Expert Review of Molecular Diagnostics, Dec 9, 2014
Activating mutation of KRAS plays a significant role in the pathogenesis of common human malignan... more Activating mutation of KRAS plays a significant role in the pathogenesis of common human malignancies and molecular testing of KRAS mutation has emerged as an essential biomarker in the current practice of clinical oncology. The presence of KRAS mutation is generally associated with clinical aggressiveness of the cancer and reduced survival of the patient. Therapeutically, KRAS mutation testing has maximum utility in stratifying metastatic colorectal carcinoma and lung cancer patients for treatment with targeted therapy. Diagnostically, KRAS mutation testing is useful in the workup of pancreaticobiliary and thyroid cancers, particularly using cytological specimens. In the era of precision medicine, the role of KRAS mutation testing is poised to expand, likely in a setting of combinatorial therapeutic strategy and requiring additional mutation testing of its upstream and/or downstream effectors.
Background: MRCC is a devastating disease with poor long-term outcomes. Bevacizumab (BEV) added t... more Background: MRCC is a devastating disease with poor long-term outcomes. Bevacizumab (BEV) added to chemotherapy (CT) improves significantly overall survival (OS) in MRCC patients (pts). Aim: to characterize clinic-pathologic features associated to CR and its impact on pts outcome. Methods: Single-institution chart review of MRCC pts who were treated with 1 st line CT between 2005 and 2016. CR was defined by Response Evaluation Criteria in Solid Tumors (RECIST v1). The prognostic and predictive value of clinic-pathologic features, was evaluated. Results: Seventy-two pts (62% squamous; 30% adenocarcinoma; 8% others); with median age of 48 years (28-77) were selected. Forty-five pts (62%) had prior CT-radiation; 55 pts (79%) had recurrent/persistent disease (27 pts > 12 months (m) disease free interval) and 15 pts (21%) were stage IVb (90% visceral involvement). Moore risk distribution: 7/44/21 pts were high/medium/low risk, respectively. Eleven pts (15%) received BEV þ CT; 57 pts (79%) platinum-based-CT (PCT) (54% Cisplatin; 26% Carboplatin) and 4 (6%) non-PCT. After a median follow-up of 33 m, ORR 51%, median OS 13 m (9.5-NA) and median PFS 6 m (4.6-7.7) were observed for overall population. Moore criteria correlated with prognosis (high-risk pts had significantly worse OS (HR ¼ 0.04, p<0.001). No differences in ORR, PFS or OS were detected between BEV and non-BEV group (p > 0.2 all comparisons). Higher ORR was observed among low and intermediate risk pts (51%, 67%; p ¼ 0.006). CRs occurred in 13/71 (18%) evaluable pts (BEV group 2/11; non-BEV 11/60, p ¼ 1). Clinic-pathologic features, including Moore criteria, did not correlate with CR in univariate analysis. Median time to CR was 3.5 m (3-NA) and median duration of CR was 7 m (4.3-NA). Five pts (7%) had CR in the irradiated field. CR significantly impacted on PFS (9.7 m vs 4.7 m non-CR, p ¼ 0.002) and OS (31 m vs 9.5 m non-CR, p ¼ 0.001). Eight pts discontinued treatment due to toxicity.
NUT midline carcinomas (NMC) are a rare, recently described class of poorly-differentiated tumors... more NUT midline carcinomas (NMC) are a rare, recently described class of poorly-differentiated tumors that exhibit rapid onset and highly aggressive clinicopathologic behavior. These tumors are defined by rearrangement of the nuclear protein in testis (NUT) gene on chromosome 15q14, most commonly in a balanced translocation with the BRD4 gene on chromosome 19p13.1, resulting in the characteristic BRD4-NUT fusion gene and protein which blocks epithelial differentiation through chromatin binding. NMC frequently involve midline structures of adolescents and young adults and affect the head and neck region in 50% of cases. To our knowledge, only one case has been previously reported involving a salivary gland. Here, we present a case of a NMC of the salivary gland in an adolescent male presenting with an intermittently painful left submandibular mass of 3 months duration.
