Carlos Parra-Herran

Cervical squamocolumnar junction-specific markers define distinct, clinically relevant subsets of low-grade squamous intraepithelial lesions

Low-grade cervical squamous abnormalities (low-grade squamous intraepithelial lesions [LSIL, CIN1]) can be confused with or followed by high-grade (HSIL, CIN2/3) lesions, expending considerable resources. Recently, a cell of origin for cervical neoplasia was proposed in the squamocolumnar junction (SCJ); HSILs are almost always SCJ+, but LSILs include SCJ+ and SCJ− subsets. Abnormal cervical biopsies from 214 patients were classified by 2 experienced pathologists (panel) as LSIL or HSIL using published criteria. SILs were scored SCJ+ and SCJ− using SCJ-specific antibodies (keratin7, AGR2, MMP7, and GDA). Assessments of interobserver agreement, p16ink4 staining pattern, proliferative index, and outcome were compared. The original diagnostician agreed with the panel diagnosis of HSIL and SCJ− LSIL in all cases (100%). However, for SCJ+ LSIL, panelists disagreed with each other by 15% and with the original diagnostician by 46.2%. Comparing SCJ− and SCJ+ LSILs, 60.2% and 94.9% were p16ink4 positive, 23% and 74.4% showed strong (full-thickness) p16ink4 staining, and 0/54 (0%) and 8/33 (24.2%) with follow-up had an HSIL outcome, respectively. Some SCJ+ LSILs are more likely to both generate diagnostic disagreement and be associated with HSIL. Conversely, SCJ− LSILs generate little observer disagreement and, when followed, have a very low risk of HSIL outcome. Thus, SCJ biomarkers in conjunction with histology may segregate LSILs with very low risk of HSIL outcome and conceivably could be used as a management tool to reduce excess allocation of resources to the follow-up of these lesions.

Inflammatory myofibroblastic tumor of the uterus: clinical and pathologic review of 10 cases including a subset with aggressive clinical course.

Inflammatory myofibroblastic tumor is currently regarded as a neoplasm with intermediate biological potential and a wide anatomic distribution. Inflammatory myofibroblastic tumors of the female genital tract are rare, and to date reported cases behaved indolently. We describe, herein, 10 cases of uterine inflammatory myofibroblastic tumor, 3 of which had an aggressive clinical course. Subject age ranged from 29 to 73 years. Tumors were composed of spindle and epithelioid myofibroblastic cells admixed with lymphoplasmacytic infiltrates in a variably myxoid stroma. Two growth patterns, myxoid and fascicular (leiomyoma-like), were noted. All tumors were positive for ALK expression by immunohistochemistry, which was stronger in the myxoid areas. Smooth muscle marker and CD10 expression was variable in extent, but typically positive. Fluorescence in situ hybridization for ALK rearrangements was positive in both fascicular and myxoid areas in all 8 cases tested. Three subjects showed clinical evidence of tumor aggressiveness as defined by extrauterine spread, local recurrence, or distant metastasis. Aggressive tumors were larger, had a higher proportion of myxoid stroma, and higher mitotic activity than indolent tumors. Tumor cell necrosis was seen only in cases with adverse outcome. This is the first report to describe aggressive biological behavior in uterine inflammatory myofibroblastic tumor. This diagnosis is often underappreciated and merits inclusion in the differential diagnosis of myxoid mesenchymal lesions of the uterus, particularly because patients with an aggressive course may benefit from targeted therapy.

CLIPPERS COMPLICATING MULTIPLE SCLEROSIS CAUSING CONCERNS OF CNS LYMPHOMA

Melissa R. Ortega, MD Nida Usmani, MD Carlos Parra-Herran, MD David J. Adams, MD Brian Steingo, MD Kottil W. Rammohan, MD CLIPPERS COMPLICATING MULTIPLE SCLEROSIS CAUSING CONCERNS OF CNS LYMPHOMA Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is a recently described inflammatory disorder of the brainstem and cerebellum with distinct clinical and radiologic features.1 Coexistence of CLIPPERS and multiple sclerosis (MS) has never been previously described. We present a patient with MS who developed CLIPPERS shortly after natalizumab withdrawal, and the presentation raised concerns of primary CNS lymphoma which led to a brain biopsy. Awareness of this benign disorder may well have averted this biopsy. Recognition of CLIPPERS is crucial when one encounters perivascular enhancing lesions of the brainstem that are atypical for MS, but typical for this disorder.

