Reproducibility in PD-L1 Immunohistochemistry Quantification through the Tumor Proportion Score and the Combined Positive Score: Could Dual Immunostaining Help Pathologists?

We studied the pathologists' agreements in quantifying PD-L1 expression through the tumor proportion score (TPS) and the combined positive score (CPS) using single PD-L1 immunohistochemistry (S-IHC) and double immunohistochemistry (D-IHC) combining PD-L1 staining and tumor cell markers. S-IHC and D-IHC were applied to 15 cancer samples to generate 60 digital IHC slides (30 whole slides images and 30 regions of interest of 1 mm2) for PD-L1 expression quantification using both TPS and CPS, twice by four pathologists. Agreements were estimated calculating intraclass correlation coefficients (ICC). Both S-IHC and D-IHC slides analyses resulted in excellent (for TPS, ICC > 0.9) to good (for CPS, ICC > 0.75) inter- and intra-pathologist agreements with slightly higher ICC with D-IHC than with S-IHC. S-IHC resulted in higher TPS and CPS than D-IHC (+5.6 and +6.1 mean differences, respectively). High reproducibility in the quantification of PD-L1 expression is attainable using S-IHC and D-IHC.

Detection of NTRK fusions in glioblastoma: fluorescent in situ hybridisation is more useful than pan-TRK immunohistochemistry as a screening tool prior to RNA sequencing.

Glioblastomas are frequent malignant brain tumours with a very poor prognosis and a need for new and efficient therapeutic strategies. With the approval of anti-TRK targeted therapies to treat patients with advanced NTRK-rearranged cancers, independent of the type of cancer, potential new treatment opportunities are available for the 0.5-5% of patients with NTRK-rearranged glioblastomas. Identification of these rare NTRK-rearranged glioblastomas requires efficient diagnostic tools and strategies which are evaluated in this study. We compared the results of NTRK1, NTRK2 and NTRK3 fluorescent in situ hybridisation (FISH) assays to those of pan-TRK immunohistochemistry (IHC) using two EPR17341 and A7H6R clones in a set of 196 patients with glioblastomas. Cases with at least 15% of positive nuclei using FISH analyses were further analysed using RNA sequencing. Above the 15% threshold, seven positive glioblastomas (3.57%) were identified by FISH assays (4 NTRK1, 3 NTRK2, no NTRK3). NTRK rearrangements were confirmed by RNA sequencing analyses in four cases [1 LMNA-NTRK1, 1 PRKAR2A-NTRK2, 1 SPECC1L-NTRK2 and 1 NACC2-NTRK2 fusions, i.e., 4/196 (2%) of NTRK-rearranged tumours in our series] but no rearrangement was detected in three samples with less than 30% of positive tumour nuclei as determined by NTRK1 FISH. Pan-TRK immunostaining showed major discrepancies when using either the EPR17341 or the A7H6R clones for the following criteria: main intensity, H-Score based scoring and homogeneity/heterogeneity of staining (Kappa values <0.2). This led to defining adequate criteria to identify NTRK-rearranged gliomas exhibiting strong and diffuse immunostaining contrasting to the variable and heterogeneous staining in non-NTRK-rearranged gliomas (p<0.0001). As assessing NTRK rearrangements has become crucial for glioma therapy, FISH seems to be a valuable tool to maximise access to TRK testing in patients with glioblastomas. In contrast to other cancers, pan-TRK IHC appears of limited interest in this field because there is no 'on/off' IHC positivity criterion to distinguish between NTRK-rearranged and non-NTRK-rearranged gliomas. RNA sequencing analyses are necessary in FISH positive cases with less than 30% positive nuclei, to avoid false positivity when scoring is close to the detection threshold.

An atypical lower limb pain revealing a possible systemic venous vasculitis.

We describe a possible systemic vasculitis involving electively large veins. The patient presented with severe febrile lower limb pain. Diagnosis was made by color Doppler ultrasound (CDU) and confirmed by anatomopathological examination of the long saphenous vein, but not by examination of the temporal artery which was normal. CDU found a unilateral halo sign of one temporal artery and a major wall swelling of the lower limb proximal deep veins. The etiology of this possible vasculitis is still unknown. It could be an unusual clinical presentation of giant cell arteritis with vein involvement but without proven arterial involvement. To confirm this hypothesis, it would be interesting to look systematically for lower limb vein thickening with CDU in patients newly diagnosed with giant cell arteritis who have lower limb pain.

