Novel ALK immunohistochemistry assay (clone OTI1A4, Dako) is a sensitive, reliable marker for identifying ALK rearrangements in lung adenocarcinomas: A validation study.

OBJECTIVES: We present the first study validating the recent Dako ALK assay (clone OTI1A4, in vitro diagnostic) for detecting ALK rearrangements in lung adenocarcinoma. METHODS: Lung adenocarcinoma cases between 2011 and 2023 were retrospectively collected to create a cohort of 203 samples. Cases were stained with Dako ALK OTI1A4 and Ventana ALK D5F3 and reviewed by 3 pathologists independently. Correlation between assays, including their sensitivity and specificity, was evaluated. RESULTS: The cohort (n = 203) consisted of resections, core needle biopsies, and cell blocks. Agreement between Dako ALK OTI1A4 and Ventana ALK D5F3 assays was "almost perfect," with κ = 0.89. The sensitivity and specificity of the Dako ALK OTI1A4 assay were 93.3% and 96%, respectively, in a subgroup of 55 molecularly confirmed cases (n = 30 with and n = 25 without ALK rearrangement). CONCLUSIONS: Immunohistochemistry-based assays provide a valid and reasonably priced alternative, especially in settings where molecular confirmatory tests are neither offered nor accessible. Given high interassay and molecular concordance, we propose that the novel Dako OTI1A4 assay can be reliably used to identify cases with ALK rearrangement.

Novel NONO::TFE3 fusion and ALK co-expression identified in a subset of cutaneous microcystic/reticular schwannoma.

Microcystic/reticular schwannoma (MRS) is a benign variant of schwannoma with a predilection for the gastrointestinal tract and skin. To date, genetic characterization of this tumor is limited. Prompted by the identification of TFE3::NONO fusion and ALK overexpression in an index case of MRS, a cohort of tumors was collected from institutional and consultation archives of two institutions. Next-generation sequencing (NGS), TFE3 fluorescence in situ hybridization (FISH), and TFE3 and ALK immunohistochemistry were performed, while clinicopathologic variables were documented. Eighteen MRS cases were identified (35 to 85 years) arising in the skin (n=8), gastrointestinal tract (n=5), adrenal gland (n=3), abdominal wall (n=1), and unknown site (n=1). Tumors showed a circumscribed to multinodular to plexiform low-power architecture with variable amounts of microcystic/reticular and solid schwannian components. Mitotic figures were scarce (0-1/10 HPFs), and atypia was absent. S100 protein and/or SOX10 immunoreactivity was noted in the microcystic/reticular and schwannian areas of all cases. NGS performed on two cutaneous tumors yielded NONO exon 12 fusion with TFE3 exon 4, and these lesions also showed HMB45 and ALK expression. Two additional cases showed ALK expression (1 weak), while a third was positive for TFE3, but these cases failed to show ALK or TFE3 rearrangement by FISH/NGS. There were no morphologic variables that correlated with the presence of NONO::TFE3. We identified a subset of microcystic/reticular schwannomas with NONO::TFE3 fusions and ALK co-expression, adding to the cohort of mesenchymal neoplasms that show ALK overexpression without rearrangement of the ALK gene.

ALK-rearranged renal cell carcinoma with TPM3::ALK gene fusion and review of the literature.

ALK-rearranged renal cell carcinoma (ALK-RCC) is a very rare novel molecularly defined entity in the recently published fifth edition of the World Health Organization classification of tumours. We describe a case of ALK-RCC in a 76-year-old female. The tumour was composed of discohesive rhabdoid cells and pleomorphic, multinucleated cells (equivalent to ISUP/WHO grade 4). The tumour showed expression with PAX8, Keratin 7 and alpha methylacyl CoA racemase. ALK (D5F3 clone) was strongly and diffusely positive. ALK-FISH showed significant split signals of ALK, confirming the diagnosis. RNA sequencing showed TPM3::ALK rearrangement. Including the current case, there are 14 reported ALK-RCC cases with the same TPM3 fusion partner gene. Review of these published cases highlights their morphological heterogeneity and stresses the importance of running ALK immunohistochemistry on difficult cases to classify renal tumours. This is important while identification of ALK-RCC has clinical significance due to the availability of targeted therapy with ALK inhibitors.

