Immunocytochemistry Profiling Research Articles

Concordancy of immunocytochemistry profiling of circulating tumor cells with immunohistochemistry for analysis of therapeutically relevant biomarkers.

3047 Background: Immunohistochemistry (IHC) profiling of tumor tissue is the present standard for evaluation of therapeutically relevant biomarkers such as ER, PR, HER2, AR, ARv7, PD-L1 and MMR for selection of targeted, endocrine and checkpoint inhibitor therapy selection. However, this critical analysis is dependent on availability of tumor tissue obtained by an invasive biopsy. Challenges to this analysis include insufficient tumor tissue and inability to perform a repeat biopsy to obtain fresh tumor tissue. We have previously described an approach for negative enrichment of Circulating Tumor Cells (CTCs) from peripheral blood and for Immunocytochemistry (ICC) profiling of these CTCs for detection of diagnostically relevant tissue of origin and subtype specific markers, concordant with tumor tissue analysis. Methods: In the present study, we determined concordance between tumor tissue (HPE) and CTCs (ICC) for ascertaining the status of therapeutically relevant markers ER, PR, HER2, AR, ARv7 PD-L1 and MMR. We evaluated 201 matched pairs of tumor tissue (FFPE blocks) and CTCs obtained from peripheral blood. Results: Among the 743 paired assays on matched tumor tissue and CTCs, concordance (positive or negative) was observed in 651 matched pairs (87.6%). The concordance was 82.9% for ER, 100% for PR, 90.2 % for Her2, 93.8% for AR, 90% for Arv7, 85.1% and 87.6% for PD-L1 clones 22c3 and 28-8, and 85.6% for MMR (MLH1, MSH2, MSH6, and PMS2). Conclusions: The study findings indicate that ICC analysis of CTCs may be able to substitute IHC analysis of tumor tissue for profiling of therapeutically relevant markers. This approach may have application in cases where tumor tissue may be limiting and / or where an invasive biopsy to obtain tumor tissue may be unviable.

Evaluation of circulating tumor cell clusters for pan-cancer noninvasive diagnostic triaging.

BackgroundHistopathologic examination (HPE) of tumor tissue obtained by invasive biopsy is the standard for cancer diagnosis but is resource‐intensive and has been associated with procedural risks. The authors demonstrate that immunocytochemistry (ICC) profiling of circulating ensembles of tumor‐associated cells (C‐ETACs) can noninvasively provide diagnostic guidance in solid organ cancers.MethodsThe clinical performance of this approach was tested on blood samples from 30,060 individuals, including 9416 individuals with known cancer; 6725 symptomatic individuals with suspected cancer; and 13,919 asymptomatic individuals with no prior diagnosis of cancer. C‐ETACs were harvested from peripheral blood and profiled by ICC for organ‐specific and subtype‐specific markers relevant to the cancer type. ICC profiles were compared with HPE diagnoses to determine concordance.ResultsThe presence of malignancy was confirmed by the detection of C‐ETACs in 91.8% of the 9416 individuals with previously known cancer. Of the 6725 symptomatic individuals, 6025 were diagnosed with cancer, and 700 were diagnosed with benign conditions; C‐ETACs were detected in 92.6% of samples from the 6025 individuals with cancer. In a subset of 3509 samples, ICC profiling of C‐ETACs for organ‐specific and subtype‐specific markers was concordant with HPE findings in 93.1% of cases. C‐ETACs were undetectable in 95% of samples from the 700 symptomatic individuals who had benign conditions and in 96.3% of samples from the 13,919 asymptomatic individuals.ConclusionsC‐ETACs were ubiquitous (>90%) in various cancers and provided diagnostically relevant information in the majority (>90%) of cases. This is the first comprehensive report on the feasibility of ICC profiling of C‐ETACs to provide pan‐cancer diagnostic guidance with accuracy comparable to that of HPE.

Abstract B61: Encyclopedic noninvasive liquid biopsies for differential diagnosis in prostate cancer