HER2 Mutated and Nonmutated Non-small Cell Lung Carcinomas Can Harbor Heterogeneous HER2 Gene Amplification and HER2 Protein Expression.

Molecular analyses have become mandatory for treatment choices in patients with advanced non-small cell lung cancers (NSCLC). Among them, HER2 gene mutation, HER2 gene amplification, and HER2 protein expression consist in potential targets of various treatments. Tumor heterogeneity and overlapping of molecular alterations may cause dilemmas in treatment choices but to date there are few that reported about HER2 with discrepant data. We led a retrospective study evaluating HER2 protein expression and HER2 gene/chromosome 17 copy number variations across different tumor areas and samples from patients with advanced NSCLC harboring HER2 gene mutations and other oncogenic mutations. Among patients with HER2-mutated (10 patients) and nonmutated lung adenocarcinomas (10 patients), we observed frequent heterogeneous HER2 protein expression with no correlation with HER2 gene copy number variations. HER2 gene amplification was observed in 6 patients (3 HER2-mutated and 3 HER2-nonmutated), but with intrasample heterogeneity in 2 cases and intersample heterogeneity in another case. Our small case series emphasizes the potential overlapping and spatial heterogeneity of HER2 alterations in NSCLC, which must be taken into account as a limitation in building predictive strategies accompanying the development of anti-HER2 therapeutic strategies in patients with advanced NSCLC.

Ramp Lesions of the Posterior Segment of the Medial Meniscus: What Is Repaired? A Qualitative Histological Study of the Meniscocapsular and Meniscotibial Attachments.

BACKGROUND: Lesions of the posterior segment of the medial meniscus are the most common intraarticular lesions associated with ACL injuries. Ramp lesions are tears in the peripheral attachment of the posterior horn of the medial meniscus. Such injuries are difficult to detect on preoperative MRI. Arthroscopically, the prevalence of these lesions can reach 24%. Anatomical descriptions of the posterior horn of the medial meniscus are becoming clearer, however, histological descriptions are lacking, especially with regard to the presence or absence of capillaries. QUESTIONS/PURPOSES: The present qualitative histologic study focused on the posterior segment of the medial meniscus and the meniscocapsular and meniscotibial junctions. Specifically, the objective of this study was to analyze the posterior segment of the medial meniscus and the meniscosynovial junction and to determine whether the meniscus tibial ligament exists. METHODS: We dissected 10 unpaired cadaveric knees (five male, five female, age range 55 to 66 years), five left and five right, from the French "Don du corps" body donation program via a posterior approach to the posteromedial capsule. We excluded specimens with intra-articular abnormalities (ACL rupture, meniscal tear, arthrosis) preceding dissection by arthrotomy. We thus accessed the posterior segment of the medial meniscus and the meniscosynovial junction. The proximal capsule, posterior segment of the medial meniscus, entire meniscal capsular-tibial junction, and a fragment of the tibia were removed en bloc. For each knee, three sagittal spaced sections of the posterior segment of the medial meniscus (Zone 4 as defined by Smigielski) were performed. Two experienced pathologists performed qualitative histological analysis on the 30 samples after Hematoxylin and eosin staining, and Safranin O staining. RESULTS: Macroscopically, the meniscotibial attachments were pellucid and homogeneous, as were the meniscocapsular attachments; however, the meniscocapsular attachments appeared to be denser in both the anterior and posterior regions of the capsule. Microscopy of the meniscosynovial junction revealed loose collagen fibers that were partially oriented but not parallel, a cellular network featuring a few fibroblasts and adipocytes, and several capillaries. No between-attachment histologic differences were apparent; both tissues shared a site of attachment to the posterior horn of the medial meniscus. We did not detect the meniscotibial ligament, macroscopically or microscopically. CONCLUSIONS: A ramp lesion may not be a ligamentous injury because the meniscotibial ligament was not detected. Rather, it appears that a ramp lesion is a tear in the common attachment point between the posterior horn of the medial meniscus and meniscocapsular and meniscotibial junctions. This structure is vascularized, and contains nonoriented low cellularity collagen of moderate density. CLINICAL RELEVANCE: Based on our results, a better rationale for the recommendation of surgical repair of a ramp appears to be needed, given the absence of a meniscotibial ligament, and the presence of capillaries in the meniscocapsular and meniscotibial attachments.