Expert Review of Molecular Diagnostics, Mar 1, 2012
Serine/threonine-protein kinase BRAF, a downstream effector of the RAS oncogene along the MEK/ERK... more Serine/threonine-protein kinase BRAF, a downstream effector of the RAS oncogene along the MEK/ERK signaling pathway, has emerged as an important biological marker for diagnosis, prognosis and therapeutic guidance for human cancers. The high prevalence of BRAF(V600E) activating mutation in papillary thyroid carcinoma, cutaneous malignant melanoma and hairy cell leukemia implies that the mutation is an important &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;driver&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; or &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;codriver&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; in the development of a subset of these cancers. Diagnostically, the BRAF(V600E) mutation is a powerful molecular marker for papillary thyroid carcinoma and, quite possibly, hairy cell leukemia as well. Cancers with a BRAF mutation are generally more aggressive than their counterparts without the mutation. Importantly, mutant BRAF has been a highly attractive target for precision cancer therapy. Indeed, recent studies in the clinical trials of BRAF inhibitors in patients with malignant melanoma are changing the treatment paradigm of this highly lethal disease. BRAF mutation testing using highly sensitive and specific methodology in a molecular diagnostic laboratory is essential in the current clinical practice of oncology.
American Journal of Clinical Pathology, Oct 1, 2015
Objectives: Acute myeloid leukemia (AML) is a rapidly fatal disease without the use of aggressive... more Objectives: Acute myeloid leukemia (AML) is a rapidly fatal disease without the use of aggressive chemotherapy regimens. Cytogenetic and molecular studies are commonly used to classify types of AML based on prognosis, as well as to determine therapeutic regimens. Methods: Although there are several AML classifications determined by particular translocations, cytogenetically normal AML represents a molecularly, as well as clinically, heterogeneous group of diseases. Laboratory evaluation of AML will become increasingly important as new mutations with both prognostic and therapeutic implications are being recognized. Moreover, because many patients with AML are being treated more effectively, these mutations may become increasingly useful as markers of minimal residual disease, which can be interpreted in an individualized approach. Results: Current laboratory studies of gene mutations in AML include analysis of NPM1, FLT3, CEBPA, and KIT. In addition to these genes, many other genes are emerging as potentially useful in determining patients' prognosis, therapy, and disease course. Conclusions: This article briefly reviews the current most clinically relevant gene mutations and their clinical and immunophenotypic features, prognostic information, and methods used for detection. Case Scenario A 52-year-old woman seeks treatment from her primary care physician for a recent onset of fatigue, night sweats, and 10 pounds of unintentional weight loss in the past month. She has a WBC count of 33 × 10 9 /L with 88% blasts noted on a manual differential count. Morphologic evaluation of the peripheral smear is remarkable for a population of intermediate-sized blasts with round to folded nuclei, open chromatin, occasional nucleoli, and a moderate amount of cytoplasm. No Auer rods are identified. Note is made that there are "prominent nuclear invaginations." Flow cytometry evaluation of blood demonstrates that 86% of WBCs are CD45 dim-positive blasts, which express MPO, CD13, CD33, HLA-DR, and CD14, and are negative for CD34, CD19, CD20, CD3 (surface and cytoplasmic), and TdT. The flow interpretation is reported as "involvement of peripheral blood by acute myeloid leukemia." Reflex polymerase chain Upon completion of this activity you will be able to: • list the common mutations that can be detected in cases of cytogenetically normal acute myelogenous leukemia (CN-AML). • discuss the methodologies commonly used for detecting mutations in CN-AML. • describe the clinical implications of mutations in CN-AML and the indications and timing for testing for them. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module.
Tumor cell heterogeneity and tumor cell-stromal interactions are being explored as determinants o... more Tumor cell heterogeneity and tumor cell-stromal interactions are being explored as determinants of disease progression and treatment resistance in solid tumor and hematological malignancies. As such, tools simultaneously capable of highly multiplexed profiling of tissues' protein and RNA content, as well as interrogation of rare or single cells, are required to precisely characterize constituent tumor cell populations, infiltrating lymphocytes and stromal elements. Access to spatial relationships will enable more precise characterization of tumors, support patient stratification and may help to identify novel drug targets. Multiple platforms are being developed to address these critical unmet needs. The NanoString digital spatial profiling (DSP) platform enables highly multiplexed, spatial assessment of protein and/or RNA targets in tissues by detecting oligonucleotide barcodes conjugated via a photocleavable linker to primary antibodies or nucleic acid probes. Although this platform enables high-dimensional spatial interrogation of tissue protein and RNA expression, a detailed understanding of its composition, function and chemistry is advisable to guide experimental design and data interpretation. The purpose of this review is to provide an independent, comprehensive description of the DSP technology, including an overview of NanoString's capture and antibody barcode conjugation chemistries, experimental workflow, data output and analysis methods. The DSP technology will be discussed in the context of other highly multiplexed immunohistochemistry methods, including imaging mass cytometry and multiplexed ion beam imaging, to inform potential users of the advantages and limitations of each. Additional issues such as preanalytical variability, sampling and specimen adequacy will be considered with respect to the platforms to inform potential experimental design.
