Avances en patología pulmonar

María Dolores Lozano

Clínica Universidad de Navarra

Barcelona, 11 Junio 2015

Definición, criterios diagnósticos e implicaciones de STAS en resecciones de

NSCLCReferencias:

Spread through alveolar spaces: A novel pattern of invasion associated with poor prognosis in 411 small (≤ 2 cm) stage I lung adenocarcinoma . Kyuichi Kadota, Jun-ichi Nitadori, Camelia Sima, David J Jones, William Travis, Prasad Adsumilli. Memorial Sloan Cancer Center, New York, NY (Abstract 1924)

Prognostic significance of “spread through alveolar spaces” in mucinous adenocarcinomas of the lung. Adina Paulk Sandly Liu, Borislav A, Allen Burke. University of Maryland Medical Center, Baltimore, MD. (Abstract 1954)

Kadota K, Nitadori JI, Sima CS, Ujiie H, Rizk NP, Jones DR, Adudumilli PS, Travis WD. Tumor spread through air spaces is an important pattern of invasión and impacts the frequency and location of recurrences following limited resection for small stage I lung adenocarcinomas. J Thorac Oncol 2015;10: 806-14

El concepto “diseminación tumoral a través de espacios alveolares – STAS” se define como una forma de invasión caracterizada por diseminación de células tumorales aisladas o grupo tumorales a

través de los espacios alveolares (STAS). Es un patrón de invasión no existente en ningún otro órgano debido a las características

específicas y únicas de la anatomía pulmonar.

Hay tres patrones morfológicos:

Micropapilar

Nidos sólidos

Células aisladas

Spread through alveolar spaces: A novel pattern of invasion associated with poor prognosis in 411 small (≤ 2 cn) stage I lung adenocarcinoma (Abstract 1924)

411 resecciones de Ac ≤ 2 cm STAS 155 (38%)

resección atípica 120

Lobectomía 291

Resección atípica + STASPeriódo libre de recurrencia menor (52% vs 80%, p<0,001)

Riesgo de metastasis & recidiva locoregional (42,6% vs 10,9%.

P<0,001)Lobectomía + STAS No correlación con estos parámetros (p=0,50;

p=0,76)

J Thorac Oncol 2015;10: 806-14Patrón sólido

Células sueltas

Patrón micropapilar

Conclusiones:

 The presence of STAS is a significant risk factor of recur-rence in small lung adenocarcinomas treated with lim-ited resection.

These findings support our proposal that STAS should formally be recognized as a pattern of invasion in lung adenocarcinoma. J Thorac Oncol 2015;10: 806-14

Prognostic significance of “spread through alveolar spaces” in mucinous adenocarcinomas of the lung. Adina Paulk Sandly Liu, Borislav A, Allen Burke. University of Maryland Medical Center, Baltimore, MD. (Abstract 1954)

El significado clínico de STAS en AC mucinosos no está bien estudiado.

30 resecciones: 2 Ac Coloides / 28 Ac mucinosos invasivos

22 STAS células aisladas, patron sólido, túbulos (15)

patron micropapilar (7)

Conclusiones:

STAS is common in mucinous adenocarcinomas. The micropapil-lary type has prognostic significance in terms of metastasis (p=0,03) and recurrence (p=0,04)

PDL-1 es una importante molécula inmunorregula-dora que bloquea la respuesta inmune citotóxica en una variedad de procesos fisiológicos y patológicos La interacción PD1/PDL1 resulta en tolerancia inmune frente al tumor

La inhibición PD1/PDL1 reestablece la tolerancia inmune frente al tumor: Inmunoterapia

Ensayos clínicos en curso muestran resultados prometedores con dianas

dirigidas frente a PDL-1 y su receptor PD-1.

Resultados preliminares de estos ensayos clínicos indican que la sobrexpresión

de PDL-1 en las células tumorales mediante inmunohistoquímica puede predecir

la respuesta clínica frente a terapias PD-1/PDL-1.

Marcadores de respuesta inmune en NSCLC (PDL1, PD1 )

Comparison of Programmed cell death ligand-1 (PDL-1) expression in main tumor and

lymph node metastasis of stage II and III lung adenocarcinomas. (Abstract 1897)

¿La expresión de PDL-1 cambia con la progresión del tumor y/o es diferente en varias localizaciones de un tumor determinado?