Leukemic evolution of polycythemia vera and essential thrombocythemia: genomic profiles predict time to transformation.

Among myeloproliferative neoplasms, polycythemia vera (PV) and essential thrombocythemia (ET) are the 2 entities associated with the most chronic disease course. Leukemic evolution occurs rarely but has a grim prognosis. The interval between diagnosis and leukemic evolution is highly variable, from a few years to >20 years. We performed a molecular evaluation of 49 leukemic transformations of PV and ET by targeted next-generation sequencing. Using a hierarchical classification, we identified 3 molecular groups associated with a distinct time to leukemic transformation. Short-term transformations were mostly characterized by a complex molecular landscape and mutations in IDH1/2, RUNX1, and U2AF1 genes, whereas long-term transformations were associated with mutations in TP53, NRAS, and BCORL1 genes. Studying paired samples from chronic phase and transformation, we detected some mutations already present during the chronic phase, either with a significant allele burden (short-term transformation) or with a very low allele burden (especially TP53 mutations). However, other mutations were not detected even 1 year before leukemic transformation. Our results suggest that the leukemic transformation of PV and ET may be driven by distinct time-dependent molecular mechanisms.

EPR17341 and A7H6R pan-TRK Immunohistochemistry Result in Highly Different Staining Patterns in a Series of Salivary Gland Tumors.

Patients with NTRK-rearranged tumors can be now treated using anti-TRK-targeted therapies making NTRK testing important for treatment choices in patients with advanced cancers. Pan-TRK immunohistochemistry (IHC) could be a valuable premolecular screening strategy in this field. The choice of 1 IHC method or another requires to investigate for intermethod comparison. A high frequency of pan-TRK positive tumors among salivary gland tumors makes these tumors particularly appropriate for such a technical study. In this work, we studied the intermethod agreement for 2 pan-TRK IHC methods (using A7H6R and EPR17341 clones) in a file of salivary gland tumors of different subtypes. Among 71 tumors, pan-TRK IHC was diagnosed as positive (ie, H score ≥5) in 23 and 18 cases using EPR17341 and A7H6R clones, respectively, with a good intermethod agreement in terms of positive/negative result (κ, 0.70) but only a moderate agreement considering the H score values themselves (intraclass correlation coefficient of 0.5399). Beyond the intensity of staining and the percentages of stained cells, major differences were also observed between the location and type of cells stained in positive cases between the 2 clones. The single NTRK-rearranged case in our series (ie, a NTRK3-rearranged salivary secretory carcinoma) was positive with the 2 pan-TRK antibodies. Future studies including molecularly proven NTRK-rearranged tumors remain required to further study and compare the performances of different pan-TRK clones in the screening of NTRK-rearranged cancers but it is now obvious that the staining patterns of A7H6R and EPR17341 clones are not strictly identical.

PD-L1 Immunohistochemistry-Discrepant Results Between Synchronous Tumor Samples May Cause More Treatment Choice Dilemmas Than Molecular Heterogeneity in Patients With Advanced Non-Small Cell Lung Cancers.