Expert Review of Molecular Diagnostics, Mar 10, 2016
Lynch syndrome, an autosomal dominant inherited disorder, is caused by inactivating mutations inv... more Lynch syndrome, an autosomal dominant inherited disorder, is caused by inactivating mutations involving DNA mismatch repair (MMR) genes. This leads to profound genetic instability, including microsatellite instability (MSI) and increased risk for cancer development, particularly colon and endometrial malignancies. Clinical testing of tumor tissues for the presence of MMR gene deficiency is standard practice in clinical oncology, with immunohistochemistry and PCR-based microsatellite instability analysis used as screening tests to identify potential Lynch syndrome families. The ultimate diagnosis of Lynch syndrome requires documentation of mutation within one of the four MMR genes (MLH1, PMS2, MSH2 and MSH6) or EPCAM, currently achieved by comprehensive sequencing analysis of germline DNA. In this review, the genetic basis of Lynch syndrome, methodologies of MMR deficiency testing, and current diagnostic algorithms in the clinical management of Lynch syndrome, are discussed.
Even before the introduction of significant disruptive technologies to the practice of pathology,... more Even before the introduction of significant disruptive technologies to the practice of pathology, resident education in the discipline had already experienced fundamental changes starting at the beginning of the 21st century. 1 After a 20-year experiment of adding a credentialing year to all pathology training, the requirement was dropped for the residency class starting in 2002, which effectively moved the curriculum for training in both anatomic pathology (AP) and clinical pathology/laboratory medicine (CP) from 5 years to 4 years and for those training in AP-only or CP-only from 4 years to 3. One consequence of the shortening of core training was a marked increase in the number of residents taking subspecialty fellowships. This was driven in part by the demands of the hiring marketplace but was also made necessary by the explosion
Background Close proximity between cytotoxic T lymphocytes and tumour cells is required for effec... more Background Close proximity between cytotoxic T lymphocytes and tumour cells is required for effective immunotherapy. However, what determines the spatial distribution of T cells in the tumour microenvironment is not well understood. Coupling digital pathology and transcriptome analysis on large ovarian tumour cohorts, here we report classification and functionally dissection of tumour-immune contexture in human ovarian cancer. Methods CD8 IHC and RNAseq analysis were performed on 370 ovarian tumours from the ICON7 phase III clinical trial. Coupling digital pathology with transcriptome analysis, a random forest machine learning algorithm was developed and independently validated for classifying tumour-immune phenotypes in ovarian cancer. Anti-tumour activity of TGFβ blockade in combination with anti-PD-L1 was evaluated in an ovarian cancer mouse model. Results We show the identified tumour-immune phenotypes are of biological and clinical importance with interconnection to molecular subtypes and association with clinical outcome in ovarian cancer. Two important hallmarks of T cell exclusion were identified: 1) loss of antigen presentation on tumour cells and 2) upregulation of TGFβ and activated stroma. We identified TGFβ as a key mediator of T cell exclusion. TGFβ reduced MHC class I expression in ovarian cancer cells and induced extracellular matrix (ECM) production and immunosuppressive molecules in human primary fibroblasts. Finally, we demonstrated that combination of anti-TGFβ and anti-PD-L1 in a mouse ovarian cancer model significantly improved the anti-tumour efficacy and survival. Conclusions This study provided the first systematic and in-depth characterization of the molecular features and mechanisms underlying the tumour-immune phenotypes in ovarian cancer. We illuminated a multi-faceted role of TGFβ in mediating consequential crosstalk between tumour cells and cancer associated fibroblasts to shape the tumour-immune contexture. Our findings support that targeting the TGFβ pathway represents a promising therapeutic strategy to overcome T cell exclusion and optimize response to cancer immunotherapy. Legal entity responsible for the study The authors. Funding Genentech/Roche. Disclosure All authors have declared no conflicts of interest.
