Developing immunoassay for absolute quantitation of protein biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. The feasibility of Quantitative Dot Blot (QDB) method for this purpose was explored in this study. We were able to measure HER2 protein levels using 0.5 µg/sample total protein lysate extracted from 2 × 5 µm FFPE slices absolutely and quantitatively using QDB method in 332 breast cancer FFPE samples. HER2 levels measured using two clinically validated antibodies for immunohistochemistry respectively were highly correlated (r = 0.963). We also achieved area under the curve (AUC) at 0.9998 ± 0.0002 (p < 0.0001, n = 224) with IHC analysis, and 0.9942 ± 0.0031 (p < 0.0001, n = 319) with combined results from IHC and Fluorescence in situ hybridization (FISH) analyses when analyzed with Receiver Operative Characteristics analysis (ROC) respectively. When the results were converted dichotomously with optimized cutoffs from ROC analyses, we achieved 99.5% concordance with IHC; and 96.9% with combined results from both IHC and FISH analyses. Therefore, we were able to demonstrate QDB method as the first immunoassay platform for absolute quantitation of protein biomarkers in FFPE samples to meet the need of daily clinical practice, especially for local laboratories or laboratories in developing countries.

Background: Developing immunoassay for Her2 assessment suitable for Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. Yet, this effort is hindered by lacking available technique. The feasibility of Quantitative Dot Blot (QDB) method to meet this demand was evaluated in this study. Methods: QDB-based Immunoassay was developed for quantitative measurement of Her2 protein levels in this retrospective study using clinically validated antibody for immunohistochemistry (IHC antibody). Total protein lysates were extracted from 2X15 um FFPE slices collected from 332 breast cancer patients sequentially and non-selectively from local hospital. The absolutely quantitated Her2 levels in these samples were compared with the results from methods of Immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH) analyses using Receiver Operative Characteristics (ROC) analysis. Results: Her2 levels measured with two IHC antibodies of EP3 and 4B5 were strongly correlated (r=0.963, p=0.0000). We also achieved area under the curve (AUC) at 0.9753±0.01026 (95% CI: 0.9551 to 0.9954, p<0.0001, n=322) using provided results from IHC and FISH analyses, with 99.6% concordance with IHC and 88.1% with FISH at optimized cutoff of 0.261 nmole/g. The concordance rate with FISH analysis was improved to 94.1% when disagreed samples were re-analyzed by third party independently. Conclusions: We introduced the first immunoassay for absolute quantitation of protein biomarker levels in FFPE samples. This method fits well with daily clinical practice with its objectivity, simplicity and consistency, especially for local hospitals and hospitals in developing countries. The quantitatively measured Her2 protein levels in FFPE samples also allow statistically analysis at population level to meet the need of precision medicine. .

AbstractSignet ring B cell lymphoma is an unusual non-Hodgkin lymphoma. It is similar to signet ring cell carcinoma and liposarcoma in morphology which should be distinguished. We treated a 63-year-old male patient who suffered from abdominal pain for two months. Multiple enlarged lymph nodes were found in the retroperitoneum by CT scan. The needle biopsy showed neoplastic cells distributed uniformly with clear cytoplasm and the nucleus squeezed to the side mimicking the appearance of signet ring in morphology. By special staining, the neoplastic cells were positive for CD45, Vimentin, Bcl-2 and CD20 but negative for AE1/AE3, S-100, CD3, EMA, CD5, CD10, Bcl-6, MUM1 , Kappa, Lambda and PAS . Ki67 proliferation index was much more than 80%. Based on the histological characters, a diagnosis of signet ring B cell lymphoma was made. Although the patient received six courses of R-CHOP therapy, he died of tumor recurrence at the 34th month after diagnosis.