Posts Tagged ‘intratumoral heterogeneity’

Can IntraTumoral Heterogeneity Be Thought of as a Mechanism of Resistance?

Curator/Reporter: Stephen J. Williams, Ph.D.

Therapeutic resistance remains one of the most challenging problems for the oncologist, despite the increase of new therapeutics in the oncologist’s toolkit. As new targeted therapies are developed, and new novel targets are investigated as potential therapies, especially cytostatic therapies which it has become evident our understanding of chemoresistance is expanding beyond mechanisms to circumvent a drug’s pharmacologic mechanism of action (i.e. increased DNA repair and cisplatin) or pharmacokinetic changes (i.e. increased efflux by acquisition of a MDR phenotype).

In a talk at the 2015 AACR National Meeting, Dr. Charles Swanton discusses the development of tumor heterogeneity in the light of developing, or acquired, drug resistance. Chemoresistance is either categorized as acquired resistance (where resistance develops upon continued exposure to drug) or inherent resistance (related to a tumor being refractory or unresponsive to drug). Dr Swanton discusses findings where development of this heterogeneity (discussed here in a posting on Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing) and here (Notes On Tumor Heterogeneity: Targets and Mechanisms, from the 2015 AACR Meeting in Philadelphia PA) on recent findings on Branched Chain Heterogeneity) is resulting in clones resistant to the initial drug treatment.

To recount a bit of background I list the overall points of the one of previous posts on tumor heterogeneity (and an interview with Dr. Charles Swanton) are as follows:

Multiple biopsies of primary tumor and metastases are required to determine the full mutational landscape of a patient’s tumor

The intratumor heterogeneity will have an impact on the personalized therapy strategy for the clinician

Metastases arising from primary tumor clones will have a greater genomic instability and mutational spectrum than the tumor from which it originates

Tumors and their metastases do NOT evolve in a linear path but have a branched evolution and would complicate biomarker development and the prognostic and resistance outlook for the patient


The following is a curation of various talks and abstracts from the 2015 AACR National Meeting in Philadelphia on effects of clonal evolution and intratumoral heterogeneity of a tumor with respect to development of chemoresistance. As this theory of heterogeneity and clonal evolution is particularly new I attempted to present all works (although apologize for the length upfront) to forgo bias and so the reader may extract any information pertinent to their clinical efforts and research. However I will give a brief highlight summary below:


From the 2015 AACR National Meeting in Philadelphia






Presentation Title: Polyclonal and heterogeneous resistance to targeted therapy in leukemia
Presentation Time: Monday, Apr 20, 2015, 10:40 AM -10:55 AM
Location: Room 201, Pennsylvania Convention Center
Author Block: Catherine C. Smith, Amy Paguirigan, Chen-Shan Chin, Michael Brown, Wendy Parker, Mark J. Levis, Alexander E. Perl, Kevin Travers, Corynn Kasap, Jerald P. Radich, Susan Branford, Neil P. Shah. University of California, San Francisco, CA, Fred Hutchinson Cancer Research Center, Seattle, WA, Pacific Biosciences, Menlo Park, CA, Royal Adelaide Hospital, Adelaide, Australia, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, Abramson Cancer Center of the University of Pennsylvania, Philadelphia, PA, University of California, San Francisco, CA
Abstract Body: Genomic studies in solid tumors have revealed significant branching intratumoral clonal genetic heterogeneity. Such complexity is not surprising in solid tumors, where sequencing studies have revealed thousands of mutations per tumor genome. However, in leukemia, the genetic landscape is considerably less complex. Chronic myeloid leukemia (CML) is the human malignancy most definitively linked to a single genetic lesion, the BCR-ABL gene fusion. Genome wide sequencing of acute myeloid leukemia (AML) has revealed that AML is the most genetically straightforward of all extensively sequenced adult cancers to date, with an average of 13 coding mutations and 3 or less clones identified per tumor.
In CML, tyrosine kinase inhibitors (TKIs) of BCR-ABL have resulted in high rates of remission. However, despite excellent initial response rates with TKI monotherapy, patients still relapse, including virtually all patients with Philadelphia-positive acute lymphoblastic leukemia and blast crisis CML. Studies of clinical resistance highlight BCR-ABL as the sole genetic driver in CML as secondary kinase domain (KD) mutations that prevent drug binding are the predominant mechanism of relapse on BCR-ABL TKIs.
In AML, a more diverse panel of disease-defining genetic mutations has been uncovered. However, in individual patients, a single oncogene can still drive disease. This is the case in FLT3 mutant AML, in which the investigational FLT3 TKI quizartinib achieved an initial response rate of ~50% in relapsed/refractory AML patients with activating FLT3 internal tandem duplication (ITD) mutations, though most patients eventually relapsed. Confirming the importance of FLT3 in disease maintenance, we showed that 8 of 8 patients who relapsed on quizartinib did so due to acquired drug-resistant FLT3 KD mutations.
Studies in CML have revealed that sequential TKI therapy is associated with additional complexity where multiple mutations can coexist separately in an individual patient (“polyclonality”) or in tandem on a single allele (“compound mutations”). In AML, we observed polyclonal FLT3-ITD KD mutations in 2 of 8 patients examined in our initial study of quizartinib resistance.
In light of the polyclonal KD mutations observed in CML and AML at the time of TKI relapse, we undertook next generation sequencing studies to determine the true genetic complexity in CML and AML patients at the time of relapse on targeted therapy. We used Pacific Biosciences RS Single Molecule Real Time (SMRT) third generation sequencing technology to sequence the entire ABL KD or the entire FLT3 juxtamembrane and KD on a single strand of DNA. Using this method, we assessed a total of 103 samples from 79 CML patients on ABL TKI therapy and 36 paired pre-treatment and relapse samples from 18 FLT3-ITD+ AML patients who responded to investigational FLT3 TKI therapy.
In CML, using SMRT sequencing, we detected all mutations previously detected by direct sequencing. Of samples in which multiple mutations were detectable by direct sequencing, 85% had compound mutant alleles detectable in a variety of combinations. Compound mutant alleles were comprised of both dominant and minor mutations, some which were not detectable by direct sequencing. In the most complex case, 12 individual mutant alleles comprised of 7 different mutations were identified in a single sample.
For 12 CML patients, we interrogated longitudinal samples (2-4 time points per patient) and observed complex clonal relationships with highly dynamic shifts in mutant allele populations over time. We detected compound mutations arising from ancestral single mutant clones as well as parallel evolution of de novo polyclonal and compound mutations largely in keeping with what would be expected to cause resistance to the second generation TKI therapy received by that patient.
We used a phospho-flow cytometric technique to assesses the phosphorylation status of the BCR-ABL substrate CRKL in as a method to test the ex vivo biochemical responsiveness of individual mutant cell populations to TKI therapy and assess functional cellular heterogeneity in a given patient at a given timepoint. Using this technique, we observed co-existing cell populations with differential ex vivo response to TKI in 2 cases with detectable polyclonal mutations. In a third case, we identified co-existence of an MLL-AF9 containing cell population that retained the ability to modulate p-CRKL in response to BCR-ABL TKIs along with a BCR-ABL containing only population that showed biochemical resistance to all TKIs, suggesting the co-existence of BCR-ABL independent and dependent resistance in a single patient.
In AML, using SMRT sequencing, we identified acquired quizartinib resistant KD mutations on the FLT3-ITD (ITD+) allele of 9 of 9 patients who relapsed after response to quizartinib and 4 of 9 patients who relapsed after response to the investigational FLT3 inhibitor, PLX3397. In 4 cases of quizartinib resistance and 3 cases of PLX3397 resistance, polyclonal mutations were observed, including 7 different KD mutations in one patient with PLX3397 resistance. In 7 quizartinib-resistant cases and 3 PLX3397-resistant cases, mutations occurred at the activation loop residue D835. When we examined non-ITD containing (ITD-) alleles, we surprisingly uncovered concurrent drug-resistant FLT3 KD mutations on ITD- alleles in 7 patients who developed quizartinib resistance and 4 patients with PLX3397 resistance. One additional PLX3397-resistant patient developed a D835Y mutation only in ITD- alleles at the time of resistance, suggesting selection for a non-ITD containing clone. All of the individual substitutions found on ITD- alleles were the same substitutions identified on ITD+ alleles for each individual patient.
Given that the same individual mutations found on ITD- alleles were also found on ITD+ alleles, we sought to determine whether these mutations were found in the same cell or were indicative of polyclonal blast populations in each patient. To answer this question, we performed single cell sorting of viably frozen blasts from 3 quizartinib-resistant patients with D835 mutations identified at the time of relapse and genotyped single cells for the presence or absence of ITD and D835 mutations. This analysis revealed striking genetic heterogeneity. In 2/3 cases, polyclonal D835 mutations were found in both ITD+ and ITD- cells. In all cases, FLT3-ITD and D835 mutations were found in both heterozygous and homozygous combinations. Most surprisingly, in all 3 patients, approximately 30-40% of FLT3-ITD+ cells had no identified quizartinib resistance-causing FLT3 KD mutation to account for resistance, suggesting the presence of non-FLT3 dependent resistance in all patients.
To determine that ITD+ cells lacking FLT3 KD mutations observed in patients relapsed on quizartinib are indeed consistent with leukemic blasts functionally resistant to quizartinib and do not instead represent a population of differentiated or non-proliferating cells, we utilized relapse blasts from another patient who initially achieved clearance of bone marrow blasts on quizartinib and developed a D835Y mutation at relapse. We performed a colony assay in the presence of 20nM quizartinib. As expected, this dose of quizartinib was unable to suppress the colony-forming ability of blasts from this relapsed patient when compared to DMSO treatment. Genotyping of individual colonies grown from this relapse sample in the presence of 20nM quizartinib again showed remarkable genetic heterogeneity, including ITD+ and ITD- colonies with D835Y mutations in homozygous and heterozygous combinations as well as ITD+ colonies without D835Y mutations, again suggesting the presence of blasts with non-FLT3 dependent resistance. Additionally, 4 colonies with no FLT3 mutations at all were identified in this sample, suggesting the presence of a quizartinib-resistant non-FLT3 mutant blast population. To see if we could identify specific mechanisms of off-target resistance, we performed targeted exome sequencing 33-AML relevant genes from relapse and pre-treatment DNA from all four patients and detected no new mutations in any genes other than FLT3 acquired at the time of disease relapse. Clonal genetic heterogeneity is not surprising in solid tumors, where multiple driver mutations frequently occur, but in CML and FLT3-ITD+ AML, where disease has been shown to be exquisitely dependent on oncogenic driver mutations, our studies suggest a surprising amount of clonal diversity. Our findings show that clinical TKI resistance in these diseases is amazingly intricate on the single allele level and frequently consists of both polyclonal and compound mutations that give rise to an complicated pool of TKI-resistant alleles that can change dynamically over time. In addition, we demonstrate that cell populations with off-target resistance can co-exist with other TKI-resistant populations, underscoring the emerging complexity of clinical TKI resistance. Such complexity argues strongly that monotherapy strategies in advanced CML and AML may be ultimately doomed to fail due to heterogeneous cell intrinsic resistance mechanisms. Ultimately, combination strategies that can address both on and off target resistance will be required to effect durable therapeutic responses.
Session Title: Tumor Heterogeneity and Evolution
Session Type: Educational Session
Session Start/End Time: Saturday, Apr 18, 2015, 1:00 PM – 3:00 PM
Location: Terrace Ballroom II-III (400 Level), Pennsylvania Convention Center
CME: CME-Designated
CME/CE Hours: 2
Session Description: One of the major challenges for both the measurement and management of cancer is its heterogeneity. Recent studies have revealed both extensive inter- and intra-tumor heterogeneity at the genotypic and phenotypic levels. Leaders in the field will discuss this challenge, its origins, dynamics and clinical importance. They will also review how we can best measure and deal with tumor heterogeneity, particularly intra-tumor heterogeneity.
Saturday, Apr 18, 2015, 1:00 PM – 3:00 PM
Carlo C. Maley. UCSF Helen Diller Family Comp. Cancer Center, San Francisco, CA
Universal biomarkers: How to handle tumor heterogeneity
Saturday, Apr 18, 2015, 1:00 PM – 1:25 PM
Carlo C. Maley. UCSF Helen Diller Family Comp. Cancer Center, San Francisco, CA
Saturday, Apr 18, 2015, 1:25 PM – 1:30 PM
Heterogeneity of resistance to cancer therapy
Saturday, Apr 18, 2015, 1:30 PM – 1:55 PM
Ivana Bozic. HARVARD UNIV., Cambridge, MA
Saturday, Apr 18, 2015, 1:55 PM – 2:00 PM
Determinants of phenotypic intra-tumor heterogeneity: integrative approach
Saturday, Apr 18, 2015, 2:00 PM – 2:25 PM
Andriy Marusyk, Michalina Janiszewska, Doris Tabassum. Dana-Farber Cancer Institute, Boston, MA, Dana-Farber Cancer Institute, Boston, MA
Saturday, Apr 18, 2015, 2:25 PM – 2:30 PM
Cancer clonal complexity and evolution at the macro- and microheterogeneity scale
Saturday, Apr 18, 2015, 2:30 PM – 2:55 PM
Marco Gerlinger. Institute of Cancer Research, London, United Kingdom
Saturday, Apr 18, 2015, 2:55 PM – 3:00 PM