Comparar la expresión de PDL-1 en el tumor primario y las metástasis ganglionares en Ac de pulmón estadios II y III

Examinar la asociación entre expresión de PDL-1 con aspectos clinicopatológicos 109 Ac sin neoadyuvancia (74 en el abstract)

PDL-1 (clone: E1L3N,Cell Signalling Technology)

Positivo si ≥ 5% de las células tumorales expresan tinción de membrana de cualquier intensidad

Inmunohistoquímica con Ac antiCD8 (4B11,RTU,Leica biosystems)

PDL1 se asocia con presencia de linfocitos intratumorales CD8+ en el tumor primario

75% de ganglios N1 y N2 muestran la misma expresión de PDL1 que el tumor primario, por tanto la expresión de PDL1 parece no estar alterada con la progresión del tumor

Clinicopathological and molecular features associated with programmed cell death

ligand-1 (PDL-1) expression in resected lung squamous cell carcinomas. Massachusetts

General Hospital, Boston, MA. (Abstract 1916)

Pocos datos sobre características clinicopatológicas y moleculares de

carcinomas escamosos que expresan PDL-1.

TMAs de 159 pacientes con Ca. escamosos de pulmón. Estadio I: 94; II: 40; III: 22; IV: 3

PDL-1 (clone: E1L3N,Cell Signalling Technology) es positivo si se observa tinción de membrana en ≥5% de las células tumorales.

Estudian los TIL con CD8. .(4B11 mouse mAb (leica b)

Estudio molecular mediante SNaPshot. (153 casos)

Diseño:

Sobrexpresión de PDL-1 en 42 (26%) Mutaciones: PIK3CA: 11; TP53: 11. Amplificaciones: FGFR: 15; PDGFR: 4 Observan relación entre expresión de PDL-1 y presencia de

TIL (p=0,007) y estadios avanzados (I vs II – IV, p=0,0058). De 11 tumores con mutaciones en PIK3CA, 7 expresaban PDL-

1 (p=0.012)

Resultados:

Sobrexpresión de PDL-1 por IHQ se asocia significativamente a la presencia de TIL y a estadios avanzados en Carcinomas escamosos de pulmón resecados

Existe asociación con mutaciones en PIK3CA

Conclusiones:

Pocos datos sobre características clinicopatológicas y moleculares de

adenocarcinomas de pulmón que expresan PDL-1.

Clinicopathological and molecular features associated with programmed cell death

ligand-1 (PDL-1) expression in resected lung adenocarcinomas. Massachusetts General

Hospital, Boston, MA. (Abstract 1917)

TMAs de 141* pacientes con Ac de pulmón. Estadio 0: 2; I: 93; II: 23; III: 11; IV: 12

Patrones: lepídico-48; acinar-49; papilar-16; micropapilar-6; sólido-18; variantes-4.

PDL-1 (clone: E1L3N,Cell Signalling Technology) es positivo si se observa tinción de membrana en ≥5% de las células tumorales.

Estudian los TIL con CD8.(4B11 mouse mAb (leica b)

Estudio molecular mediante SNaPshot.

Diseño:

Sobrexpresión de PDL-1 en (15,7%) Mutaciones KRAS: 56; EGFR: 34; otras en 5 casos* Observan relación entre expresión de PDL-1 y

hábito tabáquico (>10 paq/año; p=0,0019), patrones sólido (p=0,018) y acinar de alto grado

(p=0,0024) presencia de TIL (p=0,014).

Expresión de PDL-1 se asocia a mutaciones en KRAS (27%, p=0.0003)

No hay asociación estadísticamente significativa entre expresión de PDL-1 y periodo libre de enfermedad (p=0,9)

Resultados:

Sobrexpresión de PDL-1 por IHQ se asocia significativamente a la presencia de TIL

Grado nuclear alto, invasión vascular, tamaño del tumor

Existe asociación con mutaciones en KRAS

Conclusiones:

Novedades USCAP en tumores neuroendocrinos de pulmón

Referencias:

Beyond mitosis and necrosis: Additional histo-Cytologic differences between typical and

atypical carcinoids of the lung and their prognostic significance (Abstract 1963). Lauren Rosen,

Ihab Lamzabi, Vijaya Reddy, Paolo Gattuso. Rush University Medical Center, Chicago, IL.

