2 IHC was negative (score 0 or 1+) in 12 tumors (67%) and equivocal (score 2+) in 4 tumors (33%), whereas none of the tumors were scored as HER2 3+. Score 2+ was associated with R678Q, L755S, I767M mutations, and ERBB2/HER2 rearrangement with a breakpoint in exon 23. Concurrent ERBB2/HER2 amplification was identified in 2 endometrial carcinomas, with HER2/CEP17 ratios of 3.1 and 3.5. We also queried the cBioportal database, which revealed 70 ERBB2/HER2-mutant gynecologic tumors with a total of 77 ERBB2/HER2 mutations, most often involving the active site of the tyrosine kinase domain (n=36; 46.8%), and the most common specific mutation was S310F (n=20; 26%), located in the extracellular domain. Our results provide important details regarding the clinicopathological and molecular associations of potentially actionable ERBB2/HER2 mutations in endometrial carcinoma and other gynecological cancer types and contribute to addressing clinical treatment needs and improving pathology testing recommendations in the future.

Scattering-Based Light-Sheet Microscopy Imaging of Human Papillomavirus-Associated Squamous Lesions of the Anal Canal: A Proof-of-Principle Study.

Demand for anal cancer screening is expected to rise following the recent publication of the Anal Cancer-HSIL Outcomes Research trial, which showed that treatment of high-grade squamous intraepithelial lesions significantly reduces the rate of progression to anal cancer. While screening for human papillomavirus-associated squamous lesions in the cervix is well established and effective, this is less true for other sites in the lower anogenital tract. Current anal cancer screening and prevention rely on high-resolution anoscopy with biopsies. This procedure has a steep learning curve for providers and may cause patient discomfort. Scattering-based light-sheet microscopy (sLSM) is a novel imaging modality with the potential to mitigate these challenges through real-time, microscopic visualization of disease-susceptible tissue. Here, we report a proof-of-principle study that establishes feasibility of dysplasia detection using an sLSM device. We imaged 110 anal biopsy specimens collected prospectively at our institution's dysplasia clinic (including 30 nondysplastic, 40 low-grade squamous intraepithelial lesion, and 40 high-grade squamous intraepithelial lesion specimens) and found that these optical images are highly interpretable and accurately recapitulate histopathologic features traditionally used for the diagnosis of human papillomavirus-associated squamous dysplasia. A reader study to assess diagnostic accuracy suggests that sLSM images are noninferior to hematoxylin and eosin images for the detection of anal dysplasia (sLSM accuracy = 0.87; hematoxylin and eosin accuracy = 0.80; P = .066). Given these results, we believe that sLSM technology holds great potential to enhance the efficacy of anal cancer screening by allowing accurate sampling of diagnostic tissue at the time of anoscopy. While the current imaging study was performed on ex vivo biopsy specimens, we are currently developing a handheld device for in vivo imaging that will provide immediate microscopic guidance to high-resolution anoscopy providers.

KRAS mutation in primary ovarian serous borderline tumors correlates with tumor recurrence.

Oncogenic activation of the mitogen-activated protein kinase (MAPK) pathway due to KRAS or BRAF gain-of-function mutation is frequently found in ovarian serous borderline tumor (SBT) and their extraovarian implants. We investigated mutational status of KRAS and BRAF of the primary ovarian SBTs that had a high stage presentation in correlation with clinical outcome. Among 39 consecutive primary SBTs with either invasive implants (20 cases) or non-invasive implants (19 cases), KRAS and BRAF mutational analysis was informative in 34 cases. Sixteen cases (47%) harbored a KRAS mutation, while 5 cases (15%) had a BRAF V600E mutation. High-stage disease (IIIC) was seen in 31% (5/16) of patients with a KRAS mutation and 39% (7/18) of patients without a KRAS mutation (p = 0.64). KRAS mutations were present in 9/16 (56%) tumors with invasive implants/LGSC versus 7/18 (39%) tumors with non-invasive implants (p = 0.31). BRAF mutation was seen in 5 cases with non-invasive implants. Tumor recurrence was seen in 31% (5/16) of patients with a KRAS mutation, compared to 6% (1/18) of patients without a KRAS mutation (p = 0.04). A KRAS mutation predicted an adverse disease-free survival (31% survival at 160 months) compared to those with wild-type KRAS (94% at 160 months; log-rank test, p = 0.037; HR 4.47). In conclusion, KRAS mutation in primary ovarian SBTs is significantly associated with a worse disease-free survival, independent of the high tumor stage or histological subtypes of extraovarian implant. KRAS mutation testing of primary ovarian SBT may servce as a useful biomarker for tumor recurrence.