Histopathology of Placenta Creta: Chorionic Villi Intrusion into Myometrial Vascular Spaces and Extravillous Trophoblast Proliferation are Frequent and Specific Findings With Implications for Diagnosis and Pathogenesis

Placenta creta is characterized by invasion of placental villi into the myometrium in the setting of a dysfunctional or absent decidua. Histopathologic diagnosis of placenta creta is important, particularly in cases of hysterectomy because of unanticipated intractable postpartum hemorrhage. Previous studies have documented a higher amount and depth of myometrial infiltration by the implantation site intermediate trophoblast compared with controls. In addition, we have anecdotally observed chorionic villi in myometrial vascular spaces in specimens with placenta creta. The aim of this study was to explore the prevalence and specificity of these features. Sixty-one postpartum hysterectomies, 44 with placenta creta and 17 without were reviewed. Villous intrusion into vascular spaces was recorded. Using immunohistochemistry for GATA3, the amount of intermediate trophoblast (number of positive cells in five 40× fields) and depth of trophoblast myometrial infiltration were assessed. Mean gestational ages of the creta group (34.4 yr; range, 20–43 yr) and control group (35 yr; range, 25–51 yr) were comparable. Presence of chorionic villi in myometrial vascular spaces was frequent in placenta creta: 31/44 versus 1/17 controls (70.4% vs. 5.8%, P<0.0001). This finding was more common in the percreta (87.5%) and increta (84%) than in the accreta (27.2%, P=0.0008). Mean depth of trophoblast myometrial invasion was greater in cretas (47.9%) than in controls (14.5%, P=0.004). Likewise, mean distance of deepest trophoblast to serosa was shorter in the cretas (7.3 mm) than in controls (23.8 mm, P<0.0001). These differences were, however, attributable to placentas increta and percreta. When only accretas and controls were compared, the myometrial depth of trophoblast was similar. The mean intermediate trophoblast cell count in the placental bed was greater in cretas (664) than in controls (288, P<0.0001). Such difference was seen in all creta cases despite the type (accreta 639, increta 676, percreta 661). A trophoblast count of ≥100 cells/high-power field was seen in 75.8% of cretas and 11.1% of controls (P=0.0009). For the first time, we document the finding of chorionic villi intrusion into myometrial vascular spaces, which is highly specific of placenta creta. In addition, assessment of the amount of intermediate trophoblast using GATA3 immunohistochemistry can assist in the diagnosis. We hypothesize that placental invasion in placenta creta is due, at least partially, to transformation of low-resistance myometrial vessels leading to subsequent protrusion of villi into their lumens, in the context of absent decidua.

Immunohistochemistry in the Diagnosis of Mucinous Neoplasms Involving the Ovary: The Added Value of SATB2 and Biomarker Discovery Through Protein Expression Database Mining.

