Quantitative assessment of PD-L1 as an analyte in immunohistochemistry diagnostic assays using a standardized cell line tissue microarray.

Programmed death 1 ligand 1 (PD-L1) Immunohistochemistry (IHC) is the key FDA-approved predictive marker to identify responders to anti-PD1 axis drugs. Multiple PD-L1 IHC assays with various antibodies and cut points have been used in clinical trials across tumor types. Comparative performance characteristics of these assays have been extensively studied qualitatively but not quantitatively. Here we evaluate the use of a standardized PD-L1 Index tissue microarray (TMA) to objectively determine agreement between antibody assays for PD-L1 applying quantitative digital image analysis. Using a specially constructed Index TMA containing a panel of ten isogenic cell lines in triplicate, we tested identical but independently grown batches of isogenic cells to prove Index TMAs can be produced in large quantities and hence serve as a standardization tool. Then the Index TMAs were evaluated using quantitative immunofluorescence (QIF) to validate the TMA itself and also to compare antibodies including E1L3N, SP142 and SP263. Next, an inter-laboratory and inter-assay comparison of 5 PD-L1 chromogenic IHC assays (US Food and Drug Administration (FDA) approved and lab developed test (LDT)) were performed at 12 sites around the USA. As previously reported, the SP142 FDA assay failed to detect low levels of PD-L1 in cell lines distinguished by the other four assays. The assays for 22C3 FDA, 28-8-FDA, SP263 FDA, and E1L3N LDT were highly similar across sites and all laboratories showed a high consistency over time for all assays using this Index TMA. In conclusion, we were able to objectively quantify PD-L1 expression on a standardized Index TMA using digital image analysis and we confirmed previous subjective assessments of these assays, but now in a multi-institutional setting. We envision commercial use of this Index TMA or similar smaller version as a useful standardization mechanism to compare results between institutions and to identify abnormalities while running routine clinical samples.

Using American Type Culture Collection Cell Lines to Evaluate Interlaboratory Variables for Estrogen Receptor and Human Epidermal Growth Factor Receptor 2 Immunostaining.

CONTEXT.­: Most laboratories currently use patient tissues for validating immunohistochemical stains. OBJECTIVE.­: To explore advantages of using cell lines with known antigenicity as a validation method. DESIGN.­: Five American Type Culture Collection (ATCC) cell lines with known negative, low positive, and moderate to strong estrogen receptor (ER) expression as well as negative, equivocal, and positive human epidermal growth factor receptor 2 (HER2) expression were cultured and made into cell blocks. One block from each cell line was fixed in formalin and another in ethanol before cell block preparation. Two sets of paired unstained slides from each block were sent to 10 different laboratories for HER2 and ER staining to be stained on runs from different days according to each laboratory's defined protocol. RESULTS.­: The 10 study participants evaluated 40 slides in a blinded fashion. For ER expression, all 80 interpretations for the ER strong and moderate positive cell lines had the target ER-positive result, and 74 of 80 ER-negative cell lines (92.5%) had agreement with the intended negative result. The ER low positive cell line showed varied but positive expression among all observers. The HER2 (3+)-positive cell lines yielded a target interpretation of 3+ in 65 of 80 interpretations (81.2%). For the HER2-negative cell line 69 of 78 interpretations (88.5%) were consistent with the target response (0 or 1+). No significant variation was observed between the ethanol- and non-ethanol-exposed cell lines, or between runs by the same laboratory. Variation from target results clustered within laboratories. CONCLUSIONS.­: This study indicates that variability between laboratories can be identified by using cell lines for quantitative or semiquantitative immunohistochemistry when using cultured cell lines of known antigenicity. These cell lines could potentially play a role in aiding anatomic pathology laboratories in validating immunohistochemistry tests for formalin- and ethanol-fixed tissues.

Follicular and Hürthle cell lesions of the thyroid: can inconclusive results be minimized?