A novel fusion between CDC42BPB and ALK in a patient with quadruple wild-type gastrointestinal stromal tumor.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the most common type of mesenchymal tumor in gastrointestinal tract, with striking features of morphology and immunohistochemistry. But GISTs in pregnancy could seldom be found. Pathogenic activating mutations of the proto-oncogene KIT and PDGFRA are detected in majority GISTs, and adjuvant imatinib therapy targeting KIT and PDGFRA mutations is recommended for patients with high-risk GIST. However, some rare subgroups with distinct molecular features remain uncovered and more therapeutic targets need to be revealed. METHODS: The DNA/RNA samples were detected using the NGS-based YuanSu450 gene panel. After identifying the CDC42BPB-ALK fusion by NGS, this novel fusion was further confirmed by Sanger sequencing. Subsequently, FISH analysis was performed using the Vysis ALK Break Apart FISH Probe kit to testify the ALK status. ALK protein expression was confirmed by IHC (D5F3 and 5A4). RESULTS: Herein, we reported the first case of quadruple wild-type (WT) GIST with ALK-CDC42BPB fusion and ALK (D5F3) overexpression. In this study, we described a 33-year-old pregnant patient in lactation who had a massive space occupying lesion (with the maximum diameter of 22 cm) in the stomach and was eventually diagnosed as quadruple WT GIST (KITWT /PDGFRAWT /SDHWT /RAS-PWT ). CONCLUSION: We unexpectedly found that this GIST patient showed ALK (D5F3) overexpression and harbored a novel fusion CDC42BPB exon 24-ALK in exon 20.

Analyzing the morphological spectrum of epithelioid fibrous histiocytoma and the immunohistochemical performance of the ALK D5F3 and ALK1 clones.

Epithelioid fibrous histiocytoma (EFH) is a cutaneous neoplasm driven by translocations of the anaplastic lymphoma kinase (ALK) gene, which can be demonstrated by immunohistochemical (IHC) analysis. We analyzed the performance of two ALK clones, D5F3 and ALK1, in a cohort of EFHs and described the range of architectural variation of these lesions. TFE3 IHC was performed in ALK-negative EFHs. We identified 21 cases of EFH, 76.2% of which showed an exophytic appearance and 19% displayed flat architecture. A well-developed epidermal collarette was present in 48% of all cases with just more than a third of all the exophytic lesions presenting as dermal-based nodules. ALK D5F3 expression was identified in 76.2% (16/21) of all cases, but only 68.8% were concordantly positive with the ALK1 clone, indicative of a false-negative stain with ALK1 in 31.2% of the cases. For the subset of cases showing positivity for the ALK1 clone, a marked decrease in the percentage of immunolabelled cells was identified when compared with D5F3 (5-50% vs. 100%, respectively). Five cases (23.8%) did not demonstrate ALK expression for either clone, with 3 of those cases showing nuclear positivity for TFE3 IHC and the remaining 2 cases being double negative (ALK-/TFE3-). In summary, we identified that the prototypically described exophytic appearance with epidermal collarette is present in only less than half of the cases. We also demonstrated that the ALK1 antibody is suboptimal in EFH and should not be utilized in this setting. A subset of ALK-negative cases express TFE3, but double-negative cases occur.

A study of ALK-positive pulmonary squamous-cell carcinoma: From diagnostic methodologies to clinical efficacy.