Architectural differences between Typical and atypical carcinoids of the lung and their prognosis

significance. Ihab Lamzabi et al. Rush University Medical Center, Chicago, IL (Abstract 1929)

Tumores neuroendocrinos bien diferenciados de pulmón

Grado I: Carcinoide típico (CT)

Grado II: Carcinoide atípico (CA)Mitosis y necrosis

Hasta 20% fallecen a los 5 años

Objetivo: mejorar el diagnóstico de “subtipos de alto riesgo” de CT y CA usando criterios histológicos objetivos

Datos morfológico

s

Mitosis

Necrosis

Multinucleación

Relación N/C

Mitosis

Necrosis

Tamaño Nuclear

Cromatina

grumos

Patrón en nidos

p 0,01 0,008 0,004 0,01 <0,001

<0,001 0,01 0,001 0,001

Revisión de 50 casos (35 CT y 15 CA). Criterios cito-histológicos asociados a metástasis

Metástasis ganglionares

Metástasis a distancia

Patrón en nidos

Permeación del

pulmón adyacente

Infiltración Cápsula Crec. trabecular

TC (35) 8/35 (22%)

11/35 (31%)

18/35 (51%)

10/35 (28%)

22/35 (63%)

AC (15) 10/15 (67%)

12/15 (80%)

13/15 (87%)

1/15 (6%) 3/15 (20%)

TC vs AC P=0,001 P=0,002 P=0,01 P=0,02 P=0,004

Revisión de 50 casos (35 CT y 15 CA). Criterios arquitecturales

Pleomorfismo nuclear

Núcleos grandes

Relación N/C aumentada

Cromatina en grumos gruesos

Estratificación de pacientes con CT y CA en categorías de bajo y alto riesgo

Patrón en nidos

Permeación del pulmón adyacente

Mitosis y necrosis (p<0,001)

Metástasis ganglionares y a distancia

Criterios citohistológicos y arquitecturales en CT y CA de significado pronóstico

Expression of PAK1 and PAK2 is associated with grade of pulmonary neuroendocrine tumors. Stephen

Smith, Adam Bissonnette, David Cohen, Cynthia Timmers, Jin Jen, Junya Fukuoka, Teri Franks, William

Travis, David Carbone, Konstantin Shilo. Ohio State University, Columbus, OH; Mayo Clinic, Rochester,

MN; Nagasaki University, Nagasaki, Japan; Joint Pathology Center, Silver Spring, MD; Memorial Sloan

Kettering Cancer Center, New York, NY. (Abstract 1967)

P21-activated kinasas (PAKs), contribuyen a regular supervivencia celular y proliferación

Sobreexpresión de PAK1 y PAK2 documentada en ca escamoso y papilomas

Objetivo: estudiar al sobreexpresión IHQ de PAK 1 y PAK 2 en varios tipos de tumores de pulmón (especialmente TNE)

392 tumores usando microarrays de tejidos

Score: negativo, baja expresión, alta expresión (citoplasma)

Resultados y Conclusiones:

Expresión en varios tipos de tumores de pulmón con diferentes frecuencias

PAK1 / tumores carcinoides

PAK2 / neuroendocrinos de alto grado.

60% SCLC expresan PAK2. Potencial diana terapeútica???

Revisión de las traslocaciones en NSCLC y métodos de detección. Papel de la inmunohistoquímica.

Biomarcadores en citología.

Legend Figure 1 PDL-1 immunostaining performed using the E1LN3N clone anti-PD-L1 from Cell Signaling Technology (Boston, USA) with standard detection techniques. A: Squamous cell carcinoma showing a strong, uniform positive reaction in tumour cells. B: Despite being negative in tumor cells in the centre of the image, there is a positive reaction in macrophages and other immune cells in the tumour stroma. C: Most alveolar macrophages are positive

for PD-L1. D: This adenocarcinoma is negative for PD-L1. It should be noted that this IHC clone was not used for PD-L1 detection in any of the trials discussed in this review.