Over the past decade, invention and adoption of novel multiplexing technologies for tissues have ... more Over the past decade, invention and adoption of novel multiplexing technologies for tissues have made increasing impacts in basic and translational research and, to a lesser degree, clinical medicine. Platforms capable of highly multiplexed immunohistochemistry or in situ RNA measurements promise evaluation of protein or RNA targets at levels of plex and sensitivity logs above traditional methodsall with preservation of spatial context. These methods promise objective biomarker quantification, markedly increased sensitivity, and single-cell resolution. Increasingly, development of novel technologies is enabling multi-omic interrogations with spatial correlation of RNA and protein expression profiles in the same sample. Such sophisticated methods will provide unprecedented insights into tissue biology, biomarker science, and, ultimately, patient health. However, this sophistication comes at significant cost, requiring extensive time, practical knowledge, and resources to implement. This review will describe the technical features, advantages, and limitations of currently available multiplexed immunohistochemistry and spatial transcriptomic platforms.
Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) ha... more Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional chemokine CXCL10. By extending those initial findings to pre-clinical models of hepatocellular carcinoma and breast cancer, we discovered a distinct mechanism by which inhibition of DPP4 improves anti-tumor responses. Administration of the DPP4 inhibitor sitagliptin resulted in higher concentrations of the chemokine CCL11 and increased migration of eosinophils into solid tumors. Enhanced tumor control was preserved in mice lacking lymphocytes and was ablated after depletion of eosinophils or treatment with degranulation inhibitors. We further demonstrated that tumor-cell expression of the alarmin IL-33 was necessary and sufficient for eosinophil-mediated anti-tumor responses and that this mechanism contributed to the efficacy of checkpoint-inhibitor therapy. These findings provide insight into IL-33-and eosinophil-mediated tumor control, revealed when endogenous mechanisms of DPP4 immunoregulation are inhibited.
Expert Review of Molecular Diagnostics, Dec 9, 2014
Activating mutation of KRAS plays a significant role in the pathogenesis of common human malignan... more Activating mutation of KRAS plays a significant role in the pathogenesis of common human malignancies and molecular testing of KRAS mutation has emerged as an essential biomarker in the current practice of clinical oncology. The presence of KRAS mutation is generally associated with clinical aggressiveness of the cancer and reduced survival of the patient. Therapeutically, KRAS mutation testing has maximum utility in stratifying metastatic colorectal carcinoma and lung cancer patients for treatment with targeted therapy. Diagnostically, KRAS mutation testing is useful in the workup of pancreaticobiliary and thyroid cancers, particularly using cytological specimens. In the era of precision medicine, the role of KRAS mutation testing is poised to expand, likely in a setting of combinatorial therapeutic strategy and requiring additional mutation testing of its upstream and/or downstream effectors.
Background: MRCC is a devastating disease with poor long-term outcomes. Bevacizumab (BEV) added t... more Background: MRCC is a devastating disease with poor long-term outcomes. Bevacizumab (BEV) added to chemotherapy (CT) improves significantly overall survival (OS) in MRCC patients (pts). Aim: to characterize clinic-pathologic features associated to CR and its impact on pts outcome. Methods: Single-institution chart review of MRCC pts who were treated with 1 st line CT between 2005 and 2016. CR was defined by Response Evaluation Criteria in Solid Tumors (RECIST v1). The prognostic and predictive value of clinic-pathologic features, was evaluated. Results: Seventy-two pts (62% squamous; 30% adenocarcinoma; 8% others); with median age of 48 years (28-77) were selected. Forty-five pts (62%) had prior CT-radiation; 55 pts (79%) had recurrent/persistent disease (27 pts > 12 months (m) disease free interval) and 15 pts (21%) were stage IVb (90% visceral involvement). Moore risk distribution: 7/44/21 pts were high/medium/low risk, respectively. Eleven pts (15%) received BEV þ CT; 57 pts (79%) platinum-based-CT (PCT) (54% Cisplatin; 26% Carboplatin) and 4 (6%) non-PCT. After a median follow-up of 33 m, ORR 51%, median OS 13 m (9.5-NA) and median PFS 6 m (4.6-7.7) were observed for overall population. Moore criteria correlated with prognosis (high-risk pts had significantly worse OS (HR ¼ 0.04, p<0.001). No differences in ORR, PFS or OS were detected between BEV and non-BEV group (p > 0.2 all comparisons). Higher ORR was observed among low and intermediate risk pts (51%, 67%; p ¼ 0.006). CRs occurred in 13/71 (18%) evaluable pts (BEV group 2/11; non-BEV 11/60, p ¼ 1). Clinic-pathologic features, including Moore criteria, did not correlate with CR in univariate analysis. Median time to CR was 3.5 m (3-NA) and median duration of CR was 7 m (4.3-NA). Five pts (7%) had CR in the irradiated field. CR significantly impacted on PFS (9.7 m vs 4.7 m non-CR, p ¼ 0.002) and OS (31 m vs 9.5 m non-CR, p ¼ 0.001). Eight pts discontinued treatment due to toxicity.