BACKGROUND: High concordance has been observed between Ventana D5F3 ALK immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) in lung adenocarcinoma (LADC). However, whether a similar conclusion can be applied to lung squamous-cell carcinoma (LSCC) has remained unclear. We therefore evaluated the ALK (anaplastic lymphoma kinase) status and the therapeutic effect of an ALK tyrosine kinase inhibitor (TKI) in IHC- or FISH-positive LSCC. MATERIALS AND METHODS: A total of 2403 LSCC patients from three institutions were screened for ALK aberration by IHC. All IHC-positive cases were subjected to FISH (with an approximately equal number of negative cases as a control group) and next-generation sequencing (NGS). Clinical efficacy was evaluated for the patients who received TKI therapy. RESULTS: In 2403 cases of LSCC, 37 cases were identified as ALK-positive by IHC. After quality control, 28 cases were succeeded by FISH (six with insufficient tissue, three with lack of signals) and 13 by NGS (24 failed due to insufficient samples or poor DNA quality); the percentage of non-diagnostic tests was 24.3% (9/37) and 64.9% (24/37), respectively. Four cases (4/2394, 0.17%) analyzed by FISH were determined as ALK-positive. For the control group (40 ALK IHC), FISH demonstrated no samples with ALK gene fusion. The concordance between ALK IHC- and ALK FISH-positive results was 14.3% (4/28). In the 13 cases studied by NGS, two cases showed ALK-EML4 fusion (consistent with two FISH-positive results), and two cases were interpreted as harboring an ALK-association gene mutation. Among four patients (two FISH-positive and two IHC-positive only cases) receiving TKI therapy, two patients had stable disease and the other two had progressive disease. CONCLUSIONS: The positive concordance rate of ALK IHC and FISH in LSCC is far less than that reported for LADC. Therefore, ALK IHC detection in LSCC cannot be used as a diagnostic method for ALK rearrangement.

ALK (D5F3) CDx: an immunohistochemistry assay to identify ALK-positive NSCLC patients.

Screening for anaplastic lymphoma kinase (ALK) rearrangements is a very important process in treatment decision making for advanced non-small-cell lung cancer (NSCLC). Although fluorescent in situ hybridization (FISH) is considered the universally accepted reference standard, it is associated with technical difficulties and high costs that have made global implementation of this assay challenging. Conversely, ALK immunohistochemistry has shown high sensitivity and specificity compared to FISH and other molecular assays and is more cost-effective. In fact, the ALK (D5F3) CDx immunohistochemistry assay was approved by the US Food and Drug Administration as a standalone test for ALK rearrangements in lung cancer in 2015. In this review, we will discuss the overview of ALK rearrangements in NSCLC, various testing methods for ALK rearrangements, and the details of immunohistochemistry for ALK, in particular one with the ALK antibody clone D5F3.

Optimized immunohistochemistry using the D5F3 antibody provides a reliable test for identification of ALK-positive lung adenocarcinomas.

We used optimized immunohistochemistry (IHC) with the D5F3 antibody for detection of tumours in a prospective study of 307 pulmonary adenocarcinomas. Cases positive by IHC (1+, 2+, 3+) were further investigated by fluorescent in situ hybridization (FISH). Of 307 cases, 22 (7.2%) were moderately intensely positive (2+/3+); 18 of these (82%) were also positive by FISH. Of the four IHC-positive/FISH-negative cases, one was unsuitable for FISH and three had abnormalities of the ALK gene. All cases with weak reactivity with D5F3 (1+) were FISH-negative. The FISH positive/IHC-positive cases with moderately intense reactivity had the typical clinicopathologic features of ALK-positive patients (younger age, p < 0.01; higher frequency in metastatic sites, p < 0.01; cribriform/mucinous/signet histology, p < 0.01; stage IV disease, p < 0.01). In conclusion, our findings indicate that optimized IHC using the D5F3 antibody provides a reliable and inexpensive test for identification of ALK-positive adenocarcinomas. Inclusion of this information in the pathology report at the time of the histological diagnosis might significantly shorten time to treatment.

ALK protein expression in pulmonary adenocarcinoma of Tunisian patients.