Immunohistochemistry is frequently used to identify ovarian mucinous neoplasms as primary or metastatic; however, there is significant overlap in expression patterns. We compared traditional markers (CK7, CK20, CDX2, PAX8, estrogen receptor, &bgr;-catenin, MUC1, MUC2, and MUC5AC) to 2 novel proteins identified through mining of the Human Protein Atlas expression database: SATB2 and POF1B. The study cohort included 49 primary gastrointestinal (GI) mucinous adenocarcinomas (19 colorectal, 15 gastric, 15 pancreatobiliary), 60 primary ovarian mucinous neoplasms (19 cystadenomas, 21 borderline tumors, 20 adenocarcinomas), and 19 metastatic carcinomas to the ovary (14 lower and 5 upper GI primaries). Immunohistochemistry was performed on tissue microarrays, scored and interpreted as negative (absent or focal/weak) or positive. Metastatic tumors were frequently unilateral (42.8% of tumors from lower and 40% of tumors from upper tract) and ≥10 cm (85.7% of tumors from lower and 80% of tumors from upper tract). CK7 was positive in 88.5% upper GI and 88.3% primary ovarian compared with 24.3% lower GI neoplasms. CK20 and CDX2 were positive in 84.8% and 100% of lower GI tumors, respectively; however, expression was also common in upper GI (CK20 42.8%, CDX2 50%) and primary ovarian neoplasms (CK20 65.7%, CDX2 38.3%). Conversely, SATB2 was more specific for lower GI origin, being positive in 78.8% lower GI but only 11.5% upper GI and 1.7% primary ovarian neoplasms. PAX8 expression was common in primary ovarian neoplasms (75% of all neoplasms, 65% of carcinomas); only 1 (1.5%) GI tumor was positive. MUC2 and &bgr;-catenin were frequently positive in lower GI tumors (96.9% and 51.5%, respectively). Estrogen receptor expression was only seen in primary ovarian neoplasms (13.3%). Nuclear premature ovarian failure 1B (POF1B) expression was seen in malignant tumors regardless of their origin. A panel including CK7, SATB2, and PAX8 separated primary from secondary GI neoplasms with up to 77.1% sensitivity and 99% specificity, outperforming tumor laterality and size. Second-line markers such as CDX2, MUC2, estrogen receptor, MUC1, and &bgr;-catenin increased the sensitivity of immunohistochemistry in excluding lower GI origin. Biomarker search using proteomic databases has a value in diagnostic pathology, as shown with SATB2; however, as seen with POF1B, expression profiles in these databases are not always reproduced in larger cohorts.

Targeted development of specific biomarkers of endometrial stromal cell differentiation using bioinformatics: the IFITM1 model

When classifying cellular uterine mesenchymal neoplasms, histological distinction of endometrial stromal from smooth muscle neoplasms can be difficult. The only widely established marker of endometrial stromal differentiation, CD10, has marginal specificity. We took a bioinformatics approach to identify more specific markers of endometrial stromal differentiation by searching the Human Protein Atlas, a public database of protein expression profiles. After screening the database using different methods, interferon-induced transmembrane protein 1 (IFITM1) was selected for further analysis. Immunohistochemistry for IFITM1 was performed using tissue sections from the selected cases of proliferative endometrium (22), secretory endometrium (6), inactive endometrium (19), adenomyosis (10), conventional leiomyoma (11), cellular leiomyoma (16), endometrial stromal nodule (2), low-grade endometrial stromal sarcoma (16), high-grade endometrial stromal sarcoma (2) and undifferentiated uterine sarcoma (2). Stained slides were scored in terms of intensity and distribution. Normal endometrial samples uniformly showed diffuse and strong IFITM1 staining. Endometrial stromal neoplasms, particularly low-grade endometrial stromal sarcoma, showed higher IFITM1 expression compared with smooth muscle neoplasms (P<0.0001). IFITM1 immunohistochemistry has high sensitivity and specificity, particularly in the distinction between low-grade endometrial stromal sarcoma and leiomyoma (81.2 and 86.7%, respectively). Our results indicate that IFITM1 is a sensitive and specific marker of endometrial stromal differentiation across the spectrum from proliferative endometrium to metastatic stromal sarcoma. IFITM1 is a potential valuable addition to immunohistochemical panels used in the diagnosis of cellular mesenchymal uterine tumors. Further studies with larger number of cases are necessary to corroborate this impression and determine the utility of IFITM1 in routine practice. This study is a clear example of how bioinformatics, particularly tools for mining genomic and proteomic databases, can enhance and accelerate biomarker development in diagnostic pathology.

Application of a Pattern-based Classification System for Invasive Endocervical Adenocarcinoma in Cervical Biopsy, Cone and Loop Electrosurgical Excision (LEEP) Material: Pattern on Cone and LEEP is Predictive of Pattern in the Overall Tumor.