Biomarker analyses have become mandatory for treatment choices in patients with advanced non-small cell lung cancers. PD-L1 expression for immunotherapy as well as oncogenic molecular alterations for targeted therapies must be analyzed in tumor samples. Intersample heterogeneity may cause dilemmas in treatment choices when faced with discrepant biomarker results. We led a retrospective study evaluating the potential impact on guideline-based therapeutic decisions of biomarker analyses performed in several synchronous tumor samples per patient. We collected retrospective data about patients with advanced non-small cell lung cancers, and synchronous tumor paired samples were analyzed for PD-L1 immunohistochemistry (IHC) and oncogene molecular statuses. Among 34 patients, none had a discrepant result between paired samples with respect to oncogene molecular statuses. At the opposite, intersample-discrepant PD-L1 IHC results may have caused treatment choice dilemmas for 6 (17.6%) patients discussing first-line therapy options (ie, immune checkpoint inhibitor therapy versus other systemic therapy with regard to the threshold of ≥50% PD-L1 IHC-positive tumor cells). In addition, for 6 (17.6%) other patients, different PD-L1 IHC results may have also influenced the choice of one therapy or another in second-line therapy (ie, with regard to the threshold of ≥1% PD-L1 IHC-positive tumor cells). In our case series, discrepant results with regard to PD-L1 IHC between paired tumor samples could generate more treatment choice dilemmas than the molecular heterogeneity of oncogenic drivers.

Screening for NTRK-rearranged Tumors Using Immunohistochemistry: Comparison of 2 Different pan-TRK Clones in Melanoma Samples.

NTRK-rearranged tumors could be treated using promising anti-TRK-targeted therapies in patients with advanced cancers including melanomas. Different targeted therapies are being developed together with different screening strategies including pan-TRK immunohistochemistry (IHC) as first-line screening strategies. In this technical study, we compared 2 pan-TRK IHC (using A7H6R and EPR17341 clones) in tumor samples of patients with advanced melanomas. IHC-positive cases were studied using NTRK1, NTRK2, and NTRK3 fluorescent in situ hybridization tests. Among 300 melanoma samples, 4 samples were positive using A7H6R IHC, but none using EPR17341. None of the 4 samples were NTRK-rearranged using fluorescent in situ hybridization. Different staining was also noted in nontumor kidney tissue, whereas an NTRK1-rearranged tumor used as positive control was strongly stained with both A7H6R and EPR17341 clones. Future studies including more numerous NTRK-rearranged tumors are required to further study and compare the performances of different pan-TRK clones in the screening of NTRK-rearranged cancers.

Sequential mutational evaluation of CALR -mutated myeloproliferative neoplasms with thrombocytosis reveals an association between CALR allele burden evolution and disease progression.

In myeloproliferative neoplasms (MPN), JAK2V617F allele burden measurement has an impact on prognosis that helps in patient monitoring. Less is known about its usefulness in CALR-mutated cases. Additional mutations found by next-generation sequencing have also shown an impact on prognosis that may drive therapeutic choices, especially in myelofibrosis, but few studies focused on CALR-mutated patients. We performed a molecular evaluation combining next-generation sequencing with a myeloid panel and CALR allele burden measurement at diagnosis and during follow-up in a cohort of 45 patients with CALR-mutated essential thrombocythaemia. The bone marrow histology was also blindly reviewed in order to apply the WHO2016 classification. The most frequently mutated gene was TET2 (11/21 mutations). CALR type 1-like patients appear to have a more complex molecular landscape. We found an association between disease progression and CALR allele burden increase during follow-up, independently of additional mutations and WHO2016-reviewed diagnosis. Patients with disease progression at the time of follow-up showed a significant increase in CALR allele burden (+16·7%, P = 0·005) whereas patients without disease progression had a stable allele burden (+3·7%, P = 0·194). This result argues for clinical interest in CALR allele burden monitoring.

[Meningeal melanoma arising from a preexisting meningeal melanocytoma: A clinical, pathological and cytogenetic study about one case]. / Transformation d'un mélanocytome méningé en mélanome : étude clinique, histopathologique et cytogénétique.

Meningeal melanocytic tumors are rare. We report an exceptional case of transformation of a meningeal melanocytoma in a malignant melanoma. The course of the disease extents from 61-years to 85-years and ends with the death of the patient. Besides histopathological and immunohistochemical data, we also report the array CGH study of the melanocytoma and melanoma components suggesting the malignant transformation from whole chromosome gains in the melanocytoma to additional segmental aberrations in the malignant melanoma. Beyond the rarity of this tumor subtype, this case report highlights the potential interest of molecular analyses for diagnostic and prognostic purposes in the field of meningeal melanocytic tumors.