From Ivana Bozic:

A spatial model predicts that dispersal and cell turnover limit intratumour heterogeneity.

Waclaw B, Bozic I, Pittman ME, Hruban RH, Vogelstein B, Nowak MA.

Nature. 2015 Sep 10;525(7568):261-4. doi: 10.1038/nature14971. Epub 2015 Aug 26.



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Timing and heterogeneity of mutations associated with drug resistance in metastatic cancers.

Bozic I, Nowak MA.

Proc Natl Acad Sci U S A. 2014 Nov 11;111(45):15964-8. doi: 10.1073/pnas.1412075111. Epub 2014 Oct 27.



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Evolutionary dynamics of cancer in response to targeted combination therapy.

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Session Title: Mechanisms of Cancer Therapy Resistance
Session Type: Educational Session
Session Start/End Time: Saturday, Apr 18, 2015, 1:00 PM – 3:00 PM
Location: Room 204, Pennsylvania Convention Center
CME: CME-Designated
CME/CE Hours: 2
Session Description: Despite dramatic advances in the treatment of cancer, therapy resistance remains the most significant hurdle in improving the outcome of cancer patients. In this session, we will discuss many different aspects of therapy resistance, including a summary of our current understanding of therapy resistant tumor cell populations as well as analyses of the challenges associated with intratumoral heterogeneity and adaptive responses to targeted therapies.
Saturday, Apr 18, 2015, 1:00 PM – 3:00 PM
Charles Swanton. Cancer Research UK London Research Institute, London, United Kingdom
Tumor heterogeneity and drug resistance
Saturday, Apr 18, 2015, 1:00 PM – 1:30 PM
Charles Swanton. Cancer Research UK London Research Institute, London, United Kingdom

Saturday, Apr 18, 2015, 1:30 PM – 1:40 PM
Discussion Discussion, Discussion

Principles of resistance to targeted therapy
Saturday, Apr 18, 2015, 1:40 PM – 2:10 PM
Levi A. Garraway. Dana-Farber Cancer Institute, Boston, MA

Saturday, Apr 18, 2015, 2:10 PM – 2:20 PM
Discussion Discussion, Discussion

Adaptive re-wiring of signaling pathways driving drug resistance to targeted therapies
Saturday, Apr 18, 2015, 2:20 PM – 2:50 PM
Taru E. Muranen. Harvard Medical School, Boston, MA

Saturday, Apr 18, 2015, 2:50 PM – 3:00 PM
Discussion Discussion, Discussion

Presentation Abstract  




Abstract Number: 737
Presentation Title: Clonal evolution of the HER2 L755S mutation as a mechanism of acquired HER-targeted therapy resistance
Presentation Time: Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Location: Section 30
Poster Board Number: 29
Author Block: Xiaowei Xu1, Agostina Nardone1, Huizhong Hu1, Lanfang Qin1, Sarmistha Nanda1, Laura Heiser2, Nicholas Wang2, Kyle Covington1, Edward Chen1, Alexander Renwick1, Tamika Mitchell1, Marty Shea1, Tao Wang1, Carmine De Angelis1, Alejandro Contreras1, Carolina Gutierrez1, Suzanne Fuqua1, Gary Chamness1, Chad Shaw1, Marilyn Li1, David Wheeler1, Susan Hilsenbeck1, Mothaffar Fahed Rimawi1, Joe Gray2, C.Kent Osborne1, Rachel Schiff1. 1Baylor College of Medicine, Houston, TX; 2Oregon Health & Science University, Portland, OR
Abstract Body: Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant
trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response (pCR) rate was 27%. To investigate resistance mechanisms, we developed 10 HER2+ BC cell line
models resistant (R) to one or both drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell lines and a
subset of pre-treatment specimens from TBCRC006.
Methods: Parental (P) and LR/TR/LTR lines of 10 cell line models were profiled with whole exome/RNA sequencing. Mutations detected in R lines but not in P lines of the same model were identified. Mutation-specific Q-PCR was designed for sensitive quantification. Resistant cell and xenograft tumor growth were measured in response to drugs. Whole exome sequencing (>100X) and Ampliseq of 17 baseline tumor/normal pairs from TBCRC006 were performed.
Results: We found and validated the HER2 L755S mutation in the BT474/ATCC-LTR line and BT474/AZ-LR line (in ~30% of DNA/RNA), in which the HER pathway was reactivated for resistance. Overexpression of this mutation was previously shown to induce LR in HER2-negative BC cell lines, and resistant growth of BT474/AZ-LR line is significantly inhibited by HER2-L755S-specific siRNA knock-down, suggesting its role as an acquired L/LT resistance driver in HER2+ BC. Sequencing of BT474/AZ-LR single cell clones found the mutation in ~30% of HER2 copies in every cell. Using mutation-specific Q-PCR, we found statistically higher HER2 L755S levels in two BT474 parentals compared to P lines of SKBR3, AU565, and UACC812. These data suggest that HER2 L755S resistant subclones preexist in the BT474 parentals and were selected by L treatment to become the major clone in the two R lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of BT474/AZ-LR and BT474/ATCC-LTR cells (IC50: Afa 0.02µM vs. L 3 µM) and BT474/AZ-LR xenografts. Whole exome sequencing/Ampliseq of TBCRC006 found the HER2 L755S mutation in 1/17 primaries. This patient did not achieve pCR. The variant was present in 2% of DNA on both platforms, indicating a subclonal event of the resistance mutation.
Conclusion: Acquired L/LT resistance in the two BT474 R lines is due to selection of HER2 L755S subclones present in parental cells. The higher HER2 L755S
levels in BT474 parentals compared with other parentals, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group
with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC.
Presentation Number: SY07-04
Presentation Title: The evolutionary landscape of CLL: Therapeutic implications
Presentation Time: Sunday, Apr 19, 2015, 2:25 PM – 2:45 PM
Location: Grand Ballroom (300 Level), Pennsylvania Convention Center
Author Block: Catherine J. Wu. Dana-Farber Cancer Institute, Boston, MA
Abstract Body: Clonal evolution is a key feature of cancer progression and relapse. Recent studies across cancers have demonstrated the extensive degree of intratumoral heterogeneity present within individual cancers. We hypothesized that evolutionary dynamics contribute to the variations in disease tempo and response to therapy that are highly characteristic of chronic lymphocytic leukemia (CLL). We have recently investigated this phenomenon by developing a pipeline that estimates the fraction of cancer cells harboring each somatic mutation within a tumor through integration of whole-exome sequence (WES) and local copy number data (Landau et al., Cell 2013). By applying this analysis approach to 149 CLL cases, we discovered earlier and later cancer drivers, uncovered patterns of clonal evolution in CLL and linked the presence of subclones harboring driver mutations with adverse clinical outcome. Thus, our study, generated from a heterogeneous sample cohort, strongly supports the concept that CLL clonal evolution arises from mass extinction and therapeutic bottlenecks which lead to the emergence of highly fit (and treatment resistant) subclones. We further hypothesized that epigenetic heterogeneity also shapes CLL clonal evolution through interrelation with genetic heterogeneity. Indeed, in recent work, we have uncovered stochastic methylation disorder as the primary cause of methylation changes in CLL and cancer in general, and that this phenomena impacts gene transcription, genetic evolution and clinical outcome. Thus, integrated studies of genetic and epigenetic heterogeneity in CLL have revealed the complex and diverse evolutionary trajectories of these cancer cells.
Immunotherapy is exquisitely suited for specifically and simultaneously targeting multiple lesions. We have developed an approach that leverages whole-exome sequencing to systematically identify personal tumor mutations with immunogenic potential, which can be incorporated as antigen targets in multi-epitope personalized therapeutic vaccines. We are pioneering this approach in an ongoing trial in melanoma and will now expand this concept to address diverse malignancies. Our expectation is that the choice of tumor neoantigens for a vaccine bypasses thymic tolerance and thus generates highly specific and potent high-affinity T cell responses to eliminate tumors in any cancer, including both ‘trunk’ and ‘branch’ lesions.