A pattern-based classification system has been recently proposed for invasive endocervical adenocarcinoma, which is predictive of the risk of nodal metastases. Identifying cases at risk of nodal involvement is most relevant at the time of biopsy and loop electrosurgical excision procedure (LEEP) to allow for optimal surgical planning, and, most importantly, consideration of lymphadenectomy. This study aims to determine the topography of patterns of stromal invasion in invasive endocervical adenocarcinoma with emphasis on patterns in biopsy, cone, and LEEP. Invasive pattern was assessed following the pattern-based classification (Patterns A, B, and C) in 47 invasive endocervical adenocarcinomas treated with hysterectomy or trachelectomy and correlated with pattern of invasion at the tumor surface (2 mm of tumor depth) and on preoperative biopsy and cone/LEEP. Patterns A, B, and C were present in 21.3%, 36.2%, and 42.5% of cases, respectively. Most pattern A cases were Stage IA (90%), whereas most Pattern B and C cases were Stage IB (76.5% and 80%, respectively). Horizontal spread was on average larger in Pattern C (24.1 mm) than in Patterns A and B (7.7 and 12.3 mm, respectively). Pattern at the tumor surface correlated with the overall pattern in 95.7% of cases. Concordance between patterns at cone/LEEP and hysterectomy was 92.8%; the only discrepant case was upgraded from Pattern A on LEEP to C on final excision. Agreement between patterns in biopsy and the overall tumor, however, was only 37.5%. In all discrepant cases, biopsy failed to reveal destructive invasion, which was evident on excision. All discrepant biopsies with pattern A showed glandular complexity resembling exophytic papillary growth but did not meet criteria for destructive invasion. On excision, marked gland confluence with papillary architecture was evident. We conclude that the pattern of invasion on cone/LEEP is a good predictor of pattern of invasion on hysterectomy, particularly if there is destructive invasion (B or C). Thus, pattern-based classification can be successfully applied in these samples to guide definitive surgical treatment. Prediction of the overall pattern based on biopsy material alone appears to be suboptimal. However, glandular confluence and complexity on biopsy, regardless of its size, appears to be associated with destructive invasion in the overall tumor.

Pattern-based classification of invasive endocervical adenocarcinoma, depth of invasion measurement and distinction from adenocarcinoma in situ: interobserver variation among gynecologic pathologists.

A pattern-based classification for invasive endocervical adenocarcinoma has been proposed as predictive of the risk of nodal metastases. We aimed to determine the reproducibility of such classification in the context of common diagnostic challenges: distinction between in situ and invasive adenocarcinoma and depth of invasion measurement. Nine gynecologic pathologists independently reviewed 96 cases of endocervical adenocarcinoma (two slides per case). They diagnosed each case as in situ or invasive carcinoma classifying the latter following the pattern-based classification as pattern A (non-destructive), B (focally destructive) or C (diffusely destructive). Depth of invasion, when applicable, was measured (mm). Overall, diagnostic reproducibility of pattern diagnosis was good (κ=0.65). Perfect agreement (9/9 reviewers) was seen in only 11 cases (11%), all destructively invasive (10 pattern C and 1 pattern B). In all, ≥5/9 reviewer concordance was achieved in 82/96 cases (85%). Distinction between in situ and invasive carcinoma, regardless of the pattern, showed only slight agreement (κ=0.37). Likewise, distinction restricted to in situ versus pattern A was poor (κ=0.23). Distinction between non-destructive (in situ+pattern A) and destructive (patterns B+C) carcinoma showed significantly higher agreement (κ=0.62). Estimation of depth of invasion showed excellent reproducibility (ICC=0.82). However, different measurements resulting in differing FIGO stages were common (from at least 1 reviewer in 79% cases). On the basis of interobserver agreement, the pattern-based classification is best at diagnosing destructive invasion, which carries a risk for nodal metastases. Agreement in diagnosing in situ versus invasive carcinoma, including pattern A, was poor. Given the nil risk of nodal spread in in situ and pattern A lesions, the term ‘endocervical adenocarcinoma with non-destructive growth’ can be considered when the distinction is difficult, after excluding destructive invasion. Depth of invasion measurement was highly reproducible among pathologists; thus, the pattern-based approach can complement, but should not replace, the depth of invasion metric.