OBJECTIVE: To assess the cytologic criteria for distinguishing neoplastic from nonneoplastic follicular cell and Hürthle cell thyroid lesions. STUDY DESIGN: Ten previously described and commonly used cytologic criteria were evaluated and graded on a 0-4 scale in a consecutive series of thyroid fine needle aspirations (FNAs) reported as follicular or Hürthle cell neoplasms or lesions. Scoring was compared to subsequent surgical outcome. RESULTS: A total of 93 (57fo llicular cell and 36 Hühle cell) cases was analyzed. No individual cytologic feature was helpful in distinguishing benign neoplarms from malignancy in either category (p > 0.05), but 4 or more coexistent cytologic features in combination were identified in 50.0% of follicular neoplasms, 13.6% of Hürthle cell neoplasms and none of the nonneoplastic lesions. An unexpected number (13 of 93, 14.0%) of unrecognized papillary carcinomas, some of follicular subtype, was encountered. CONCLUSION: In this series, the indeterminate thyroid FNA category could have been reduced by diagnosis of samples with 4 or more of the studied criteria as definite follicular (50% of cases) or Hürthle cell (13.6% of cases) neoplasms and by more astute recognition of papillary carcinomas (14.0% of cases), which blend into this category, often as a result of less-than-optimal sampling or preservation.

Transition to Subspecialty Sign-Out at an Academic Institution and Its Advantages.

Many pathology departments are introducing subspecialty sign-out in surgical pathology. In 2014, the University of Vermont Medical Center transitioned from general sign-out to partial subspecialty sign-out to include gastrointestinal and breast/cervix subspecialty benches; other specimens remained on general benches. Our experiences with the transition are described, including attending pathologist, trainee, support staff, and clinician satisfaction. A survey was e-mailed to all University of Vermont Medical Center anatomic pathology attendings, pathology trainees, pathologist assistants and grossing technicians, and clinicians who send surgical pathology specimens, immediately before and 1 year after transitioning to partial subspecialty sign-out. Quality assurance metrics were obtained for the 18 months prior to and following the transition. Gastrointestinal and breast/cervix attendings were more satisfied with partial subspecialty sign-out compared to those on the general benches. Overall, trainees were more satisfied with general sign-out because of the rotation schedule but preferred partial subspecialty sign-out due to improved teaching and more focused learning while on subspecialty benches. Clinicians remained very satisfied with our department and our reports; no differences were observed. Turnaround time was unchanged. After switching to partial subspecialty sign-out, there were significantly fewer discrepancies following multidisciplinary conference review for gastrointestinal and breast/cervix cases but remained the same for general cases. Fewer formal internal consults were performed after transitioning to partial subspecialty sign-out across all areas, but more notable for gastrointestinal and breast/cervix cases. Our data show improved quality assurance metrics and trainee education in a subspecialty sign-out setting compared to general sign-out setting.

Recommendations for Pathologic Evaluation of Reduction Mammoplasty Specimens: A Prospective Study With Systematic Tissue Sampling.

CONTEXT: - Breast reduction mammaplasty (RMP) for symptomatic macromastia or correction of asymmetry is performed in more than 100 000 patients per year in the United States. The reported incidence of significant pathologic findings (SPF), that is, carcinoma and atypical hyperplasia, ranges from 0.06% to 12.8%. No standard pathology assessment for RMP exists. OBJECTIVES: - To propose standard sampling for microscopic evaluation in RMP specimens, to evaluate the incidence of occult carcinoma and atypical hyperplasia, and to identify clinical risk factors for SPF in patients undergoing RMP. DESIGN: - All RMP specimens from 2006 to 2013 at a single institution were prospectively examined. After baseline gross and microscopic evaluations, each specimen was subjected to systematic additional sampling. The incidence of SPF was tabulated, and variables such as age, specimen weight, previous history of SPF, and results of preoperative mammogram were examined. Clinical follow-up review was also subsequently undertaken. RESULTS: - A total of 595 patients were evaluated. Significant pathologic findings were present in 9.8% (58 of 595) of patients. No cancer was identified in patients younger than 40 years; the rates of carcinoma were 2.4% (14 of 595) in all patients, 3.6% (14 of 392) in patients aged 40 years or older, and 4.3% (10 of 233) in patients aged 50 years or older. No carcinoma or atypical hyperplasia was identified on preoperative mammogram. Increased sampling was associated with a significantly greater frequency of SPF only in patients aged 40 years or older. CONCLUSIONS: - In patients younger than 35 years, gross-only evaluation is sufficient. However, increased sampling may be necessary in patients older than 40 years.

Comparison of Estrogen and Progesterone Receptor Antibody Reagents Using Proficiency Testing Data.