TP53 Mutation-driven Stratified Mucin-producing Carcinoma Coexisting With Squamous Cell Carcinoma of the Vulva: A Case Study.

Associated with high-risk human papillomavirus infection, invasive stratified mucin-producing carcinoma is a recently characterized adenocarcinoma of the cervix. It often occurs in association with adjacent stratified mucin-producing intraepithelial lesion. Differentiated vulvar intraepithelial neoplasia and related invasive squamous cell carcinoma often arise in background vulvar lichen sclerosus with TP53 mutation as the underlying molecular signature. We present a unique case of vulvar invasive stratified mucin-producing carcinoma-like component coexisting with invasive squamous cell carcinoma in a 64-year-old woman. Both neoplastic components were proven TP53 -driven processes arising in the background of differentiated vulvar intraepithelial neoplasia and lichen sclerosus. The invasive stratified mucin-producing carcinoma-like component behaved aggressively in this case, evidenced by the presence of lymphovascular invasion and inguinal lymph node metastasis.

Anastomosing Hemangioma of the Ovary With Leydig Cell Hyperplasia: A Clinicopathologic Study of 12 Cases.

Anastomosing hemangioma is a recently described vascular neoplasm, initially identified in the male genitourinary tract. Since its first description, it has been reported at multiple anatomic sites, including rare cases in the female genital tract, most in the ovary. Herein we report the largest series to date of 12 ovarian anastomosing hemangiomas identified at our institution over a 15-yr period. The patients' age at the time of resection ranged from 50 to 76 yr (median: 62 yr), 3 patients presented with symptomatic pelvic masses, 3 tumors were identified by imaging studies, and the remaining 6 were incidentally discovered in ovaries removed for other indications. All tumors were unilateral, occurred at the ovarian hilum, and contained a vaguely lobulated architecture with sinusoidal-like vessels lined by hobnail endothelial cells with minimal to no cytologic atypia. A rim of luteinized/Leydig cells with abundant, eosinophilic cytoplasm and round, centrally placed nuclei surrounding the hemangioma was present in 9/12 tumors. Reinke crystals were observed in 3 of these 9 tumors. The volume of luteinized cells relative to the vascular proliferation ranged from 2% to 30%. All tumors with luteinized/Leydig cells also displayed numerous small eosinophilic, globular intracytoplasmic inclusions within the endothelial cells. The 3 tumors without luteinized/Leydig cells were exclusively intravascular lesions. Despite the frequent presence of luteinization/Leydig cells none of the patients experienced hormonal manifestations. Awareness of this rare benign ovarian entity is important, as its association with luteinized cells/Leydig cell hyperplasia (often exuberant) may be misinterpreted as a steroid cell tumor, Leydig-cell tumor, or as a mixed stromal-vascular tumor.

Detection of SARS-CoV-2 in tissue: the comparative roles of RT-qPCR, in situ RNA hybridization, and immunohistochemistry.

INTRODUCTION: The emergence of SARS-CoV-2, the causative agent the COVID-19 pandemic, has led to a rapidly expanding arsenal of molecular diagnostic assays for the detection of viral material in tissue specimens. AREAS COVERED: We review the value and shortcomings of available tissue-based assays for SARS-CoV-2 detection in formalin-fixed paraffin-embedded (FFPE) tissue, including immunohistochemistry, in situ hybridization, and quantitative reverse transcription PCR (RT-qPCR). The validation, accuracy, and comparative utility of each method is discussed. Subsequently, we identify commercially available antibodies which render the greatest specificity and reproducibility of staining in FFPE specimens. EXPERT OPINION: We offer expert opinion on the efficacy of such techniques and guidance for future implementation, both clinical and experimental.

Human herpesvirus 8-negative effusion-based large B-cell lymphoma: a distinct entity with unique clinicopathologic characteristics.