Abstract Differential diagnosis of prostatic adenocarcinomas is based on histopathologic analysis of tumor tissue obtained by transrectal ultrasound (TRUS)-guided biopsies. However, such invasive biopsies are associated with pain, procedural expenses, sequelae, and risk of complications. Though immunocytochemistry (ICC) profiling of circulating tumor cells (CTCs, defined as EpCAM+, panCK+, CD45-) has been previously attempted for diagnosis in some cancers, it has been unviable due to limited yields of CTCs. For the first time we used enrichment of CTCs that permit meaningful ICC profiling for diagnosis of prostate cancers in suspected cases as contradistinguished from cases of benign enlargement of prostate (BEP). We obtained 15 mL of venous blood draw from 650 individuals, of whom 65 had a confirmed diagnosis of either prostate adenocarcinoma and 585 were diagnosed with benign prostate hyperplasia. CTCs were enriched from PBMCs fraction using an epigenetically acting enrichment process that is lethal towards normal (nonmalignant) cells but confers survival privilege on apoptosis-resistant CTCs. Harvested CTCs were confirmed by immunostaining for EpCAM and pan-CK. ICC profiling of these CTCs was also performed with organ-specific markers PSMA and AMACR. Viable CTCs could be obtained from 58 samples (89.2%) out of 65 confirmed prostate cancer cases. Among the 40 samples that were characterized by deep ICC profiling, all samples (100%) were positive for PSMA and AMACR. Among the benign cases, CTCs were observed in 10 samples (1.7%), of which all 10 were positive for PSMA but negative for AMACR. These patients are being followed up. Possibility of other cancers is not ruled out in these patients. Our results show that ICC-based characterization of CTCs can provide necessary diagnostic information noninvasively to substitute for conventional procedures dependent on tissue extraction. Citation Format: Darshana A. Patil, Dadasaheb Akolkar, Revati Patil, Pooja Fulmali, Pradip Fulmali, Akshay Ainwale, Navin Srivastava, Vineet Datta, Cynthe Sims, Ajay Srinivasan, Rajan Datar. Encyclopedic noninvasive liquid biopsies for differential diagnosis in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B61.

Abstract B16: Noninvasive liquid biopsies for guideline-compliant diagnostic assessment in ovarian cancers

Abstract Diagnosis of ovarian adenocarcinomas is based on histopathologic analysis of tumor tissue obtained by invasive biopsies. However, invasive biopsies are associated with procedural expenses, sequelae, and risk of complications. Though immunocytochemistry (ICC) profiling of circulating tumor cells (CTCs, defined as EpCAM+, panCK+, CD45-) has been previously considered for diagnosis in some cancers, the attempts have been unsuccessful. We used a novel approach for enrichment of sufficient viable CTCs that permit meaningful ICC profiling for diagnosis of ovarian cancers in suspected cases. We obtained 15 mL of venous blood draw from 248 patients with confirmed diagnosis of ovarian cancer. PMBCs were isolated by centrifugation. CTCs were enriched from PBMC using an epigenetically acting enrichment process that is lethal towards normal (nonmalignant) cells but confers survival privilege on apoptosis-resistant CTCs. Harvested CTCs were confirmed by immunostaining for EpCAM and pan-CK. Deep ICC profiling of these CTCs was performed with organ-specific markers CA125 and WT1 as well as P63 as a nonspecific marker. Viable CTCs could be obtained from 225 samples (90.7%) out of 248. Among the 100 samples characterized by deep ICC profiling, 70 samples (70%) were positive for CA125 and WT1. None of the samples was positive for P63. Our results show that ICC-based characterization of CTCs can provide necessary diagnostic information noninvasively to substitute conventional procedures dependent on tissue extraction. Citation Format: Dadasaheb B. Akolkar, Darshana Patil, Revati Patil, Pradip Devhare, Pradip Fulmali, Pooja Fulmali, Shoeb Patel, Navin Srivastava, Vineet Datta, Cynthe Sime, Ajay Srinivasan, Rajan Datar. Noninvasive liquid biopsies for guideline-compliant diagnostic assessment in ovarian cancers [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B16.

Abstract B62: Wholesome non-invasive liquid biopsies for pharmacodiagnostic work-up in breast cancer

Abstract Background: Diagnosis and treatment of breast cancers is based on histopathological analysis of tumor tissue obtained by invasive biopsies. However invasive biopsies cause pain, anxiety and carry the risk of needle seeding. Though Immunocytochemistry (ICC) profiling of Circulating Tumor Cells (CTCs, defined as EpCAM+, panCK+, CD45-) has been previously attempted for diagnosis, these efforts have been unsuccessful. We used an epigenetically modified medium for enrichment of sufficient viable CTCs. These CTCs were used for diagnosis and characterization. Methods: We obtained 15 ml of venous blood draw from 2500 patients with confirmed diagnosis of breast cancer. CTCs were enriched from PBMCs fraction using an epigenetically acting enrichment process that is lethal towards normal (non-malignant) cells, but confers survival privilege on apoptosis-resistant CTCs. Harvested CTCs were confirmed by immunostaining for EpCAM and pan-CK. Deep ICC profiling of these CTCs was performed with organ specific marker GCDFP15. Theranostic ICC Profiling was performed with ER and Her2. Results: CTCs could be obtained from 2232 samples (89.3%) out of 2500. A subset of 100 samples were characterised by deep ICC profiling, among which 80% of samples were positive for GCDFP15. 14 and 17 samples out of 100 were positive for ER and Her2 respectively. Conclusions: Our results show that ICC based characterization of CTCs can provide necessary diagnostic information non-invasively to substitute conventional procedures dependent on tissue extraction. Citation Format: Dadasaheb Akolkar. Wholesome non-invasive liquid biopsies for pharmacodiagnostic work-up in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B62.