BACKGROUND: It is now necessary to determine ALK status in order to use targeted therapy. AIM: herein, we assess immunohistochemical profile of ALK protein in a series of Tunisian patients with pulmonary adenocarcinoma. MATERIALS AND METHODS: ALK protein expression was studied applying the D5F3 antibody with a fully automated Ventana CDx technique on a series of 19 patients. RESULTS: Positive ALK expression was found in one case (5.2%) corresponding to a papillary adenocarcinoma which showed a strong granular and homogenous cytoplasmic staining. The patient was a 30-years-old woman. CONCLUSION: The frequency of positive ALK expression based on immunohistochemistry in our series was similar to that reported in the world literature.

Clinical value of Antibody D5F3 and Survivin in patients with non- small cell lung cancer / 中国生化药物杂志

Objective To investigate the clinical value of D5F3 and Survivin in patients with non-small cell lung cancer. Methods 100 patients with non - small cell lung cancer admitted in our hospital from January 2016 to June 2017 were examined by immunohistochemical method, to analyze the clinical value of D5F3 and Survivin antibody in non - small cell lung cancer (NSCLC). Results The coincidence rate of D5F3 was (20％), (++) was 33.33％, and the coincidence rate was (87.50％), and the difference was statistically significant (P<0.05). The positive rate of Survivin protein expression was correlated with the differentiation degree of non-small cell lung cancer, TNM stage and lymph node metastasis (P<0.05). Conclusion D5F3 has high accuracy, sensitivity and specificity, and has important clinical diagnostic value; The detection of Survivin expression is helpful for clinical differentiation, clinical staging and lymph node metastasis in patients with non-small cell lung cancer.

Clinical value of antibody D5F3 and Survivin in non small cell lung cancer / 中国生化药物杂志

Objective To investigate the clinical value of antibody D5F3 and Survivin in patients with non small cell lung cancer.Methods 200 paraffin embedded specimens of patients with non small cell lung cancer tested by RT-PCR(including EML4-ALK mutant and wild type)from October 2012 to June 2014 were selected.The gene protein expression were tested by ALK new antibody D5F3 and the sensitivity and specificity were compared by negative and positive(+～3+).ResultsTest results show(+)the coincidence rate is 15.78％,(+ +)the coincidence rate is 27.27％and(+ + +)compliance rate was 87.5％,the difference was statistically significant(P<0.05).The expression of Survivin protein in NSCLC tissues was correlated with clinicopathological features.The positive rate of Survivin protein expression was correlated with the degree of differentiation(P<0.05),but not with other clinical and pathological features,with the decrease of differentiation,the positive rate of Survivin protein expression was significantly increased.Conclusion D5F3 and Survivin antibodies are highly sensitive and specific in patients with NSCLC,with the screening value,save social resources,for the majority of patients with lung cancer services.

Efficacy of D5F3 IHC for detecting ALK gene rearrangement in NSCLC patients: a systematic review and meta-analysis.

We conducted a pooled analysis comparing the efficacy of an immunohistochemistry (IHC) assay using the D5F3 antibody with that of fluorescence in situ hybridization (FISH) for detecting ALK gene rearrangement in NSCLC patients. A total of 32 studies involving 5805 samples were included in this review. Pooled sensitivity for D5F3 IHC was 0.97 (95%CI: 0.93-0.98), specificity was 0.99 (95%CI: 0.98-1.00), PLR was 119.20 (95%CI: 57.79-245.89), NLR was 0.03 (95%CI: 0.02-0.07), DOR was 3526.66 (95%CI: 1344.71-9249.03), and AUROC was 1.00 (95%CI: 0.99-1.00). Meta-regression revealed that specimen type was a source of heterogeneity for specificity, and specimen type and FISH signal distance were sources of heterogeneity in the joint model. Subgroup analysis revealed that sensitivity and specificity were higher when the FISH signal distance standard was ≥ 2 than when it was ≥ 1. Sensitivity was higher for tumor specimens than for cell specimens; specificity was higher for cell specimens than for tumor specimens. In conclusion, the D5F3 IHC assay was nearly as effective as FISH for detection of ALK gene rearrangement in NSCLC patients.