Rare cases of human herpesvirus 8 (HHV8)-negative effusion-based large B-cell lymphoma (EB-LBCL) occur in body cavities without antecedent or concurrent solid mass formation. In contrast to HHV8 + primary effusion lymphoma (PEL), EB-LBCL has no known association with HIV or HHV8 infection. However, the small sample sizes of case reports and series worldwide, especially from non-Japanese regions, have precluded diagnostic uniformity. Therefore, we conducted a retrospective, multi-institutional study of 55 cases of EB-LBCL and performed a comprehensive review of an additional 147 cases from the literature to identify distinct clinicopathologic characteristics. In our study, EB-LBCL primarily affected elderly (median age 80 years), immunocompetent patients and manifested as lymphomatous effusion without a solid component. The lymphomatous effusions mostly occurred in the pleural cavity (40/55, 73%), followed by the pericardial cavity (17/55, 31%). EB-LBCL expressed CD20 (53/54, 98%) and PAX5 (23/23, 100%). Most cases (30/36, 83%) were of non-germinal center B-cell subtype per the Hans algorithm. HHV8 infection was absent (0/55, 0%), while Epstein-Barr virus was detected in 6% (3/47). Clinically, some patients were managed with drainage alone (15/34, 44%), while others received rituximab alone (4/34, 12%) or chemotherapy (15/34, 44%). Eventually, 56% (22/39) died with a median overall survival (OS) of 14.9 months. Our findings were similar to those from the literature; however, compared to the non-Japanese cases, the Japanese cases had a significantly higher incidence of pericardial involvement, a higher rate of chemotherapy administration, and longer median OS. Particularly, we have found that Japanese residence, presence of pericardial effusion, and absence of MYC rearrangement are all favorable prognostic factors. Our data suggest that EB-LBCL portends a worse prognosis than previously reported, although select patients may be managed conservatively. Overall, EB-LBCL has distinct clinicopathologic characteristics, necessitating the establishment of separate diagnostic criteria and consensus nomenclature.

Placenta Teratoma or Acardiac Fetus Amorphous: A Case Study by DNA Genotyping.

To date, 40 cases of placental teratoma and 21 cases of umbilical cord teratoma have been reported in the literature. Such entities are purportedly described as originating from ectopically derived totipotential germ cells forming 1 or more of 3 germ layers, similar to teratomas arising in other sites. These entities have been described as distinct from acardiac twins based on the absence of both an axial skeleton and/or separate umbilical cord attachment. We present a case that would be compatible with placental teratoma according to these criteria. However, DNA genotyping analysis of the "teratoma" and its corresponding normal placental tissue revealed an identical genetic profile at all microsatellite polymorphic loci with exception of one locus demonstrating loss of heterozygosity involving 1 of 2 "teratoma" samples tested. Our finding established that the "teratoma" in fact represented a monozygotic acardiac (amorphous) twin with aberrant division of embryogenesis as a continuum of the monozygotic twinning phenomenon. In summary, this is the first case study of so-called placental teratoma by DNA genotyping investigation. We conclude that the diagnostic term "placental teratoma" should be discouraged unless evidence of monozygotic twining can be ruled out by molecular genotyping.

High Counts of CD68+ and CD163+ Macrophages in Mantle Cell Lymphoma Are Associated With Inferior Prognosis.

BACKGROUND: Lymphoma-associated macrophages (LAMs) are key components in the lymphoma microenvironment, which may impact disease progression and response to therapy. There are two major subtypes of LAMs, CD68+ M1 and CD163+ M2. M2 LAMs can be transformed from M1 LAMs, particularly in certain diffuse large B-cell lymphomas (DLBCL). While mantle cell lymphoma (MCL) is well-known to contain frequent epithelioid macrophages, LAM characterization within MCL has not been fully described. Herein we evaluate the immunophenotypic subclassification, the expression of immune checkpoint molecule PD-L1, and the prognostic impact of LAMs in MCL. MATERIALS AND METHODS: A total of 82 MCL cases were collected and a tissue microarray block was constructed. Immunohistochemical staining was performed using CD68 and CD163, and the positive cells were recorded manually in four representative 400× fields for each case. Multiplexed quantitative immunofluorescence assays were carried out to determine PD-L1 expression on CD68+ M1 LAMs and CD163+ M2 LAMs. In addition, we assessed Ki67 proliferation rate of MCL by an automated method using the QuPath digital imaging analysis. The cut-off points of optimal separation of overall survival (OS) were analyzed using the X-Tile software, the SPSS version 26 was used to construct survival curves, and the log-rank test was performed to calculate the p-values. RESULTS: MCL had a much higher count of M1 LAMs than M2 LAMs with a CD68:CD163 ratio of 3:1. Both M1 and M2 LAMs were increased in MCL cases with high Ki67 proliferation rates (>30%), in contrast to those with low Ki67 (<30%). Increased number of M1 or M2 LAMs in MCL was associated with an inferior OS. Moreover, high expression of PD-L1 on M1 LAMs had a slightly better OS than the cases with low PD-L1 expression, whereas low expression of PD-L1 on M2 LAMs had a slightly improved OS, although both were not statistically significant. CONCLUSIONS: In contrast to DLBCL, MCL had a significantly lower rate of M1 to M2 polarization, and the high levels of M1 and M2 LAMs were associated with poor OS. Furthermore, differential PD-L1 expressions on LAMs may partially explain the different functions of tumor-suppressing or tumor-promoting of M1 and M2 LAMs, respectively.