CONTEXT: - Immunohistochemical analysis of estrogen receptor (ER) and progesterone receptor (PgR) expression in breast cancer is the current standard of care and directly determines therapy. In 2010 the American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) published guidelines for ER and PgR predictive testing, encompassing preanalytic, analytic, postanalytic factors; antibody validation; and proficiency testing. OBJECTIVE: - To compare the performance of different antibody reagents for ER and PgR immunohistochemical analysis by using CAP proficiency testing data. DESIGN: - The CAP PM2 survey uses tissue microarrays of ten 2-mm cores per slide. We analyzed survey data from 80 ER and 80 PgR cores by antibody clone from more than 1200 laboratories. RESULTS: - Laboratories used the ER antibodies SP1 (72%), 6F11 (17%), 1D5 (3%), and the PgR antibodies 1E2 (61%), 16 (12%), PgR-636 (13%), PgR-1294 (8%) in 2015. While 63 of 80 ER cores (79%) were scored similarly using each of the 3 antibodies, there were significant differences for others, with SP1 yielding more positive interpretations. Four cores were scored as ER negative by more than half of the laboratories using 1D5 or 6F11, while SP1 produced positive results in more than 70% of laboratories using that antibody. Despite the greater variety of PgR antibody reagents and greater PgR tumor heterogeneity, 61 of 80 cores (76%) were scored similarly across the 4 PgR antibodies. CONCLUSIONS: - Accurate ER and PgR testing in breast cancer is crucial for appropriate treatment. The CAP proficiency testing data demonstrate differences in staining results by ER clone, with SP1 yielding more positive results.

Increased utilization, verification, and clinical implications of immunocytochemistry: Experience in a northern New England hospital.

BACKGROUND: We report our experience in utilization, verification, and clinical implications of antibodies for use in diagnostic immunocytochemistry (ICC). METHODS: A computer search identified cytology cases utilizing ICC and corresponding surgical pathology material. Alcohol-fixed liquid based cytology (LBC) specimens were generated from surgical pathology bench specimens. ICC on LBC and immunohistochemistry on formalin fixed paraffin embedded tissue (FFPE) were performed in parallel for 71 commonly used antibodies. Cytology and corresponding surgical pathology reports were reviewed for all cases in which antibodies failed verification studies but had been used in the four years prior to implementation of our verification process. RESULTS: From 2007 to 2011, the number of cytology cases in which ICC was performed increased from 98 (or 5% of all non-Pap test/nonurine cytology cases in our laboratory) to 306 (or 15%). Verification studies revealed calretinin, CD5, c-kit/CD117, inhibin, napsin A, OCT 3/4, and PAX-5 to be nonreliable in LBC despite consistent immunoreactivity in concurrent IHC on surgical specimens. No antibodies were found to be immunoreactive on LBC but nonreactive on FFPE. No adverse clinical outcomes resulted from the use of nonverified antibodies. CONCLUSIONS: Utilization of ICC at our institution has increased dramatically in recent years. Our verification process confirmed reliability in the majority of antibodies, but did identify several inconsistent antibodies. Although, in our series, no adverse clinical outcomes resulted from preverification use of these inconsistent antibodies, we encourage other institutions to confirm reliability of antibodies prior to use for diagnostic ICC.

IgG4-Related Neuromyopathy Associated With Recurrent Pleural Effusion.

OBJECTIVE: Immunoglobulin G4-related disease (IgG4-RD) is an increasingly recognized idiopathic systemic disorder associated with elevated serum IgG4 level and tissue infiltration by IgG4-positive plasma cells. Multiple neurological manifestations, including peripheral neuropathy, have been described in IgG4-RD. Our objective is to discuss a case report and review of the literature, which would expand the spectrum of IgG4-RD. METHODS: We describe the clinical features and biopsy findings in a patient with IgG4-RD who presented with features suggestive of neuromyopathy in the setting of recurrent pleural effusion and weight loss. RESULTS: Electrodiagnostic findings were suggestive of an irritable myopathy and polyradiculoneuropathy with primary demyelination and secondary axonal degeneration. Pleural biopsy and laboratory studies confirmed the diagnosis. Improvement was sustained with steroid therapy. CONCLUSIONS: We describe the first case, to our knowledge, of IgG4-related neuromyopathy associated with recurrent pleural effusion. Our case expands the clinical spectrum of IgG4-RD. Neurologists should be aware of this treatable disorder and in the appropriate clinical context consider it in the differential diagnosis of neuromyopathy.