Abstract Number: LB-056
Presentation Title: TP53 and RB1 alterations promote reprogramming and antiandrogen resistance in advanced prostate cancer
Presentation Time: Sunday, Apr 19, 2015, 4:50 PM – 5:05 PM
Location: Room 122, Pennsylvania Convention Center
Author Block: Ping Mu, Zhen Cao, Elizabeth Hoover, John Wongvipat, Chun-Hao Huang, Wouter Karthaus, Wassim Abida, Elisa De Stanchina, Charles Sawyers. Memorial Sloan Kettering Cancer Center, New York, NY
Abstract Body: Castration-resistant prostate cancer (CRPC) is one of the most difficult cancers to treat with conventional methods and is responsible for nearly all prostate cancer deaths in the US. The Sawyers laboratory first showed that the primary mechanism of resistance to antiandrogen therapy is elevated androgen receptor (AR) expression. Research based on this finding has led to the development of next-generation antiandrogen: enzalutamide. Despite the exciting clinical success of enzalutamide, about 60% of patients exhibit various degrees of resistance to this agent. Highly variable responses to enzalutamide limit the clinical benefit of this novel antiandrogen, underscoring the importance of understanding the mechanisms of enzalutamide resistance. Most recently, an unbiased SU2C-Prostate Cancer Dream Team metastatic CRPC sequencing project led by Dr. Sawyers and Dr. Chinnaiyan revealed that mutations in the TP53 locus are the most significantly enriched alteration in CRPC tumors when compared to primary prostate cancers. Moreover, deletions and decreased expressions of the TP53 and RB1 loci (co-occurrence and individual occurrence) are more commonly associated with CRPC than with primary tumors. These results established that alteration of the TP53 and RB1 pathways are associated with the development of antiandrogen resistance.
By knockdowning TP53 or/and RB1 in the castration resistant LNCaP/AR model, we demonstrate that the disruption of either TP53 or RB1 alone confers significant resistance to enzalutamide both in vitro and in vivo. Strikingly, the co-inactivation of these pathways confers the most dramatic resistance. Since up-regulation of either AR or AR target genes is not observed in the resistant tumors, loss of TP53 and RB1 function confers enzalutamide resistance likely through an AR independent mechanism. In the clinic, resistance to enzalutamide is increasingly being associated with a transition to a poorly differentiated or neuroendocrine-like histology. Interestingly, we observed significant up-regulations of the basal cell marker Ck5 and the neuroendocrine-like cell marker Synaptophysin in the TP53 and RB1 inactivated cells, as well as down-regulation of the luminal cell marker Ck8. The differences between these markers became even greater after enzalutamide treatment. By using the p53-stabilizing drug Nutlin, level of p53 is rescued and consequently the the decrease of AR protein caused by RB1 and TP53 knockdown is reversed. These results strongly suggest that interference of TP53 and RB1 pathways confers antiandrogen resistance by “priming” prostate cancer cells to reprogramming or transdifferentiation, likely neuroendocrine-like differentiation, in response to treatment. Futher experiments will be performed to assess the molecular mechanism of TP53/RB1 alterations in mediating cell programming and conferring antiandrogen resistance.