Complete Hydatidiform Mole and Coexisting Fetus With Gastroschisis: A Case Report Highlighting the Importance of Diagnostic Genotyping.

Twin pregnancy with a complete hydatidiform mole and a coexisting fetus (CHMCF) is an extremely rare occurrence, described only by a handful of published series and cases reports. The majority of the literature on CHMCF examines prenatal care and follow-up in relation to the increased risk of gestational trophoblastic neoplasia (GTN). At present, few reports elaborate on the diagnostic process and differential diagnosis, especially in the context of recent molecular advances in risk stratification for GTN. Here, we describe the first known case of a CHMCF with gastroschisis with liveborn delivery at 35 weeks gestation. This report aims to review the pre- and postnatal differential diagnosis and discuss recent updates on the importance of ancillary studies in the diagnosis of gestational trophoblastic disease.

HAI-1 is an independent predictor of lung cancer mortality and is required for M1 macrophage polarization.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Though immune checkpoint inhibitors (ICIs) have revolutionized lung cancer therapy in recent years, there are several factors limiting the therapeutic efficacy of ICI-based immunotherapy in lung cancer. Recent evidence suggests that one such mechanism is the phenotypic shift of tumor-infiltrating macrophages away from an anti-tumor M1 phenotype and towards an anti-inflammatory and tumor-permissive M2 phenotype. Though this phenomenon is well documented, the means through which the lung tumor microenvironment (TME) usurps macrophage function are poorly described. Hepatocyte growth factor (HGF) is a known driver of both lung cancer pathobiology as well as M2 polarization, and its signaling is antagonized by the tumor suppressor gene HAI-1 (SPINT1). Using a combination of genomic databases, primary NSCLC specimens, and in vitro models, we determined that patients with loss of HAI-1 have a particularly poor prognosis, hallmarked by increased HGF expression and an M2-dominant immune infiltrate. Similarly, conditioned media from HAI-1-deficient tumor cells led to a loss of M1 and increased M2 polarization in vitro, and patient NSCLC tissues with loss of HAI-1 showed a similar loss of M1 macrophages. Combined, these results suggest that loss of HAI-1 is a potential means through which tumors acquire an immunosuppressive, M2-dominated TME, potentially through impaired M1 macrophage polarization. Hence, HAI-1 status may be informative when stratifying patients that may benefit from therapies targeting the HGF pathway, particularly as an adjuvant to ICI-based immunotherapy.

Reproducibility of scoring criteria for HER2 immunohistochemistry in endometrial serous carcinoma: a multi-institutional interobserver agreement study.

Targeted anti-human epidermal growth factor receptor 2 (HER2) therapy has recently been proven to improve progression-free and overall survival of patients with advanced stage or recurrent endometrial serous carcinoma. To date, no specific pathology HER2 testing or scoring guidelines exist for endometrial cancer. However, based on evidence from the recent successful clinical trial and comprehensive pre-trial pathologic studies, a new set of HER2 scoring criteria have been proposed for endometrial serous carcinoma-distinct from the existing breast and gastric cancer-specific criteria. We present the first study assessing interobserver agreement of HER2 scores using the proposed serous endometrial cancer-specific scoring system. A digitally scanned set of 40 HER2-immunostained slides of endometrial serous carcinoma were sent to seven gynecologic pathologists, who independently assigned HER2 scores for each slide following a brief tutorial. Follow-up fluorescent in situ hybridization (FISH) for HER2 gene amplification was performed on cases with interobserver disagreement when a 2+ HER2 score was assigned by at least one observer. Complete agreement of HER2 scores among all 7 observers was achieved on 15 cases, and all but one case had an agreement by at least 4 observers. The overall agreement was 72.3% (kappa 0.60), 77.5% (kappa 0.65), and 83.3% (kappa 0.65), using four (0 to 3+ ), three (0/1+ , 2+ , 3+ ), or two (0/1+ , 2/3+ ) HER2 scoring categories, respectively. Based on the combination of HER2 immunostaining scores and FISH, the interobserver disagreement may have potentially resulted in a clinically significant difference in HER2 status only in three tumors. We conclude, that the proposed serous endometrial cancer-specific HER2 scoring criteria are reproducible among gynecologic pathologists with moderate to substantial interobserver agreement rates comparable to those of previously reported in breast and gastric carcinomas. Our findings significantly strengthen the foundation for establishing endometrial cancer-specific HER2 scoring guidelines in the future.