Antibody 1A4 with routine immunohistochemistry demonstrates high sensitivity for ALK rearrangement screening of Chinese lung adenocarcinoma patients: A single-center large-scale study.

OBJECTIVES: The rearrangement of echinoderm microtubule-associated protein-like 4-analplastic lymphoma kinase (EML4-ALK) in non-small cell lung cancer (NSCLC) cells might be a promising therapeutic target. However, the low positive rate seeks a reliable and cost-effective method for ALK rearrangement prescreening. This study aimed to evaluate the application of a novel primary antibody 1A4 for routine ALK immunohistochemistry (IHC) test. MATERIALS AND METHODS: Primary antibody 1A4 and D5F3 were used for the screening of 595 formalin-fixed, paraffin-embedded tissues of consecutive patients with lung adenocarcinoma for ALK-positive candidates. Ventana detection system and fluorescence in-situ hybridization (FISH) were used as reference methods. RESULTS: Among 595 cases, the protein expression statuses of 1A4 were 3+ (18), 2+ (50), 1+ (153), and 0+ (374), and those of D5F3 were 3+ (17), 2+ (18), 1+ (20), and 0+ (540). Ventana detection system and FISH test results were successfully obtained from 482 cases. A total of 298 specimens with 1A4 (-) showed 100% concordance with standard FISH results. All 58 FISH (+) cases were identified by antibody 1A4. Meanwhile, 14 and 5 were missed by antibody D5F3 with routine IHC and Ventana system, respectively. 1A4 with routine IHC had better sensitivity (100%, 75.9%, and 91.4%, respectively), but lower specificity (70.3%, 99.8%, and 100%, respectively), than D5F3 with routine IHC and Ventana system. CONCLUSION: The novel antibody 1A4 used as a prescreening method may help to reduce the false-negative rearranged ALK status if FISH or reverse transcription polymerase chain reaction results were used for validation.

Screening for ALK in non-small cell lung carcinomas: 5A4 and D5F3 antibodies perform equally well, but combined use with FISH is recommended.

OBJECTIVES: Immunohistochemistry (IHC) has become a promising method for pre-screening ALK-rearrangements in non-small cell lung carcinomas (NSCLC). Various ALK antibodies, detection systems and automated immunostainers are available. We therefore aimed to compare the performance of the monoclonal 5A4 (Novocastra, Leica) and D5F3 (Cell Signaling, Ventana) antibodies using two different immunostainers. Additionally we analyzed the accuracy of prospective ALK IHC-testing in routine diagnostics. MATERIALS AND METHODS: Seventy-two NSCLC with available ALK FISH results and enriched for FISH-positive carcinomas were retrospectively analyzed. IHC was performed on BenchMarkXT (Ventana) using 5A4 and D5F3, respectively, and additionally with 5A4 on Bond-MAX (Leica). Data from our routine diagnostics on prospective ALK-testing with parallel IHC, using 5A4, and FISH were available from 303 NSCLC. RESULTS: All three IHC protocols showed congruent results. Only 1/25 FISH-positive NSCLC (4%) was false negative by IHC. For all three IHC protocols the sensitivity, specificity, positive (PPV) and negative predictive values (NPV) compared to FISH were 96%, 100%, 100% and 97.8%, respectively. In the prospective cohort 3/32 FISH-positive (9.4%) and 2/271 FISH-negative (0.7%) NSCLC were false negative and false positive by IHC, respectively. In routine diagnostics the sensitivity, specificity, PPV and NPV of IHC compared to FISH were 90.6%, 99.3%, 93.5% and 98.9%, respectively. CONCLUSIONS: 5A4 and D5F3 are equally well suited for detecting ALK-rearranged NSCLC. BenchMark and BOND-MAX immunostainers can be used for IHC with 5A4. True discrepancies between IHC and FISH results do exist and need to be addressed when implementing IHC in an ALK-testing algorithm.