NUT midline carcinomas (NMC) are a rare, recently described class of poorly-differentiated tumors... more NUT midline carcinomas (NMC) are a rare, recently described class of poorly-differentiated tumors that exhibit rapid onset and highly aggressive clinicopathologic behavior. These tumors are defined by rearrangement of the nuclear protein in testis (NUT) gene on chromosome 15q14, most commonly in a balanced translocation with the BRD4 gene on chromosome 19p13.1, resulting in the characteristic BRD4-NUT fusion gene and protein which blocks epithelial differentiation through chromatin binding. NMC frequently involve midline structures of adolescents and young adults and affect the head and neck region in 50% of cases. To our knowledge, only one case has been previously reported involving a salivary gland. Here, we present a case of a NMC of the salivary gland in an adolescent male presenting with an intermittently painful left submandibular mass of 3 months duration.
Expert Review of Molecular Diagnostics, Mar 1, 2012
Serine/threonine-protein kinase BRAF, a downstream effector of the RAS oncogene along the MEK/ERK... more Serine/threonine-protein kinase BRAF, a downstream effector of the RAS oncogene along the MEK/ERK signaling pathway, has emerged as an important biological marker for diagnosis, prognosis and therapeutic guidance for human cancers. The high prevalence of BRAF(V600E) activating mutation in papillary thyroid carcinoma, cutaneous malignant melanoma and hairy cell leukemia implies that the mutation is an important &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;driver&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; or &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;codriver&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; in the development of a subset of these cancers. Diagnostically, the BRAF(V600E) mutation is a powerful molecular marker for papillary thyroid carcinoma and, quite possibly, hairy cell leukemia as well. Cancers with a BRAF mutation are generally more aggressive than their counterparts without the mutation. Importantly, mutant BRAF has been a highly attractive target for precision cancer therapy. Indeed, recent studies in the clinical trials of BRAF inhibitors in patients with malignant melanoma are changing the treatment paradigm of this highly lethal disease. BRAF mutation testing using highly sensitive and specific methodology in a molecular diagnostic laboratory is essential in the current clinical practice of oncology.