Abstract Number: LB-146
Presentation Title: TGF-β-induced tumor heterogeneity and drug resistance of cancer stem cells
Presentation Time: Monday, Apr 20, 2015, 1:00 PM – 5:00 PM
Location: Section 41
Author Block: Naoki Oshimori1, Daniel Oristian1, Elaine Fuchs2. 1Rockefeller University, New York, NY; 2HHMI/Rockefeller University, New York, NY
Abstract Body: Among the most common and life-threatening cancers world-wide, squamous cell carcinoma (SCC) exhibit high rates of tumor recurrence following anti-cancer therapy. Subsets of cancer stem cells (CSCs) often escape anti-cancer therapeutics and promote recurrence. However, its sources and mechanisms that generate tumor heterogeneity and therapy-resistant cell population are largely unknown. Tumor microenvironment may drive intratumor heterogeneity by transmitting signaling factors, oxygen and metabolites to tumor cells depending on their proximity to the local sources. While the hypothesis is attractive, experimental evidence is lacking, and non-genetic mechanisms that drive functional heterogeneity remain largely unknown. As a potential non-genetic factor, we focused on TGF-β because of its multiple roles in tumor progression.
Here we devise a functional reporter system to monitor, track and modify TGF-β signaling in mouse skin SCC in vivo. Using this approach, we found that perivascular TGF-β in the tumor microenvironment generates heterogeneity in TGF-β signaling in neighboring CSCs. This heterogeneity is functionally important: small subsets of TGF-β-responding CSCs proliferate more slowly than their non-responding counterparts. They also exhibit invasive morphology and a malignant differentiation program compared to their non-responding neighbors. By lineage tracing, we show that although TGF-β-responding CSCs clonally expand more slowly they gain a growth advantage in a remarkable ability to escape cisplatin-induced apoptosis. We show that indeed it is their progenies that make a substantial contribution in tumor recurrence. Surprisingly, the slower proliferating state of this subset of CSCs within the cancer correlated with but did not confer the survival advantage to anti-cancer drugs. Using transcriptomic, biochemical and genetic analyses, we unravel a novel mechanism by which heterogeneity in the tumor microenvironment allows a subset of CSCs to respond to TGF-β, and evade anti-cancer drugs.
Our findings also show that TGF-β established ability to suppress proliferation and promote invasion and metastasis do not happen sequentially, but rather simultaneously. This new work build upon the roles of this factor in tumor progression, and sets an important paradigm for a non-genetic factor that produces tumor heterogeneity.
Abstract Number: LB-129
Presentation Title: Identifying tumor subpopulations and the functional consequences of intratumor heterogeneity using single-cell profiling of breast cancer patient-derived xenografts
Presentation Time: Monday, Apr 20, 2015, 1:00 PM – 5:00 PM
Location: Section 41
Author Block: Paul Savage1, Sadiq M. Saleh1, Ernesto Iacucci1, Timothe Revil1, Yu-Chang Wang1, Nicholas Bertos1, Anie Monast1, Hong Zhao1, Margarita Souleimanova1, Keith Szulwach2, Chandana Batchu2, Atilla Omeroglu1, Morag Park1, Ioannis Ragoussis1. 1McGill University, Montreal, QC, Canada; 2Fluidigm Corporation, South San Francisco, CA
Abstract Body: Human breast tumors have been shown to exhibit extensive inter- and intra-tumor heterogeneity. While recent advances in genomic technologies have allowed us to deconvolute this heterogeneity, few studies have addressed the functional consequences of diversity within tumor populations. Here, we identified an index case for which we have derived a patient-derived xenograft (PDX) as a renewable tissue source to identify subpopulations and perform functional assays. On pathology, the tumor was an invasive ductal carcinoma which was hormone receptor-negative, HER2-positive (IHC 2+, FISH average HER2/CEP17 2.4), though the FISH signal was noted to be heterogeneous. On gene expression profiling of bulk samples, the primary tumor and PDX were classified as basal-like. We performed single cell RNA and exome sequencing of the PDX to identify population structure. Using a single sample predictor of breast cancer subtype, we have identified single basal-like, HER2-enriched and normal-like cells co-existing within the PDX tumor. Genes differentially expressed between these subpopulations are involved in proliferation and differentiation. Functional studies distinguishing these subpopulations are ongoing. Microfluidic whole genome amplification followed by whole exome capture of 81 single cells showed high and homogeneous target enrichment with >75% of reads mapping uniquely on target. Variant calling using GATK and Samtools revealed founder mutations in key genes as BRCA1 and TP53, as well as subclonal mutations that are being investigated further. Loss of heterozygocity was observed in 16 TCGA cancer driver genes and novel mutations in 7 cancer driver genes. These findings may be important in understanding the functional consequences of intra-tumor heterogeneity with respect to clinically important phenotypes such as invasion, metastasis and drug-resistance.
Abstract Number: 2847
Presentation Title: High complexity barcoding to study clonal dynamics in response to cancer therapy
Presentation Time: Monday, Apr 20, 2015, 4:35 PM – 4:50 PM
Location: Room 118, Pennsylvania Convention Center
Author Block: Hyo-eun C. Bhang1, David A. Ruddy1, Viveksagar Krishnamurthy Radhakrishna1, Rui Zhao2, Iris Kao1, Daniel Rakiec1, Pamela Shaw1, Marissa Balak1, Justina X. Caushi1, Elizabeth Ackley1, Nicholas Keen1, Michael R. Schlabach1, Michael Palmer1, William R. Sellers1, Franziska Michor2, Vesselina G. Cooke1, Joshua M. Korn1, Frank Stegmeier1. 1Novartis Institutes for BioMedical Research, Cambridge, MA; 2Dana-Farber Cancer Institute, Boston, MA
Abstract Body: Targeted therapies, such as erlotinib and imatinib, lead to dramatic clinical responses, but the emergence of resistance presents a significant challenge. Recent studies have revealed intratumoral heterogeneity as a potential source for the emergence of therapeutic resistance. However, it is still unclear if relapse/resistance is driven predominantly by pre-existing or de novo acquired alterations. To address this question, we developed a high-complexity barcode library, ClonTracer, which contains over 27 million unique DNA barcodes and thus enables the high resolution tracking of cancer cells under drug treatment. Using this library in two clinically relevant resistance models, we demonstrate that the majority of resistant clones pre-exist as rare subpopulations that become selected in response to therapeutic challenge. Furthermore, our data provide direct evidence that both genetic and non-genetic resistance mechanisms pre-exist in cancer cell populations. The ClonTracer barcoding strategy, together with mathematical modeling, enabled us to quantitatively dissect the frequency of drug-resistant subpopulations and evaluate the impact of combination treatments on the clonal complexity of these cancer models. Hence, monitoring of clonal diversity in drug-resistant cell populations by the ClonTracer barcoding strategy described here may provide a valuable tool to optimize therapeutic regimens towards the goal of curative cancer therapies.
Abstract Number: 3590
Presentation Title: Resistance mechanisms to ALK inhibitors
Presentation Time: Tuesday, Apr 21, 2015, 8:00 AM -12:00 PM
Location: Section 31
Poster Board Number: 13
Author Block: Ryohei Katayama1, Noriko Yanagitani1, Sumie Koike1, Takuya Sakashita1, Satoru Kitazono1, Makoto Nishio1, Yasushi Okuno2, Jeffrey A. Engelman3, Alice T. Shaw3, Naoya Fujita1. 1Japanese Foundation for Cancer Research, Tokyo, Japan; 2Graduate School of Medicine, Kyoto University, Kyoto, Japan; 3Massachusetts General Hospital Cancer Center, Boston, MA
Abstract Body: Purpose: ALK-rearranged non-small cell lung cancer (NSCLC) was first reported in 2007. Approximately 3-5% of NSCLCs harbor an ALK gene rearrangement. The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with advanced ALK-rearranged NSCLC. Several next-generation ALK-TKIs have entered the clinic and have shown promising antitumor activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to one next-generation ALK-TKI – alectinib – and potential strategies to overcome this resistance.
Experimental Procedure: We established a cell line model of alectinib resistance, and analyzed resistant tumor specimens from patients who had relapsed on alectinib. Cell lines were also established under an IRB-approved protocol when there was sufficient fresh tumor tissue. We established Ba/F3 cells expressing EML4-ALK and performed ENU mutagenesis to compare potential crizotinib or alectinib-resistance mutations. In addition, we developed Ba/F3 models harboring ALK resistance mutations and evaluated the potency of multiple next-generation ALK-TKIs including 3rd generation ALK inhibitor in these models and in vivo. To elucidate structure-activity-relationships of ALK resistance mutations, we performed computational thermodynamic simulation with MP-CAFEE.
Results: We identified multiple resistance mutations, including ALK I1171N, I1171S, and V1180L, from the ENU mutagenesis screen and the cell line model. In addition we found secondary mutations at the I1171 residue from the Japanese patients who developed resistance to alectinib or crizotinib. Both ALK mutations (V1180L and I1171 mutations) conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Based on thermodynamics simulation, each resistance mutation is predicted to lead to distinct structural alterations that decrease the binding affinity of ALK-TKIs for ALK.
Conclusions: We have identified multiple alectinib-resistance mutations from the cell line model, patient derived cell lines, and tumor tissues, and ENU mutagenesis. ALK secondary mutations arising after alectinib exposure are sensitive to other next generation ALK-TKIs. These findings suggest a potential role for sequential therapy with multiple next-generation ALK-TKIs in patients with advanced, ALK-rearranged cancers.
Session Title: Mechanisms of Resistance: From Signaling Pathways to Stem Cells
Session Type: Major Symposium
Session Start/End Time: Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM
Location: Terrace Ballroom II-III (400 Level), Pennsylvania Convention Center
CME: CME-Designated
CME/CE Hours: 2
Session Description: Even the most effective cancer therapies are limited due to the development of one or more resistance mechanisms. Acquired resistance to targeted therapies can, in some cases, be attributed to the selective propagation of a small population of intrinsically resistant cells. However, there is also evidence that cancer drugs themselves can drive resistance by triggering the biochemical- or genetic-reprogramming of cells within the tumor or its microenvironment. Therefore, understanding drug resistance at the molecular and biological levels may enable the selection of specific drug combinations to counteract these adaptive responses. This symposium will explore some of the recent advances addressing the molecular basis of cancer cell drug resistance. We will address how tumor cell signaling pathways become rewired to facilitate tumor cell survival in the face of some of our most promising cancer drugs. Another topic to be discussed involves how drugs select for or induce the reprogramming of tumor cells toward a stem-like, drug resistant fate. By targeting the molecular driver(s) of rewired signaling pathways and/or cancer stemness it may be possible to select drug combinations that prevent the reprogramming of tumors and thereby delay or eliminate the onset of drug resistance.
Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM
David A. Cheresh. UCSD Moores Cancer Center, La Jolla, CA
Tuesday, Apr 21, 2015, 10:30 AM -10:40 AM
Resistance to tyrosine kinase inhibitors: Heterogeneity and therapeutic strategies.
Tuesday, Apr 21, 2015, 10:40 AM -10:55 AM
Jeffrey A. Engelman. Massachusetts General Hospital, Boston, MA
Tuesday, Apr 21, 2015, 10:55 AM -11:00 AM
NG04: Clinical acquired resistance to RAF inhibitor combinations in BRAF mutant colorectal cancer through MAPK pathway alterations
Tuesday, Apr 21, 2015, 11:00 AM -11:15 AM
Ryan B. Corcoran, Leanne G. Ahronian, Eliezer Van Allen, Erin M. Coffee, Nikhil Wagle, Eunice L. Kwak, Jason E. Faris, A. John Iafrate, Levi A. Garraway, Jeffrey A. Engelman. Massachusetts General Hospital Cancer Center, Boston, MA, Dana-Farber Cancer Institute, Boston, MA
Tuesday, Apr 21, 2015, 11:15 AM -11:20 AM
SY27-02: Tumour heterogeneity and therapy resistance in melanoma
Tuesday, Apr 21, 2015, 11:20 AM -11:35 AM
Claudia Wellbrock. Univ. of Manchester, Manchester, United Kingdom

Presentation Number: SY27-02
Presentation Title: Tumour heterogeneity and therapy resistance in melanoma
Presentation Time: Tuesday, Apr 21, 2015, 11:20 AM -11:35 AM
Location: Terrace Ballroom II-III (400 Level), Pennsylvania Convention Center
Author Block: Claudia Wellbrock. Univ. of Manchester, Manchester, United Kingdom
Abstract Body: Solid tumors are structurally very complex; they consist of heterogeneous cancer cell populations, other non-cancerous cell types and a distinct extracellular matrix. Interactions of cancer cells with non-cancerous cells is well investigated, and our recent work in melanoma has demonstrated that the cellular environment that surrounds cancer cells has a major impact on the way a patient responds to MAP-kinase pathway targeting therapy.
We have shown that intra-tumor signaling within a heterogeneous tumor can have a major impact on the efficacy of BRAF and MEK inhibitors. With the increasing evidence of genetic and phenotypic heterogeneity within tumors, intra-tumor signaling between individual cancer-cell subpopulations is therefore a crucial factor that needs to be considered in future therapy approaches. Our work has identified the ‘melanocyte-lineage survival oncogene’ MITF as an important player in phenotypic heterogeneity (MITFhigh and MITFlow cells) in melanoma, and MITF expression levels are crucial for the response to MAP-kinase pathway targeted therapy. We found that ‘MITF heterogeneity’ can be caused by cell-autonomous mechanisms or by the microenvironment, including the immune-microenvironment.
We have identified various mechanisms underlying MITF action in resistance to BRAF and MEK inhibitors in melanoma. In MITFhigh expressing cells, MITF confers cell-autonomous resistance to MAP-kinase pathway targeted therapy. Moreover, it appears that in melanomas heterogeneous for MITF expression (MITFhigh and MITFlow cells), individual subpopulations of resistant and sensitive cells communicate and MITF can contribute to overall tumor-resistance through intra-tumor signaling. Finally, we have identified a novel approach of interfering with MITF action, which profoundly sensitizes melanoma to MAP-kinase pathway targeted therapy.
Tuesday, Apr 21, 2015, 11:35 AM -11:40 AM
SY27-03: Breast cancer stem cell state transitions mediate therapeutic resistance
Tuesday, Apr 21, 2015, 11:40 AM -11:55 AM
Max S. Wicha. University of Michigan, Comprehensive Cancer Center, Ann Arbor, MI
Tuesday, Apr 21, 2015, 11:55 AM -12:00 PM
SY27-04: Induction of cancer stemness and drug resistance by EGFR blockade
Tuesday, Apr 21, 2015, 12:00 PM -12:15 PM
David A. Cheresh. UCSD Moores Cancer Center, La Jolla, CA