Combining ALK Fluorescent In Situ Hybridization and Immunohistochemistry to Analyze Multiple Non-Small Cell Lung Carcinoma Samples per Patient Reveals Intermethod and Intersample-discrepant Results.

ALK inhibitors have improved the therapeutic management of patients with ALK-rearranged advanced non-small cell lung cancers (NSCLC). Several diagnostic methods, mainly fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC), can be used as single or combined tests to detect the so-called "ALK-positive" NSCLC. Intersample and intermethod discrepancies could cause issues with therapeutic consequences in ALK testing. In this article, we report a case series of our real-life experience and issues in combining FISH and IHC in multiple tumor samples per patient to diagnose and treat a subset of "ALK-positive" patients with NSCLC. Among analyses conducted in 40 samples of 18 patients with advanced lung adenocarcinomas, we retrospectively encountered 10 patients with intersample-concordant ALK FISH and IHC results. Discrepant results about FISH and/or IHC were noted between different samples in 8 patients. Therapeutic responses were observed in 5 of 10 crizotinib-treated patients including 1 patient with ALK FISH+ IHC- status and 1 patient with ALK FISH- IHC+ status. Our data highlight the difficulty to predict the response/nonresponse to crizotinib therapy, in patients with advanced NSCLC, not only on the basis of single and multiple tumor samples, but also on the basis of single and combined diagnostic methods.

Histopathological Diagnosis of Prosthetic Joint Infection: Does a Threshold of 23 Neutrophils Do Better than Classification of the Periprosthetic Membrane in a Prospective Multicenter Study?

No gold standard exists for histopathological diagnosis of a prosthetic joint infection (PJI). The historical criterion considers the presence of neutrophil infiltration upon examination of periprosthetic tissue. Morawietz et al. proposed a classification of periprosthetic membranes (Morawietz et al., Clin Pathol 59:591-597, 2006, https://doi.org/10.1136/jcp.2005.027458) and a more recently described classification with a new cutoff value of 23 neutrophils in 10 high-power fields (Morawietz et al., Histopathology 54:847-853, 2009. https://doi.org/10.1111/j.1365-2559.2009.03313.x). We performed a multicenter prospective study, which compared both methods for the diagnosis of PJI. All suspicions of PJI (n = 264) between December 2010 and March 2012 in seven centers were prospectively included. Five perioperative specimens were collected per patient for cultures, and one was collected for histology. Diagnosis of PJI was made according to the Infectious Diseases Society of America (IDSA) guidelines. Histopathological analysis classified the patients according to the threshold of 23 neutrophils and according to the classification of Morawietz. Performances of both methods were compared by using clinical and/or bacteriological criteria as the gold standard. Among 264 patients with suspected PJI, a diagnosis of infection was confirmed in 215 and unconfirmed in 49 patients. Histopathological analysis was available for 150 confirmed PJI and 40 unconfirmed PJI cases. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 78.7%, 90.0%, 96.7%, 52.9%, and 81.1%, respectively, for the Morawietz classification, and 82.0%, 90.0%, 96.9%, 57.1%, and 83.7%, respectively, for the 23-neutrophil threshold. The new algorithm using a threshold of 23 neutrophils can be proposed as a new gold standard for the histopathological diagnosis of PJI.

Outcome of Ph negative myeloproliferative neoplasms transforming to accelerated or leukemic phase.

Myeloproliferative neoplasms (MPN) are chronic disorders that can sometimes evolve into accelerated or leukemic phases. We retrospectively identified 122 patients with such blastic phases. The overall median survival was four months: 10.2 months for patients treated with intensive treatments compared to three months for best supportive care (p = .005). Azacytidine, intensive chemotherapies, or allogeneic stem cell transplantation gave the highest median survivals with 9, 10.2, and 19.4 months, respectively. Accelerated phases (AP) had a longer median survival compared to acute leukemia (4.8 months vs. 3.1 months; p = .02). In this retrospective and observational study, we observe that the longest survivals are seen in patients eligible for intensive treatments. Azacytidine shows interesting results in patients non-fit for intensive chemotherapy. Supportive care should probably be restricted to elderly patients and those with unfavorable karyotype. An early diagnosis of AP could also result in a better survival rate.