High-grade Müllerian Adenosarcoma: Genomic and Clinicopathologic Characterization of a Distinct Neoplasm With Prevalent tp53 Pathway Alterations and Aggressive Behavior

Müllerian adenosarcoma harbors low malignant potential, except in cases with myometrial invasion or sarcomatous overgrowth. The presence of a high-grade stromal component has been proposed as an important pathologic predictor of outcome. We hypothesized that high-grade adenosarcoma has distinct clinical and molecular features, distinct from low-grade adenosarcoma. We analyzed the clinicopathologic features and follow-up of 9 high-grade adenosarcomas and a control group of 9 low-grade adenosarcomas. Comprehensive genomic analysis of the high-grade group was performed targeting exons of 409 oncogenes and tumor suppressor genes. In 1 case, the high-grade and low-grade components were separately sequenced. High-grade and low-grade adenosarcomas were comparable in patient age, myometrial invasion, and stage at presentation. Sarcomatous overgrowth was observed in 2/9 (22%) low-grade and 8/9 (89%) high-grade adenosarcomas. Six of 9 (67%) patients with high-grade adenosarcoma developed rapid recurrence; 1 died of her disease. Conversely, no low-grade tumors recurred or metastasized. Sequencing of high-grade adenosarcomas revealed frequent TP53 pathway alterations, identified in 7/9 (78%) cases. p53 expression by immunohistochemistry highly correlated with mutation status. Copy number variations occurred at a mean of 28.8 per tumor; most frequently involved genes included CDK4, MDM2, GNAS, SGK1, and DICER1. High-grade adenosarcoma is an aggressive neoplasm with propensity for short-interval recurrence and metastasis. The proportion of copy number alterations is similar to that reported for adenosarcoma with sarcomatous overgrowth. However, the high frequency of TP53 abnormalities is a novel finding, indicating that high-grade adenosarcoma is a distinct subset with driver TP53 pathway alterations. p53 immunohistochemistry can be used to confirm the presence of a high-grade component. Given its aggressive potential, the presence of any high-grade component in an adenosarcoma should be reported, even in the absence of sarcomatous overgrowth.

Genomic abnormalities in invasive endocervical adenocarcinoma correlate with pattern of invasion: biologic and clinical implications

The pattern-based classification system for HPV-related endocervical adenocarcinoma, which classifies tumors based on the destructiveness of stromal invasion, is predictive of the risk of nodal metastases and adverse outcome. Previous studies have demonstrated clinically important molecular alterations in endocervical adenocarcinoma, including KRAS and PIK3CA mutations; however, correlation between the molecular landscape and pathological variables including pattern of invasion has not been thoroughly explored. In this study, 20 endocervical adenocarcinomas were classified using the pattern-based classification system and were subjected to targeted sequencing using the Ion AmpliSeq Cancer Hotspot Panel v2 (ThermoFisher Scientific, Waltham, MA, USA) that surveys hotspot regions of 50 oncogenes and tumor suppressor genes. Single-nucleotide polymorphisms were correlated with clinical and pathologic variables including pattern of invasion. Five (25%), six (30%), and nine (45%) cases were classified as patterns A, B, and C respectively. Lymph node metastases, advanced stage at presentation and mortality from disease were exclusively seen in destructively invasive tumors (patterns B or C). Prevalent mutations in the cohort involved PIK3CA (30%), KRAS (30%), MET (15%), and RB1 (10%). Most (94%) relevant genomic alterations were present in destructively invasive tumors with PIK3CA, KRAS, and RB1 mutations seen exclusively in pattern B or C subgroups. KRAS mutations correlated with advanced stage at presentation (FIGO stage II or higher). Our findings indicate that the pattern of stromal invasion correlates with genomic abnormalities detected by next-generation sequencing, suggesting that tumors without destructive growth (pattern A) are biologically distinct from those with destructive invasion (patterns B and C), and that pattern B endocervical adenocarcinoma is more closely related to its pattern C counterpart. The pattern-based classification may be used as a triage tool when considering molecular testing for prognostic or therapeutic purposes.