#TwitterHomework During Pathology Electives: Transforming Pathology Pedagogy.

CONTEXT.­: Twitter has become a popular platform for pathologists, where they share cases and educational content, arrange journal club meetings, network, and collaborate. OBJECTIVE.­: To determine if composing original tweets or retweeting existing content can be used as an educational and networking tool for medical students during pathology electives. DESIGN.­: In this retrospective study, a survey was sent to assess if medical students who used Twitter during their pathology electives found the platform useful for the attainment of medical knowledge, and for networking and professional development. A similar survey was sent to rotating students who did not use Twitter, asking if they thought using the platform could be beneficial. Additionally, we used Twitter analytical software (Symplur Signals) to determine the potential for networking by analyzing the number of retweets and impressions. RESULTS.­: Most respondents who used Twitter described using the platform as helpful in increasing their medical knowledge and useful for networking and professional development. From August 1, 2017, to January 2, 2019, thirty-seven elective medical students composed a total of 527 original tweets. The tweets were retweeted a total of 3399 times by 810 nonstudent users, and this engagement resulted in 6 360 731 impressions. Most of the retweeting was done by pathologists and pathology residents. CONCLUSIONS.­: The responses from the survey suggest that Twitter can be an educational tool during pathology electives and be useful for networking purposes. The number of retweets and impressions, and the demographics of the users who retweeted the students confirm the networking potential of Twitter.

Treatment of granuloma annulare and suppression of proinflammatory cytokine activity with tofacitinib.

BACKGROUND: Granuloma annulare (GA) is a common cutaneous inflammatory disorder characterized by macrophage accumulation and activation in skin. Its pathogenesis is poorly understood, and there are no effective treatments. The potential health implications of severe GA are unknown. OBJECTIVE: We sought to better understand GA pathogenesis and evaluate a molecularly targeted treatment approach for this disease. METHODS: We used single-cell RNA sequencing to study the immunopathogenesis of GA and also evaluated the efficacy of tofacitinib (a Janus kinase 1/3 inhibitor) in 5 patients with severe, long-standing GA in an open-label clinical trial. RESULTS: Using single-cell RNA sequencing, we found that in GA lesions IFN-γ production by CD4+ T cells is upregulated and is associated with inflammatory polarization of macrophages and fibroblasts. In particular, macrophages upregulate oncostatin M, an IL-6 family cytokine, which appears to act on fibroblasts to alter extracellular matrix production, a hallmark of GA. IL-15 and IL-21 production appears to feed back on CD4+ T cells to sustain inflammation. Treatment of 5 patients with recalcitrant GA with tofacitinib inhibited IFN-γ and oncostatin M, as well as IL-15 and IL-21, activity and resulted in clinical and histologic disease remission in 3 patients and marked improvement in the other 2. Inhibition of these effects at the molecular level paralleled the clinical improvement. Evidence of systemic inflammation is also present in some patients with severe GA and is mitigated by tofacitinib. CONCLUSIONS: The Janus kinase-signal transducer and activator of transcription pathway is activated in GA, likely in part through the activity of IFN-γ and oncostatin M, and Janus kinase inhibitors appear to be an effective treatment.

Does cholesterol suppress the antimicrobial peptide induced disruption of lipid raft containing membranes?

The activity of antimicrobial peptides has been shown to depend on the composition of the target cell membrane. The bacterial selectivity of most antimicrobial peptides has been attributed to the presence of abundant acidic phospholipids and the absence of cholesterol in bacterial membranes. The high amount of cholesterol present in eukaryotic cell membranes is thought to prevent peptide-induced membrane disruption by increasing the cohesion and stiffness of the lipid bilayer membrane. While the role of cholesterol on an antimicrobial peptide-induced membrane disrupting activity has been reported for simple, homogeneous lipid bilayer systems, it is not well understood for complex, heterogeneous lipid bilayers exhibiting phase separation (or "lipid rafts"). In this study, we show that cholesterol does not inhibit the disruption of raft-containing 1,2-dioleoyl-sn-glycero-3-phosphocholine:1,2-dipalmitoyol-sn-glycero-3-phosphocholine model membranes by four different cationic antimicrobial peptides, MSI-78, MSI-594, MSI-367 and MSI-843 which permeabilize membranes. Conversely, the presence of cholesterol effectively inhibits the disruption of non-raft containing 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyol-sn-glycero-3-phosphocholine lipid bilayers, even for antimicrobial peptides that do not show a clear preference between the ordered gel and disordered liquid-crystalline phases. Our results show that the peptide selectivity is not only dependent on the lipid phase but also on the presence of phase separation in heterogeneous lipid systems.