Cellular Reprogramming in Carcinogenesis: Implications for Tumor Heterogeneity, Prognosis, and Therapy
Session Type: Major Symposium
Session Start/End Time: Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM
Location: Room 103, Pennsylvania Convention Center
CME: CME-Designated
CME/CE Hours: 2
Session Description: Cancers, both solid and liquid, consist of phenotypically heterogeneous cell types that make up the full cellular complement of disease. Deep sequencing of bulk cancers also frequently reveals a genetic intratumoral heterogeneity that reflects clonal evolution in space and in time and under the influence of treatment. How the distinct phenotypic and genotypic cells contribute to individual cancer growth and progression is incompletely understood. In this symposium, we will discuss issues of cancer heterogeneity and effects on growth and treatment resistance, with emphasis on cancer cell functional properties and influences of the microenvironment, interclonal genomic heterogeneity, and lineage relationships between cancer cells with stem cell and differentiated properties. Understanding these complex cellular relationships within cancers will have critical implications for devising more effective treatments.
Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM
Peter B. Dirks. Univ. of Toronto Hospital for Sick Children, Toronto, ON, Canada

Tuesday, Apr 21, 2015, 10:30 AM -10:40 AM

Origins, evolution and selection in childhood leukaemia
Tuesday, Apr 21, 2015, 10:40 AM -11:00 AM
Tariq Enver. Cancer Research UK, London, United Kingdom

Tuesday, Apr 21, 2015, 11:00 AM -11:05 AM

Cytokine-controlled stem cell plasticity inintestinal tumorigenesis
Tuesday, Apr 21, 2015, 11:05 AM -11:25 AM
Florian Greten. Georg-Speyer-Haus, Frankfurt, Germany

Tuesday, Apr 21, 2015, 11:25 AM -11:30 AM

SY23-03: Intratumoural heterogeneity in human serous ovarian carcinoma
Tuesday, Apr 21, 2015, 11:30 AM -11:50 AM
John P. Stingl. Cancer Research UK Cambridge Research Inst., Cambridge, United Kingdom

Tuesday, Apr 21, 2015, 11:50 AM -11:55 AM

Functional and genomic heterogeneity in brain tumors
Tuesday, Apr 21, 2015, 11:55 AM -12:15 PM


Proc Natl Acad Sci U S A. 2015 Jan 20;112(3):851-6. doi: 10.1073/pnas.1320611111. Epub 2015 Jan 5.

Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity.

Meyer M1, Reimand J2, Lan X3, Head R1, Zhu X1, Kushida M1, Bayani J4, Pressey JC5, Lionel AC6, Clarke ID7, Cusimano M8, Squire JA9, Scherer SW6, Bernstein M10, Woodin MA5, Bader GD11, Dirks PB12.

Author information


Glioblastoma (GBM) is a cancer comprised of morphologically, genetically, and phenotypically diverse cells. However, an understanding of the functional significance of intratumoral heterogeneity is lacking. We devised a method to isolate and functionally profile tumorigenic clones from patient glioblastoma samples. Individual clones demonstrated unique proliferation and differentiation abilities. Importantly, naïve patient tumors included clones that were temozolomide resistant, indicating that resistance to conventional GBM therapy can preexist in untreated tumors at a clonal level. Further, candidate therapies for resistant clones were detected with clone-specific drug screening. Genomic analyses revealed genes and pathways that associate with specific functional behavior of single clones. Our results suggest that functional clonal profiling used to identify tumorigenic and drug-resistant tumor clones will lead to the discovery of new GBM clone-specific treatment strategies.



739: Tumor cell plasticity with transition to a mesenchymal phenotype is a mechanism of chemoresistance that is reversed by Notch pathway inhibition in lung adenocarcinoma
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Khaled A. Hassan. University Of Michigan, Ann Arbor, MI

745: Oncostatin M receptor activation leads to molecular targeted therapy resistance in non-small cell lung cancer
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Kazuhiko Shien1, Vassiliki A. Papadimitrakopoulou1, Dennis Ruder1, Nana E. Hanson1, Neda Kalhor1, J. Jack Lee1, Waun Ki Hong1, Ximing Tang1, Roy S. Herbst2, Luc Girard3, John D. Minna3, Jonathan M. Kurie1, Ignacio I. Wistuba1, Julie G. Izzo1. 1University of Texas MD Anderson Cancer Center, Houston, TX; 2Yale Cancer Center, Yale School of Medicine, New Haven, CT; 3Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX

746: Activation of EGFR bypass signaling through TGFα overexpression induces acquired resistance to alectinib in ALK-translocated lung cancer cells
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Tetsuo Tani, Hiroyuki Yasuda, Junko Hamamoto, Aoi Kuroda, Daisuke Arai, Kota Ishioka, Keiko Ohgino, Ichiro Kawada, Katsuhiko Naoki, Hayashi Yuichiro, Tomoko Betsuyaku, Kenzo Soejima. Keio University, Tokyo, Japan

752: Elucidating the mechanisms of acquired resistance in lung adenocarcinomas
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Sandra Ortiz-Cuarán1, Lynnette Fernandez-Cuesta1, Christine M. Lovly2, Marc Bos1, Matthias Scheffler3, Sebastian Michels3, Kerstin Albus4, Lydia Meyer4, Katharina König4, Ilona Dahmen1, Christian Mueller1, Luca Ozretić4, Lars Tharun4, Philipp Schaub1, Alexandra Florin4, Berit Pinther1, Nike Bahlmann1, Sascha Ansén3, Martin Peifer1, Lukas C. Heukamp4, Reinhard Buettner4, Martin L. Sos1, Jürgen Wolf3, William Pao2, Roman K. Thomas1. 1University of Cologne, Cologne, Germany; 2Department of Medicine, Vanderbilt University, Nashville, TN; 3Department of Internal Medicine, Center for Integrated Oncology Köln-Bonn, University Hospital Cologne, Cologne, Germany; 4Institute of Pathology, University Hospital Cologne, Cologne, Germany

760: On the evolution of erlotinib-resistant NSCLC subpopulations
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Michael E. Ramirez1, Robert J. Steininger, III1, Lani F. Wu2, Steven J. Altschuler2. 1UT Southwestern, Dallas, TX; 2UCSF, San Francisco, CA
763: Implications of resistance patterns with NSCLC targeted agents
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
David J. Stewart, Paul Wheatley-Price, Rob MacRae, Jason Pantarotto. University of Ottawa, Ottawa, ON, Canada


768: A kinome-wide siRNA screen identifies modifiers of sensitivity to the EGFR T790M-targeted tyrosine kinase inhibitor (TKI), AZD9291, in EGFR mutant lung adenocarcinoma
Sunday, Apr 19, 2015, 1:00 PM – 5:00 PM
Eiki Ichihara1, Joshua A. Bauer2, Pengcheng Lu3, Fei Ye3, Darren Cross4, William Pao1, Christine M. Lovly1. 1Vanderbilt University School of Medicine, Nashville, TN; 2Vanderbilt Institute of Chemical Biology High-Throughput Screening Facility, Nashville, TN; 3Vanderbilt University Medical Center, Nashville, TN; 4AstraZeneca Oncology Innovative Medicines, United Kingdom

LB-055: Clinical acquired resistance to RAF inhibitor combinations in BRAF-mutant colorectal cancer through MAPK pathway alterations
Sunday, Apr 19, 2015, 4:35 PM – 4:50 PM
Leanne G. Ahronian1, Erin M. Sennott1, Eliezer M. Van Allen2, Nikhil Wagle2, Eunice L. Kwak1, Jason E. Faris1, Jason T. Godfrey1, Koki Nishimura1, Kerry D. Lynch3, Craig H. Mermel1, Elizabeth L. Lockerman1, Anuj Kalsy1, Joseph M. Gurski, Jr.1, Samira Bahl4, Kristin Anderka4, Lisa M. Green4, Niall J. Lennon4, Tiffany G. Huynh3, Mari Mino-Kenudson3, Gad Getz1, Dora Dias-Santagata3, A. John Iafrate3, Jeffrey A. Engelman1, Levi A. Garraway2, Ryan B. Corcoran1. 1Massachusetts General Hospital Cancer Center, Boston, MA; 2Dana Farber Cancer Institute, Boston, MA; 3Massachusetts General Hospital Department of Pathology, Boston, MA; 4Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA


Other Articles on this Site Related to Tumor Heterogeneity Include

Notes On Tumor Heterogeneity: Targets and Mechanisms, from the 2015 AACR Meeting in Philadelphia PA

Issues in Personalized Medicine: Discussions of Intratumor Heterogeneity from the Oncology Pharma forum on LinkedIn

Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

CANCER COMPLEXITY: Heterogeneity in Tumor Progression and Drug Response – 2015 Annual Symposium @Koch Institute for Integrative Cancer Research at MIT – W34, 6/12/2015 9:00 AM EDT – 4:30 PM EDT

In vitro Models of Tumor Microenvironment for New Cancer Target and Drug Discovery, 11/17 – 11/19/2014, Hyatt Boston Harbor

What can we expect of tumor therapeutic response?



Read Full Post »

Issues in Personalized Medicine: Discussions of Intratumor Heterogeneity from the Oncology Pharma forum on LinkedIn

Curator and Writer: Stephen J. Williams, Ph.D.