American Journal of Clinical Pathology, Oct 1, 2015
Objectives: Acute myeloid leukemia (AML) is a rapidly fatal disease without the use of aggressive... more Objectives: Acute myeloid leukemia (AML) is a rapidly fatal disease without the use of aggressive chemotherapy regimens. Cytogenetic and molecular studies are commonly used to classify types of AML based on prognosis, as well as to determine therapeutic regimens. Methods: Although there are several AML classifications determined by particular translocations, cytogenetically normal AML represents a molecularly, as well as clinically, heterogeneous group of diseases. Laboratory evaluation of AML will become increasingly important as new mutations with both prognostic and therapeutic implications are being recognized. Moreover, because many patients with AML are being treated more effectively, these mutations may become increasingly useful as markers of minimal residual disease, which can be interpreted in an individualized approach. Results: Current laboratory studies of gene mutations in AML include analysis of NPM1, FLT3, CEBPA, and KIT. In addition to these genes, many other genes are emerging as potentially useful in determining patients' prognosis, therapy, and disease course. Conclusions: This article briefly reviews the current most clinically relevant gene mutations and their clinical and immunophenotypic features, prognostic information, and methods used for detection. Case Scenario A 52-year-old woman seeks treatment from her primary care physician for a recent onset of fatigue, night sweats, and 10 pounds of unintentional weight loss in the past month. She has a WBC count of 33 × 10 9 /L with 88% blasts noted on a manual differential count. Morphologic evaluation of the peripheral smear is remarkable for a population of intermediate-sized blasts with round to folded nuclei, open chromatin, occasional nucleoli, and a moderate amount of cytoplasm. No Auer rods are identified. Note is made that there are "prominent nuclear invaginations." Flow cytometry evaluation of blood demonstrates that 86% of WBCs are CD45 dim-positive blasts, which express MPO, CD13, CD33, HLA-DR, and CD14, and are negative for CD34, CD19, CD20, CD3 (surface and cytoplasmic), and TdT. The flow interpretation is reported as "involvement of peripheral blood by acute myeloid leukemia." Reflex polymerase chain Upon completion of this activity you will be able to: • list the common mutations that can be detected in cases of cytogenetically normal acute myelogenous leukemia (CN-AML). • discuss the methodologies commonly used for detecting mutations in CN-AML. • describe the clinical implications of mutations in CN-AML and the indications and timing for testing for them. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module.
Tumor cell heterogeneity and tumor cell-stromal interactions are being explored as determinants o... more Tumor cell heterogeneity and tumor cell-stromal interactions are being explored as determinants of disease progression and treatment resistance in solid tumor and hematological malignancies. As such, tools simultaneously capable of highly multiplexed profiling of tissues' protein and RNA content, as well as interrogation of rare or single cells, are required to precisely characterize constituent tumor cell populations, infiltrating lymphocytes and stromal elements. Access to spatial relationships will enable more precise characterization of tumors, support patient stratification and may help to identify novel drug targets. Multiple platforms are being developed to address these critical unmet needs. The NanoString digital spatial profiling (DSP) platform enables highly multiplexed, spatial assessment of protein and/or RNA targets in tissues by detecting oligonucleotide barcodes conjugated via a photocleavable linker to primary antibodies or nucleic acid probes. Although this platform enables high-dimensional spatial interrogation of tissue protein and RNA expression, a detailed understanding of its composition, function and chemistry is advisable to guide experimental design and data interpretation. The purpose of this review is to provide an independent, comprehensive description of the DSP technology, including an overview of NanoString's capture and antibody barcode conjugation chemistries, experimental workflow, data output and analysis methods. The DSP technology will be discussed in the context of other highly multiplexed immunohistochemistry methods, including imaging mass cytometry and multiplexed ion beam imaging, to inform potential users of the advantages and limitations of each. Additional issues such as preanalytical variability, sampling and specimen adequacy will be considered with respect to the platforms to inform potential experimental design.
Expert Review of Molecular Diagnostics, Mar 10, 2016
Lynch syndrome, an autosomal dominant inherited disorder, is caused by inactivating mutations inv... more Lynch syndrome, an autosomal dominant inherited disorder, is caused by inactivating mutations involving DNA mismatch repair (MMR) genes. This leads to profound genetic instability, including microsatellite instability (MSI) and increased risk for cancer development, particularly colon and endometrial malignancies. Clinical testing of tumor tissues for the presence of MMR gene deficiency is standard practice in clinical oncology, with immunohistochemistry and PCR-based microsatellite instability analysis used as screening tests to identify potential Lynch syndrome families. The ultimate diagnosis of Lynch syndrome requires documentation of mutation within one of the four MMR genes (MLH1, PMS2, MSH2 and MSH6) or EPCAM, currently achieved by comprehensive sequencing analysis of germline DNA. In this review, the genetic basis of Lynch syndrome, methodologies of MMR deficiency testing, and current diagnostic algorithms in the clinical management of Lynch syndrome, are discussed.