Abstract B15: Circulating tumor cells express tissue specific antigens in multiple cancers

Abstract Definitive diagnosis of solid organ cancers is based on histopathologic analysis of tumor tissue obtained by invasive biopsies. However, invasive biopsies are associated with procedural expenses, sequelae, and risks. Though immunocytochemistry (ICC) profiling of circulating tumor cells (CTCs) has been previously attempted for diagnosis in some cancers, they have been unviable due to limited yields of CTCs. We used a novel approach for enrichment of sufficient viable CTCs that permit meaningful ICC profiling for diagnosis of cancers in suspected cases. We obtained 15 mL of venous blood draw from 965 patients with confirmed diagnosis of various solid organ cancers. CTCs were enriched from PBMCs fraction using an epigenetically activated enrichment process that is lethal towards normal (nonmalignant) cells but confers survival privilege on apoptosis-resistant CTCs. Harvested CTCs were confirmed by immunostaining for EpCAM and pan-CK. Deep ICC profiling of these CTCs was performed with organ-specific marker for various organs such as lungs (TTF-1 or Napsin), breast (GCDFP15 or GATA3), ovarian (CA125 or WT-1), pancreatic (CK19 or CA19-9), cervical (P63), liver (Hep Par-1 or Glypican), colorectal (CK20 or CDX-2), head and neck (P63), kidney (Pax-8 or RCC), and prostate (PSMA or AMACR). CTCs (EpCAM+, pan-CK+) could be obtained from 94.4% of all samples. Organ-specific markers were positive in 100% of lung cancer, 79.3% of breast cancers, 70.0% of ovarian cancers, 93.3% of pancreatic cancers, 100% of cervical cancers, 61% of hepatocellular carcinomas, 92.6% ofcColorectal cancers, 98.8% of head and neck cancers, 100% of renal cancers, and 100% of prostate cancers. Our results show that deep ICC profiling of CTCs can determine tissue of origin in solid organ cancers noninvasively to substitute conventional procedures dependent on tissue extraction. Citation Format: Dadasaheb Akolkar, Revati Patil, Darshana Patil, Pradip Fulmali, Pooja Fulmali, Shoeb Patel, Pradip Devhare, Akshay Ainwale, Vineet Datta, Cynthe Sims, Ajay Srinivasan, Rajan Datar. Circulating tumor cells express tissue specific antigens in multiple cancers [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B15.

PD-L1 profiling of circulating tumor cells for immune checkpoint inhibitor therapy in gastroesophageal cancers.

29 Background: Selection of Immune Checkpoint Inhibitor (ICI) therapies in Gastroesophageal cancers are based on IHC based detection of PD-L1 expression in tumor tissue. Invasive biopsy to obtain tumor tissue for IHC is associated with procedural risks, sequelae and expenses. Prior efforts at PD-L1 profiling of Circulating Tumor Cells (CTCs) have been constrained by low CTC yields. We employed a novel approach for harvesting sufficient CTCs from Gastroesophageal cancers that permit PD-L1 profiling by Immunocytochemistry (ICC). Methods: 15 ml peripheral blood was collected from 106 patients among whom 20 were diagnosed with gastric adenocarcinomas (AD) and 86 with either esophageal AD or esophageal squamous cell carcinomas (SCC). CTCs were enriched from PBMCs via an epigenetically acting stabilization process which induced apoptosis in non-malignant cells and conferred survival privilege on apoptosis-resistant CTCs, which were harvested and confirmed by immunostaining for EpCAM and pan-CK positivity. Deep ICC profiling of CTCs was performed with organ-specific markers as well as PD-L1:22c3 and PD-L1:28-8 clones. Results: Viable CTCs could be enriched and harvested in 103 out of 106 samples (97.2%) regardless of metastatic or treatment status. Deep ICC and PD-L1 profiling could be performed in all 103 samples (100%). PD-L1 expression was quantitatively assigned as ‘Low’, ‘Moderate’ or ‘High’. Among the 19 gastric ADs, 5 (26.3%) samples were positive for PD-L1:22c3 and 2 (10.5%) were positive for PD-L1:28-8; all gastric ADs were ‘Low’ for either PD-L1 subtype. Among the 84 esophageal carcinomas (AD+SCC), 32 samples (38.1%) were positive for PD-L1:22c3 (26 Low + 5 Moderate + 1 High) and 16 (19.1%) were positive for PD-L1:28-8 (11 Low + 5 Moderate). Conclusions: CTCs in Gastroesophageal cancers can be considered for evaluating PD-L1 expression in patients where ICI therapies are otherwise viable.