In an earlier post entitled “Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing” the heterogenic nature of solid tumors was discussed.  There resulted an excellent discussion in the Oncology Pharma forum on LinkedIn so I curated the comments (below article) to foster further discussion. To summarize the original post, this was a discussion of Dr. Charles Swanton’s paper[1] in which he and colleagues had noticed that individual biopsies from primary renal tumors displayed a variety of mutations of the same and different tumor suppressor genes (TSG), thereby not only revealing the heterogeneity of individual tumors but also how tumors can evolve.  Thus it was suggested that individual cells of a primary tumor can represent individual clones, each evolving on a distinct pathway to tumorigenicity and metastasis as each clone would have accumulated different passenger mutations.  It is these passenger mutations which have been posited to be responsible for a tumor’s continued growth (as discussed in the following post Rewriting the Mathematics of Tumor Growth; Teams Use Math Models to Sort Drivers from Passengers).  Indeed, as Dr. Swanton mentioned in the posting that it is very likely a solid tumor contains discrete clones with different driver and passenger mutations and possibly different mutated TSG but also this intra-tumor heterogeneity would have great implications for personalized chemotherapeutic strategies, not only against the primary tumor but against resistant outgrowth clones, and to the metastatic disease, as Swanton and colleagues had found that the metastatic disease displayed tremendously increased genomic instability than the underlying primary disease.

Therefore it may behoove the clinical oncologist to view solid tumors as a collection of multiple clones, each having their own mutagenic spectrum and tumorigenic phenotype.  Each of these clones may acquire further mutations which provide growth advantage over other clones in the early primary tumor.  In addition, branched evolution of a clone most likely depends more on genomic instability and epigenetic factors than on solely somatic mutation.

This is echoed in a  report in Carcinogenesis back in 2005[3] Lorena Losi, Benedicte Baisse, Hanifa Bouzourene and Jean Benhatter had shown some similar results in colorectal cancer as their abstract described:

“In primary colorectal cancers (CRCs), intratumoral genetic heterogeneity was more often observed in early than in advanced stages, at 90 and 67%, respectively. All but one of the advanced CRCs were composed of one predominant clone and other minor clones, whereas no predominant clone has been identified in half of the early cancers. A reduction of the intratumoral genetic heterogeneity for point mutations and a relative stability of the heterogeneity for allelic losses indicate that, during the progression of CRC, clonal selection and chromosome instability continue, while an increase cannot be proven.”

Therefore if a tumor had evolved in time closer to the initial driver mutation multiple therapies may be warranted while tumors which had not yet evolved much from their driver mutation may be tackled with an agent directed against that driver, hence the branched evolution as shown in the following diagram:

branced chain evolution cancer

Cancer Sequencing

Unravels clonal evolution.

From Carlos Caldas. (2012).

Nature Biotechnology V.30

pp 405-410.[2] used with









An article written by Drs. Andrei Krivtsov and Scott Armstron entitled “Can One Cell Influence Cancer Heterogeneity”[4] commented on a study by Friedman-Morvinski[5] in Inder Verma’s laboratory discussed how genetic lesions can revert differentiated neorons and glial cells to an undifferentiated state [an important phenotype in development of glioblastoma multiforme].

In particular it is discussed that epigenetic state of the transformed cell may contribute to the heterogeneity of the resultant tumor.  Indeed many investigators (initially discovered and proposed by Dr. Beatrice Mintz of the Institute for Cancer Research, later to be named the Fox Chase Cancer Center) show the cellular microenvironment influences transformation and tumor development[6-8].

Briefly the Friedman-Morvinski study used intra-cerebral ventricular (ICV) injection of lentivirus to introduce oncogenes within the CNS and produced tumors of multiple cell origins including neuronal and glial cell origin (neuroblastoma and glioma).  The important takeaway was differentiated somatic cells which acquire genetic lesions can transform to form multiple tumor types.  As the authors state, “cellular differentiation and specialization are accompanied by gradual changes in epigenetic programs” and that “the cell of origin may influence the epigenetic state of the tumor”.   In essence this means that the success of therapy may depend on the cellular state (whether stem cell, progenitor cell, or differentiated specialized cell) at time of transformation.  In other words tumors arising from cells with an epigenetic state seen in stem cells would be more resistant to therapy unless given an epigenetic therapy, such as azacytididne, retinoic acid or HDAC inhibitors.


So as the Oncology Pharma forum on LinkedIn was such an excellent discussion I would like to post the comments for curation purposes and foster further discussion.  I would like to thank everyone’s great comments below.  I would especially like to thank Dr. Emanuel Petricoin from George Mason and Dr. David Anderson for supplying extra papers which will be the subject of a future post. I had curated each comment with inserted LIVE LINKS to make it easier to refer to a paper and/or company mentioned in the comment.

The comments seemed to center on three main themes:

  1. 1.      Clinicians pondering the benefit to mutational spectrum analysis to determine personalized therapy and develop biomarkers of early disease
  2. 2.      A shift in the clinicians paradigm of cancer development, diagnoses, and treatment from strictly histologic evaluation to a genetic and altered cellular pathway view
  3. 3.      Use of proteomics, microarray and epigenetics as an alternative to mutational analysis to determine aberrant cellular networks in various stages of tumor development


Victor Levenson • Thanks for posting this! To be honest, I am puzzled by the insistence on sequencing as a tool for tumor analysis – we know that expression patterns rather than mutations in a limited number of genes determine tumor physiology (or, even more, physiology of any tissue). Since the AACR-2012 we know that different tumors have similar or even identical mutations, so >functional< rather than >structural< differences are important. Frankly, I’d be much more excited learning about expression pattern heterogeneity in tumors.Granted that is much more challenging than NGS sequencing, but the value of the data would be incomparable, especially in its application to biomarker development.

Stephen J. Williams, Ph.D. • Dear Dr. Levenson, thanks for your comments. I agree with you and in no way am insisting on the releiance of sequencing mutations in cancer as the sole means for determining therapy. It is extremely true that tumors will show tremendous heterogeneity of mRNA expression. There are a number of studies (one which I will post on that individual tumor cells will have differing expression patterns based on the levels of regional hypoxia within the tumor as well as other microenvironmental factors. I do have two posts on on this matter, curating various programs around the world which are using microarray expression analysis of tumors to determine personalized strategies. I believe the reliance on mutational analysis is based on the drugs that have been developed (such as Gleevec and crizotinib) which are based on mutant forms of BCR-Abl and ALK, respectively. However (as per two posts I did based on Mike Martin on our site “Mathematical Models of Driver and Passenger mutations) where he discusses how certain driver mutations will get the senescent cell over the hump to get to fully transformed and contribute to a certain level of growth while subsequent passengers are responsible for the sustained survival and expansion of the tumor.

Victor Levenson • Dr. Williams, thanks for the comments. Driving a senescent cell into proliferative stage is a tremendous change, which >may< begin with a mutation, but involves dramatic restructuring of transcription patterns that will drive the process. Hypoxia will definitely contribute to variations in the patterns, although will probably not be the main driver of the process. As to whether a mutation or a change in transcription pattern initiate the process, I am not sure we will ever be able to determine <grin>.

Vanisree Staniforth • Thanks for posting! Certainly a thought provoking article with regard to the future of personalized cancer therapies.


Dr. Raj Batra • If we follow Dr Levenson’s proposed conceptual approach (which we also published in 2009 and 2010), we are MUCH more likely to significantly impact tumor morbidity and mortality.

Stephen J. Williams, Ph.D. • Thanks Vanisiree and Dr. Batra for your comments. Hopefully we will see, from the future cancer statistics, how personlized therapy have improved outcomes for the solid tumors, like the hematologic cancers. 26 days ago

Emanuel Petricoin • The issue about intra and inter tumor heterogeneity is very important however since it is unknown which mutations are true drivers, an explanation of the results found in these studies simply could be the variances are all in the inconsequential mutations and the commonality is the driver mutations. Moreover, at the end of the day, its not the mRNA expression that we really care about but the functional protein signaling -phosphoprotein driven signaling architecture, that we care about since these are the drug targets directly.

Mohammad Azhar Aziz,PhD • This article addresses the potential complexity of dealing with cancer which is apparently increasing proportionally with the amount of data generated. Intratumor heterogeneity will remain there and even multiple biopsies that are randomly chosen will offer no conclusive solution.Mutations,expression profiles and functional protein signaling (as discussed above) alone can not provide any breakthrough. It will be a composite picture of all these and many other components (e.g. microenvironment, alternative splicing, epigenetics,non-coding RNAs etc.) that will hold the promises in the future. We have made phenomenal advances in understanding each of these aspects separately but definitely lack the tools to integrate all these. Developing tools to integrate all these data may provide some breakthrough in understanding and thus treating cancer.

Emanuel Petricoin • I agree Mohammad in a systems biology approach however the current compendium of drugs largely are kinase inhibitors or enzymatic inhibitors. Since most studies have shown little correlation between gene mutation and protein levels and phosphoprotein levels, for example, it is no wonder why the recent spate of failed trials (e.g. stratification by PIK3CA mutation or PTEN mutation for AKT-mTOR inhibitors) should come as any shock. We will be publishing work using protein pathway activation mapping coupled to laser dissection of a number of intra and inter tumoral analysis that indicates that the signaling architecture appears much more stable.