Metastatic melanoma presenting as an isolated breast tumor: a study of 20 cases with emphasis on several primary mimickers.

CONTEXT: The mammary gland can be a site of metastasis in patients with malignant melanoma, which is easily recognized microscopically if clinical information is available. Nonetheless, metastatic melanoma presenting as an isolated mammary tumor can be more challenging to diagnose because it can simulate a primary breast carcinoma clinically and morphologically. OBJECTIVE: To review metastatic melanoma to the breast, presenting as primary breast carcinomas clinically and morphologically. DESIGN: The authors report 20 cases of metastatic melanoma clinically presenting as breast tumors. Cases with widespread metastatic presentation were excluded. RESULTS: Epithelioid and spindle cell tumors predominated, suggesting mammary ductal, papillary, or sarcomatoid carcinoma. Most cases (16 of 20) were submitted for consultation or second opinion owing to their unusual presentation in the breast, or to perform predictive/prognostic immunohistochemical assays. Seven cases had a remarkable phenotypic spectrum expanding the differential diagnosis to large cell lymphoma, leiomyosarcoma, medullary carcinoma, malignant schwannoma, and liposarcoma. Tumor cells were negative for cytokeratin stains and positive for S100 protein, HMB-45, and Melan-A. Negative staining was also observed for epithelial membrane antigen, CD45, desmin, estrogen and progesterone receptors, and human epidermal growth factor receptor 2. CONCLUSIONS: Metastatic melanoma may simulate a broad spectrum of primary breast malignancies. Although the application of a simple panel of antibodies assists in rendering the correct interpretation, lesions presenting as isolated breast tumors may introduce a significant diagnostic difficulty, especially when there is inadequate patient history and/or limited biopsy material. Further challenges are introduced by the extraordinary phenotypic plasticity of metastatic melanoma. Awareness of this pattern variance is essential to avoid inappropriate treatment, especially in cases simulating a "triple negative," poorly differentiated carcinoma of the breast.

Adult testicular granulosa cell tumor: a review of the literature for clinicopathologic predictors of malignancy.

Adult testicular granulosa cell tumors are rare sex cord-stromal tumors of which only 28 have been previously reported. As compared with their ovarian counterparts, these tumors may follow a more aggressive course because the proportion of malignant cases is higher. To date, there are no clinical or pathologic features that definitively predict malignancy. We reviewed all prior case reports for features that may predict their malignant potential. Tumor size greater than 5.0 cm is the only feature statistically associated with malignancy. Mitotic count, tumor necrosis, patient age, and the presence of gynecomastia do not, at present, predict benign versus malignant behavior.

Carcinoma and atypical hyperplasia in reduction mammaplasty: increased sampling leads to increased detection. A prospective study.

BACKGROUND: Reduction mammaplasty for symptomatic macromastia or correction of asymmetry is performed more than 100,000 times per year in the United States. The reported incidence of occult breast cancer in reduction mammaplasty ranges from 0.06 to 4.6 percent. No standard pathology assessment for reduction mammaplasty exists. The authors evaluated the incidence of occult carcinoma and atypical hyperplasia in reduction mammaplasty specimens and identified clinical risk factors. Systematic sampling of additional tissue sections was instituted to evaluate the hypothesis that increased sampling would identify more significant pathologic findings. METHODS: All reduction mammaplasty specimens over a 20-month period at a single institution were prospectively examined. All specimens had baseline gross and microscopic evaluations, and then each was subjected to systematic additional sampling. The incidence of significant pathologic findings (carcinoma and atypical hyperplasia) was tabulated. Variables such as age and preoperative mammogram were examined. RESULTS: A total of 202 cases were evaluated. Significant pathologic findings (carcinoma and atypical hyperplasia) were present in 12.4 percent. The rate of carcinoma was 4 percent in all patients (6.2 percent in patients >or=40 years and 7.9 percent in patients >or=50 years). CONCLUSIONS: A significantly higher rate (12.4 percent) of significant pathologic findings was identified in this prospective study compared with published literature. None of the lesions was identified on preoperative mammogram. Age was significantly associated with significant pathologic findings. Increased sampling was associated with significant pathologic findings only in patients 40 years or older, indicating the need for thorough sampling of reduction mammaplasty specimens in patients older than 40.