PD-L1 profiling of circulating tumor cells for immune checkpoint inhibitor therapy in head and neck cancers.

36 Background: Selection of Immune Checkpoint Inhibitor (ICI) therapies in Head and Neck cancers are based on IHC based detection of PD-L1 expression in tumor tissue. Invasive biopsy to obtain tumor tissue for IHC is associated with procedural risks, sequelae and expenses. Prior efforts at PD-L1 profiling of Circulating Tumor Cells (CTCs) have been constrained by low CTC yields. We employed a novel approach for harvesting sufficient CTCs from Head and Neck cancers that permit PD-L1 profiling by Immunocytochemistry (ICC). Methods: 15 ml peripheral blood was collected from 95 patients with head and neck squamous cell carcinomas (HNSCC). CTCs were enriched from PBMCs via an epigenetically acting stabilization process which induced apoptosis in non-malignant cells and conferred survival privilege on apoptosis-resistant CTCs, which were harvested and confirmed by immunostaining for EpCAM and pan-CK. Deep ICC profiling of CTCs was performed with organ-specific markers as well as PD-L1:22c3 and PD-L1:28-8 clones. Results: Viable CTCs could be enriched and harvested in 90 out of 95 samples (95.3%) regardless of metastatic or treatment status. Deep ICC and PD-L1 profiling could be performed in all 90 samples (100%). PD-L1 expression was quantitatively assigned as ‘Low’, ‘Moderate’ or ‘High’. 28 (31.1%) samples were positive for PD-L1:28-8 (27 Low + 1 Moderate) and 26 (28.9%) samples were positive for PD-L1:22c3 (24 Low + 2 Moderate). 15 samples were positive for both PD-L1 clones. Conclusions: CTCs in Head and Neck squamous cell carcinomas can be considered for evaluating PD-L1 expression in patients where ICI therapies are otherwise viable.

PD-L1 profiling of circulating tumour cells is a viable companion diagnostic for checkpoint inhibitor therapy in lung cancer

Abstract Background Selection of Checkpoint Inhibitor therapies in several cancers are based on PD-L1 expression in tumor tissue determined by IHC. Invasive biopsy to obtain tumor tissue is associated with procedural risks, sequelae and expenses. Though PD-L1 profiling of Circulating Tumor Cells (CTCs) has been attempted previously, limitations arise due to low yields of CTCs in conventional methods. We describe a novel approach for harvesting sufficient CTCs that can be used for PD-L1 profiling by Immunocytochemistry (ICC). Methods 15 ml peripheral blood was collected from 95 patients with histopathologically confirmed diagnosis of lung cancer. PBMCs were isolated by centrifugation. CTCs were enriched from PBMCs via an epigenetically acting stabilization process which induces apoptosis in normal (non-cancerous) cells while simultaneously conferring survival privilege on apoptosis-resistant cells of tumorigenic origin (CTCs). Surviving CTCs were confirmed by immunostaining for EpCAM and pan-CK. CTCs were further profiled for Napsin and TTF-1 (Lung-Adenocarcinoma specific), p40 (Squamous Cell Carcinoma specific), CK7 (Adenocarcinoma specific), Synaptophysin and Chromogranin (Neuroendocrine carcinoma specific) and PD-L1 (22c3). Results Viable CTCs could be enriched and harvested in all 95 samples regardless of treatment status and extent of disease (metastatic status). Deep ICC profiling including all organ- and subtype-specific markers as well as PD-L1 could be conducted in 71 out of the 95 samples (74.7%). Among the 71 samples which were evaluable, PD-L1 positivity was observed in 33 samples (46.5%). PD-L1 expression in these 33 samples was quantitatively estimated and assigned as ‘Low’, ‘Moderate’ or ‘High’. Among the 33 PD-L1 positive samples, 25 (75.8%) had low PD-L1 expression, 7 (21.2%) had moderate expression, and 1 (3%) had high expression. Conclusion ICC profiling of CTCs is a viable approach for determining suitability of patients for checkpoint inhibitor therapies. This method permits quantitative determination of PD-L1 expression which appears to have clinical relevance in determining the probability of favourable response to checkpoint inhibitor therapies. Legal entity responsible for the study The authors. Funding Datar Cancer Genetics Limited. Disclosure R. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. D. Akolkar: Full / Part-time employment: Datar Cancer Genetics Limited. D. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. V. Datta: Full / Part-time employment: Datar Cancer Genetics Limited. P. Devhare: Full / Part-time employment: Datar Cancer Genetics Limited. S. Patel: Full / Part-time employment: Datar Cancer Genetics Limited. A. Srinivasan: Full / Part-time employment: Datar Cancer Genetics Limited. R. Datar: Shareholder / Stockholder / Stock options: Datar Cancer Genetics Limited. All other authors have declared no conflicts of interest.