Stephen J. Williams, Ph.D. • Thank you Dr. Pettricoin for your comments. I eagerly await the publication of your results concerning proteomic evaluation of multiple biopsies of a tumor. I am very interested that you found limited intratuoral heterogeneity of signaling pathways given the diversity of intratumoral microenvironmental stresses (changes in regional hypoxia, blood flow, and populations of cancer stem cells). I agree with you and Mohammed that proteomic profiling will be imperative in determining personalized approaches for targeted therapy. Dr. Swanton had informed me that they had used IHC to determine if mTOR signaling had correlated with the mutational spectrum they had seen. In addition he had mentioned that there was enhanced genomic instability in the metastatic disease relative to the primary tumor and it would be very interesting to see how signaling pathways change in cohorts of matched metastatic and primary tumors. A few years ago we were looking at genes which were completely lost upon transformation of ovarian epithelial cells and worked up one of those genes (CRBP1) in cohorts of human ovarian cancer samples, using expression analysis in conjunction with laser capture microdissection and backed up by IHC analysis, and found that the expression pattern of CRBP1 was uniform in a tumor, either there was a complete loss in all cells in a tumor of CRBP1 or all the cells expressed the protein. Therefore I am curious if intratumor heterogeneity is dependent on the cell lineage and evolution of the transformed cell into a full tumor or a function of a discrete population of stem cells with varied genomic instability. Your results might suggest a more clonal evolution rather than a branched evolution which was found in this paper.
It is interesting that you mention the tough trials with the PTEN/PI3K/AKT axis of inhibitors. In high grade serous ovarian cancer we were never able to find any PI3K, PTEN, nor AKT mutations yet PI3K activity is usually overactive. If feel both your and Mohammed’s assessment that a systems biology approach instead of just relying on DNA mutational analysis will be more important in the future. In addition, there is nice work from Dr. Jefferey Peterson at Fox Chase and the development of a database of kinase inhibitors and activity effects on the kinome, showing the vast amount of crosstalk between once thought linear enzyme systems. If TKI’s will be the brunt of pharma’s development I feel they need to quickly develop as many TKI’s as they can now before we get to a clinical problem (resistance and lack of available therapeutics).

Emanuel Petricoin • Thanks Steven- yes, we are working with Charlie Swanton and Marco on the renal sets- our other studies are from breast and colon cancers. I think one of the things we do that really no one else is doing, unfortunately, is to laser capture microdissect the tumor cells from these specimens so that we have a more pure and accurate view of the signaling architecture. One confounder from the proteomic stand-point is the fact that pre-analytical variables such as post-excision delay times where the tissue is a hypoxic wound and signaling changes fluctuating as the tissue reacts to the ex-vivo condition can really effect things. When we look at tissue sets where the tissue is biopsied and immediately frozen we really dont see big differences in the signaling – the within tumor architecture is much more similar then between. We use the reverse phase array technology we invented to provide quantitative analysis on hundreds of phosphoproteins at once – so a nice view of the functional protein activation network. Your results of CRBP1 in ovarian tumors and the IHC data are very interesting. We will see how this all plays out. Of course once other confounder with the mutational data is that we really dont know what are the drivers and what are the passengers…
Yes I know Jeff Peterson’s work- its fantastic. In the end the hope I think- and in my personal opinion- will be rationally combined therapeutics based on the signaling architecture of each individual patient.

Incidentally, we just published a paper that you may be interested in from a “systems biology” standpoint-


Federici G, Gao X, Slawek J, Arodz T, Shitaye A, Wulfkuhle JD, De Maria R, Liotta LA, Petricoin EF 3rd. Mol Cancer Res. 2013 May

also- we published a paper that speaks directly to your point where we compared the signaling network activation of patient-matched primary colorectal cancers and synchronous liver mets. indeed there is huge systemic differences in the liver metastasis compared to the primary. there is no doubt in my mind that we will need to biopsy the metastasis to know how to treat. Looking at the primary tumor as a guide for therapy is a fools errand. here is the paper reference:

Protein pathway activation mapping of colorectal metastatic progression reveals metastasis-specific network alterations.

Silvestri A, Calvert V, Belluco C, Lipsky M, De Maria R, Deng J, Colombatti A, De Marchi F, Nitti D, Mammano E, Liotta L, Petricoin E, Pierobon M.

Clin Exp Metastasis. 2013 Mar;30(3):309-16. doi: 10.1007/s10585-012-9538-5. Epub 2012 Sep 29.

Center for Applied Proteomics and Molecular Medicine, George Mason University, 10900 University Blvd., Manassas, VA, 20110, USA.


The mechanism by which tissue microecology influences invasion and metastasis is largely unknown. Recent studies have indicated differences in the molecular architecture of the metastatic lesion compared to the primary tumor, however, systemic analysis of the alterations within the activated protein signaling network has not been described. Using laser capture microdissection, protein microarray technology, and a unique specimen collection of 34 matched primary colorectal cancers (CRC) and synchronous hepatic metastasis, the quantitative measurement of the total and activated/phosphorylated levels of 86 key signaling proteins was performed. Activation of the EGFR-PDGFR-cKIT network, in addition to PI3K/AKT pathway, was found uniquely activated in the hepatic metastatic lesions compared to the matched primary tumors. If validated in larger study sets, these findings may have potential clinical relevance since many of these activated signaling proteins are current targets for molecularly targeted therapeutics. Thus, these findings could lead to liver metastasis specific molecular therapies for CRC.

Adrian Anghel • I think both patterns (protein phosphorylation and mRNA) should be important in this complicated equation of heterogeneity. Let’s not forget the so-called functional miRNA-mRNA regulatory modules (FMRMs). Also I think we have different patterns of this heterogeneity for different evolutive stages of the tumour.


Alvin L. Beers, Jr., M.D. • This is a great study, but bad news for attempting to tailor treatment based on molecular markers. Dr. Swanton’s comment: “herterogeneity is likely to complicate matters” is an understatement. Intratumoral heterogeneity, branched, instead of linear, evolution of mutational events portends a nightmare in trying to predict location and volume of biopsies. I am reminded of a series of articles in Nature 491 (22 November 2012) “Physical Scientists take on Cancer”. There is a great comment by Jennie Dusheck: “Cancer researchers now recognize that taming wild cancer cells – populations of cells that evolve, cooperate, and roam freely through the body-demand a wider-angle view than molecular biology has been able to offer. Cross-disciplinary collaborations can approach cancer a greater spatial and temporal scales, using mathematical methods more typical of engineering, physics, ecology and evolutionary biology. The sense of failure so evident five years ago is giving way to the excitement of a productive intellectual partnership.” I’m not certain how well the “productive partnership” is going, but this Swanton study confirms the limitations of molecular biology.

Stephen J. Williams, Ph.D. • Thanks Dr. Beers for adding in your comment and adding in Jennie’s comment. Certainly it is something to be aware of if a cancer center’s strategy is to rely solely on gene arrays to genotype tumors. I think Dr. Pettricoin’s work on using proteomics might give some resolution to the matter however, in communicating with Dr. Swanton, I did not get the feeling of an “all hope is lost” but just that, in the case of solid tumors like renal, that careful monitoring of tumors after treatment may be warranted and, more interestingly, from a scientific standpoint, is the genetic complexity surrounding the origin of the disease, and not simple mutational spectrum of a single clone.

Burke Lillian • This is clinically a very important issue. Right now, sequencing or massive approaches such as pan-phosphorylation studies are helpful because, although we know many of the drivers, these studies are actually identifying new genes or new pathways that are activated. After a few (or several years), we truly will know which genes are typically activated and there will be panels to look for these.

Emanuel Petricoin • yes, I agree. In fact, the company that I co-founded, Theranostics Health, Inc– is launching a CLIA based protein pathway activation mapping test at ASCO that measures actionable drug targets (e.g. phospho HER2, EGFR, HER3, AKT, ERK, JAK, STAT, p70S6) and total HER2, EGFR, HER3 and PTEN. So these tests are coming even now.


Alvin L. Beers, Jr., M.D. • I do not think that “all hope is lost” nor did I have the impression that Dr. Swanton feels that way with regards to molecular profiling of cancer. I certainly applaud further research into the molecular aspects of cancer biology. But I do not believe that this will be sufficient. Integrating physicial sciences into cancer biology makes perfect sense toward better understanding of this complex disease.

Eleni Papadopoulos-Bergquist • I have enjoyed reading these comments and different ideas regarding genetic testing and profiling. As a nurse and researcher at heart, this is information that will make a huge impact on drug protocols, therefore allowing the best and most specific treatment to each individual rather than having a standard treatment protocol. Even with the scientific complexity of specifying genotypes of particular cancers, there is still the question of each individuals body responding to treatment. I’d love to have some dialogue regarding immune response.

Bradford Graves • I too have enjoyed reading this discussion. I am not a clinician but as a drug discovery researcher I have been struck by some parallels to the concept of virus fitness in virology – particularly as applied to HIV. Drug discovery cannot wait for the final answers to the many important questions being addressed in the discussion initiated by Dr. Williams. The best we can do is to pursue a broad range of therapeutics that will give the clinicians the armament they will need to either cure a given cancer or to at least turn it into a chronic as opposed to an acute disease. There has been a measure of success in the HIV field and it seems like it will be achievable for cancer. Obviously, to the extent that the labels of driver and passenger mutations can be correctly applied will help to prioritize the targets we address.

David W. Anderson • I would suggest that you look at the following publications:

Horn and Pao, (2009) JCO 26: 4232-4234.

Bunn and Doebele (2011) JCO:29:1-3

Boguski et al. (2009) Customized care 2020: how medical sequencing and network biology will enable personalized medicine. F1000 Bio Report 1:7.

Jones, S et al. (2010). Evolution of an adenocarcinoma in response to selection by targeted kinase inhibitors. Genome Biology. 11:R82. Marco Marra’s group in Toronto.

Also look at how companies and organizations like Foundation Medicine, Caris, Clarient, and CollabRx who are using genomics and sequencing on a large scale to address cancer from a personalized/individual approach.

Cancer is/will be a chronic disease requiring individualized/combinatorial therapies in many cases.

Alvin L. Beers, Jr., M.D. • David. These are excellent articles by Paul Bunn and Mark Boguski regarding integrating molecular markers into diagnostic evaluation, and I’ve seen other papers of similiar elk, and likely there will be more to come. Particularly in NSC lung cancer, the SOC is to use these markers up front. Diagnosis based on histology alone can no longer be recommended. The challenge for the future is how to integrate other aspects of cell biology with these markers. It remains daunting that not only do we see heterogeneity in molecular within tumors at a particularly point in time, but that there is often an evolution of markers over time, ie, a “plasticity” of markers, whether treatment is given or not. We know that targeted agents, TKI’s, enzyme inhibitors are not curative, but do give an improvement in PFS. A great deal of this resistance has to do with this “moving target” aspect of cancer cell biology..