Even before the introduction of significant disruptive technologies to the practice of pathology,... more Even before the introduction of significant disruptive technologies to the practice of pathology, resident education in the discipline had already experienced fundamental changes starting at the beginning of the 21st century. 1 After a 20-year experiment of adding a credentialing year to all pathology training, the requirement was dropped for the residency class starting in 2002, which effectively moved the curriculum for training in both anatomic pathology (AP) and clinical pathology/laboratory medicine (CP) from 5 years to 4 years and for those training in AP-only or CP-only from 4 years to 3. One consequence of the shortening of core training was a marked increase in the number of residents taking subspecialty fellowships. This was driven in part by the demands of the hiring marketplace but was also made necessary by the explosion
Background Close proximity between cytotoxic T lymphocytes and tumour cells is required for effec... more Background Close proximity between cytotoxic T lymphocytes and tumour cells is required for effective immunotherapy. However, what determines the spatial distribution of T cells in the tumour microenvironment is not well understood. Coupling digital pathology and transcriptome analysis on large ovarian tumour cohorts, here we report classification and functionally dissection of tumour-immune contexture in human ovarian cancer. Methods CD8 IHC and RNAseq analysis were performed on 370 ovarian tumours from the ICON7 phase III clinical trial. Coupling digital pathology with transcriptome analysis, a random forest machine learning algorithm was developed and independently validated for classifying tumour-immune phenotypes in ovarian cancer. Anti-tumour activity of TGFβ blockade in combination with anti-PD-L1 was evaluated in an ovarian cancer mouse model. Results We show the identified tumour-immune phenotypes are of biological and clinical importance with interconnection to molecular subtypes and association with clinical outcome in ovarian cancer. Two important hallmarks of T cell exclusion were identified: 1) loss of antigen presentation on tumour cells and 2) upregulation of TGFβ and activated stroma. We identified TGFβ as a key mediator of T cell exclusion. TGFβ reduced MHC class I expression in ovarian cancer cells and induced extracellular matrix (ECM) production and immunosuppressive molecules in human primary fibroblasts. Finally, we demonstrated that combination of anti-TGFβ and anti-PD-L1 in a mouse ovarian cancer model significantly improved the anti-tumour efficacy and survival. Conclusions This study provided the first systematic and in-depth characterization of the molecular features and mechanisms underlying the tumour-immune phenotypes in ovarian cancer. We illuminated a multi-faceted role of TGFβ in mediating consequential crosstalk between tumour cells and cancer associated fibroblasts to shape the tumour-immune contexture. Our findings support that targeting the TGFβ pathway represents a promising therapeutic strategy to overcome T cell exclusion and optimize response to cancer immunotherapy. Legal entity responsible for the study The authors. Funding Genentech/Roche. Disclosure All authors have declared no conflicts of interest.
Over the past decade, invention and adoption of novel multiplexing technologies for tissues have ... more Over the past decade, invention and adoption of novel multiplexing technologies for tissues have made increasing impacts in basic and translational research and, to a lesser degree, clinical medicine. Platforms capable of highly multiplexed immunohistochemistry or in situ RNA measurements promise evaluation of protein or RNA targets at levels of plex and sensitivity logs above traditional methodsall with preservation of spatial context. These methods promise objective biomarker quantification, markedly increased sensitivity, and single-cell resolution. Increasingly, development of novel technologies is enabling multi-omic interrogations with spatial correlation of RNA and protein expression profiles in the same sample. Such sophisticated methods will provide unprecedented insights into tissue biology, biomarker science, and, ultimately, patient health. However, this sophistication comes at significant cost, requiring extensive time, practical knowledge, and resources to implement. This review will describe the technical features, advantages, and limitations of currently available multiplexed immunohistochemistry and spatial transcriptomic platforms.
Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) ha... more Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional chemokine CXCL10. By extending those initial findings to pre-clinical models of hepatocellular carcinoma and breast cancer, we discovered a distinct mechanism by which inhibition of DPP4 improves anti-tumor responses. Administration of the DPP4 inhibitor sitagliptin resulted in higher concentrations of the chemokine CCL11 and increased migration of eosinophils into solid tumors. Enhanced tumor control was preserved in mice lacking lymphocytes and was ablated after depletion of eosinophils or treatment with degranulation inhibitors. We further demonstrated that tumor-cell expression of the alarmin IL-33 was necessary and sufficient for eosinophil-mediated anti-tumor responses and that this mechanism contributed to the efficacy of checkpoint-inhibitor therapy. These findings provide insight into IL-33-and eosinophil-mediated tumor control, revealed when endogenous mechanisms of DPP4 immunoregulation are inhibited.
Uploads
Papers by James Ziai