1.         Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P et al: Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. The New England journal of medicine 2012, 366(10):883-892.

2.         Caldas C: Cancer sequencing unravels clonal evolution. Nature biotechnology 2012, 30(5):408-410.

3.         Losi L, Baisse B, Bouzourene H, Benhattar J: Evolution of intratumoral genetic heterogeneity during colorectal cancer progression. Carcinogenesis 2005, 26(5):916-922.

4.         Krivtsov AV, Armstrong SA: Cancer. Can one cell influence cancer heterogeneity? Science 2012, 338(6110):1035-1036.

5.         Friedmann-Morvinski D, Bushong EA, Ke E, Soda Y, Marumoto T, Singer O, Ellisman MH, Verma IM: Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice. Science 2012, 338(6110):1080-1084.

6.         Mintz B, Cronmiller C: Normal blood cells of anemic genotype in teratocarcinoma-derived mosaic mice. Proceedings of the National Academy of Sciences of the United States of America 1978, 75(12):6247-6251.

7.         Watanabe T, Dewey MJ, Mintz B: Teratocarcinoma cells as vehicles for introducing specific mutant mitochondrial genes into mice. Proceedings of the National Academy of Sciences of the United States of America 1978, 75(10):5113-5117.

8.         Mintz B, Cronmiller C, Custer RP: Somatic cell origin of teratocarcinomas. Proceedings of the National Academy of Sciences of the United States of America 1978, 75(6):2834-2838.



Other articles on this site on “PERSONALIZED MEDICINE” and “CANCER” and “OMICS” include:

Personalized medicine-based diagnostic test for NSCLC

Personalized medicine and Colon cancer

Helping Physicians identify Gene-Drug Interactions for Treatment Decisions: New ‘CLIPMERGE’ program – Personalized Medicine @ The Mount Sinai Medical Center

Systems Diagnostics – Real Personalized Medicine: David de Graaf, PhD, CEO, Selventa Inc.

Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

Personalized Medicine: Clinical Aspiration of Microarrays

Understanding the Role of Personalized Medicine

Directions for Genomics in Personalized Medicine

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1

Rewriting the Mathematics of Tumor Growth; Teams Use Math Models to Sort Drivers from Passengers

Diagnosing Diseases & Gene Therapy: Precision Genome Editing and Cost-effective microRNA Profiling

Breast Cancer: Genomic profiling to predict Survival: Combination of Histopathology and Gene Expression Analysis

Proteomics and Biomarker Discovery


 Also please see our upcoming e-book “Genomics Orientations for Individualized Medicine” in our Medical E-book Series at











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Author, reporter: Tilda Barliya PhD

Breast cancer is the second most common cancer worldwide after lung cancer, the fifth most  common cause of cancer death, and the leading  cause of cancer death in women. the global burden of  breast cancer exceeds all other cancers and the incidence  rates of breast cancer are increasing (1,2).

The heterogeneity of breast cancers makes them both a fascinating and challenging solid tumor to diagnose and treat. Here is a great review of the molecular pathology of breast cancer progression (3).

The molecular pathology of breast cancer progression” by Alessandro Bombonati  and Dennis C Sgroi.

Breast cancer is the most frequent carcinoma in females and the second most common cause of cancer related mortality in women. Approximately 54 000 and 207 000 new cases of in situ and invasive breast carcinoma, respectively. Overall, breast cancer incidence rates have levelled off since 1990, with a decrease of 3.5%/year from 2001 to 2004.  Most notably, during this same time period, breast cancer mortality rates have declined 24%, with the largest impact among young women and women with estrogen receptor (ER)-positive disease.

The decline in breast cancer mortality has been attributed to the combination of early detection with screening programmes and the advent of more efficacious adjuvant progression have aided in the discovery of novel pathway-specific targeted therapeutics, and the emergence of such effective therapeutics is currently driving the need for molecular-based, ‘patient-tailored’ treatment planning.

Proposed models of human breast cancer progression

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Epidemiological and morp

hological observations led to the formulation of several linear models of breast cancer initiation, transformation and

progression. Figure 1

The ductal and lobular subtypes constitute the majority of all breast cancers worldwide, with the ductal subtype accounting for 40–75% of all diagnosed cases.

The classic model of breast cancer progression of the ductal type proposes thatneoplastic evolution initiates in normal epithelium (normal), progresses to flat epithelial atypia (FEA), advances to atypical ductalhyperplasia (ADH), evolves to ductal carcinoma in situ (DCIS) and culminates as invasive ductal carcinoma (IDC).

The model of lobular neoplasia proposes a multi-step progression from normal epithelium to atypicallobular hyperplasia, lobular carcinoma in situ (LCIS) and invasive lobular carcinoma (ILC).

The cell of origin of breast cancer: the clonal and stem cell hypotheses

The two leading models accounting for breast carcinogenesis are the sporadic clonal evolution model and the cancer stem cell (cSC) model. According to the sporadic clonal evolution hypothesis, any breast epithelial cell can be the target of random mutations. The cells with advantageous genetic and epigenetic alterations are selected over time to contribute to tumour progression. The third alternative cSC model postulates that only stem and progenitor cells (representing a small fraction of the tumor cells within the cancer) can initiate and maintain tumor progression. Figure 2.

Normal breast stem cells (nBSCs) are long-lived, tissue-resident cells capable of self-renewal activity and multi-lineage differentiation that can recapitulate the breast tubulolobular architecture that is composed of luminal and myoepithelial cells.

As normal breast cancer stem cells are long-time tissue residents, it has been proposed that such cells are candidates for accumulating genetic and epigenetic modifications. It has been further proposed that such molecular alterations result in deregulation of normal self-renewal, leading to the development of a cancer stem cell (cSC).

It is believed that the cSC undergoes asymmetrical division, maintaining the stem cell population while at the same time differentiating into committed progenitor(s) cells that give rise to the different breast cancer subtypes.

A second scenario, as it relates to breast cancer development, is one in which the cancer-initiating cells are derived from committed progenitor cells that spawn different breast cancer subtypes. Both scenarios are highly supported.

Molecular analysis of the different stages of breast cancer progression

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Genomic and transcriptomic data in combination with morphological and immunohistochemical data stratify the majority of breast cancers into a “low-grade-like” molecular pathway and a “high-grade-like” molecular pathway. Figure 3. The low-grade-like pathway (left hand side) is characterized by recurrent chromosomal loss of 16q, gains of 1q, a low-grade-like gene expression signature, and the expression of estrogen and progesterone receptors (ER+ and PR+). The progression (vertical arrows) along this pathway (green rectangles) culminates with the formation of low and intermediate grade invasive ductal, (LG IDC and IG IDC) and invasive lobular carcinomas including both the classic (ILC) and the pleomorphic variant (pILC). The tumors arising from the low grade pathway are classified as luminal consisting of a continuum of gene expression frequently associated with the absence (luminal A) or presence of HER2 expression (luminal B). The vast majority of ILCs and pILCs and their precursors cluster together within the luminal subtype. The high grade-like gene expression molecular pathway (right hand side) is characterized by recurrent gain of 11q13 (+11q13), loss of 13q (13q−), expression of a high-grade-like gene expression signature, amplification of 17q12 (17q12AMP), and lack of estrogen and progesterone receptors expression (ER− and PR−). The progression along this pathway (red rectangles) includes intermediate and high grade ductal carcinomas that are stratified as HER2, or basal-like, depending on the expression/amplification of HER2. The molecular apocrine subtype, characterized by the lack of ER expression and presence of AR expression, arises from the high grade pathway. The model also depicts intra-pathway tumor grade progression (horizontal arrows).

Although the genomic and transcriptomic data presented in this review support the divergent model of breast cancer progression, the clinical experience indicates that tumors within each pathway are still fairly heterogeneous with respect to clinical outcome suggesting that even this advanced molecular progression scheme is oversimplified.

The future application of massively parallel sequencing technologies to the preinvasive stages of breast cancer will assist in assessing intratumoral heterogeneity during the transition from preinvasive to invasive breast cancer, and may assist in identifying early tumor initiating genetic events.


Over the past decade the integration of numerous genomic and transcriptomic analyses of the various stages of breast cancer has generated multiple novel insights in the complex process of breast cancer progression.

  • First, human breast cancer appears to progress along two distinct molecular genetic pathways that strongly associate with tumor grade.
  • Second, in the epithelial and non-epithelial components of the tumor microenvironment, the greatest molecular alterations (at the gene expression level) occur prior to local invasion.
  • Third, in the epithelial compartment, no major additional gene expression changes occur between the preinvasive and invasive stages of breast cancer.
  • Fourth, the non-epithelial compartment of the tumor micromilieu undergoes dramatic epigenetic and gene expression alterations occur during the transition form preinvasive to invasive disease. Despite these significant advances, we have only begun to scratch the surface of this multifaceted biological process. With the advent of additional novel high-throughput genetic, epigenetic and proteomic technologies, it is anticipated that the next decade of breast cancer research will gain an equally paralleled appreciation for the complexity breast cancer progression. It is with great hope that knowledge gained from such studies will provide for more effective strategies to not only treat, but also prevent breast cancer.



2. Jemal, a. et al. CA Cancer J. Clin. 60, 277–300; 2010

3. Alessandro Bombonati and Dennis C Sgro. The molecular pathology of breast cancer progression. J Pathol 2011; 223: 307–317.

4. Rodney C. Richie and John O. Swanson. Breast Cancer: A Review of the Literature. J Insur Med 2003;35:85–101.


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