The IASLC Pathology Committee raises the prospect of trying to harmonize and standardize testing for PD-L1 by IHC, at least at a technical level, but also, ideally, as a predictive marker, in order to facilitate availability of this test and a promising treatment for patients with NSCLC.

Proposal for multicentre international standardization project

A multicenter, international standardization effort could address many of these questions and help develop one “standardized” assay, for each of this family of drugs that comes into clinical use as well as analyze additional immunotherapy-related predictive markers.

ALK IQH como screening

ALK IHQ negatico / no FISH

ALK IHQ positivo / confirmación por FISH

Algunos ensayos clínicos aceptan ALK IHQ / VENTANA

+ neumocitos reactivos y CGMN

NRG1 (Neuregulin) “The newest kid on the block”

Codifica una proteína de la familia EGF 7-27% de Ac mucinosos invasivos Mujeres no fumadoras Ensayos clínicos en marcha frente posible diana (ERBB3 inhibitor)

Predictive biomarker testing in cytology ans small biopsy specimens

Nastasha Rekhman, MD, PhD

MSKCC, New York

• Limitada celularidad

Alto % de pacientes se diagnostican mediante estas muestras

Su uso adecuado permitiría mayor beneficio.

Retos y oportunidades

Infrautilización.

Preanalítica variable.

Optimización.

Obtención y manejo de las muesras

REFERENCES1. Brustung OT, Khattak AM, Tromborg AK, Beigi M, Beiske K, Lund-Iversen M, Helland A. BRAF-mutations in non-small cell lung cáncer. Lung Cancer 2014;84:36-82. Li S, Li L, Zhu Y, Huang C, Qin Y, Liu H, Ren-Heidenreich L, Shi B, Ren H, Chu X, Kang J, Wang W, Xu J, Tang K, Yang H, Zheng Y, He J, Yu G, Liang N. Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatig mutations in lung cáncer: a comprehensive mutation profiling from 5125 Chinese cohorts. Br J Cancer 2014;110:2812-203. Villaruz LC, Socinski MA, Abberbock S, Berry LD, Jonhson BE, Kwiatkowski DJ, Iafrate AJ, Varella-Garcia M, Franklin WA, Camidge DR, Sequist LV, Haura EB, Ladanyi M, Kurland BF, Kugler K, Minna JD, Bunn PA, Kris MG. Clinicopathological features and outcomes of patients with lung adeocarcinomas harboring BRAF mutations in the Lung Cancer Mutation Consortium. Cancer 2015;121:448-564. Marchetti A, Felicione L, Malatesta S, Grazia Sciarrotta M, Guetti L, Viola P, Pullara C, Mucilli F, Buttitta F. Clinical features and outcome of patients with non-small-cell lung cáncer harboring BRAF mutations. J Clin Oncol 2011;29:3574-9

BRAF Mutations in NSCLC: Clinical Features and Outcome of a Clinical Series of Patients Diagnosed by Cytology María D. Lozano M.D.1, Tania Labiano M.D. 1,José I Echeveste M.D.1 , Alfonso Gúrpide PhD 2, Nerea Gómez CLT1, . P. Preciado MD2, José Luis Solorzano M.D.1 , Hernán Quiceno M.D.1 ,

Mariam Maset CLT1, Salvador Martín-Algarra - Algarra MD2.1. Department of Pathology, 2. Department of Medical Oncology. University Clinic of Navarra.

Pamplona, SPAIN

BACKGROUNDPresence of genetic alterations in various kinases is known as predictive markers in non-small cell lung carcinoma (NSCLC). The classification schema is based on specific so-called driver mutations in frequencies exceeding 1%. BRAF is one of three members of the RAF kinase family. BRAF-mutations are known as malignant drivers in a number of cancers. While BRAF mutations in NSCLC have been described for several years, the actual prevalence and clinical features of patients with NSCLC who harbor BRAF mutations are not well defined. Therapies against the specific V600-mutated BRAF-variant are developed and show promising results.We report a series of 205 consecutive NSCLC patients diagnosed by FNA on which BRAF mutational analysis was performed as a part of routine molecular analysis.

PATIENTS AND METHODS• Analysis of BRAF was introduced during last year as a part of routine molecular studies in NSCLC patients together with EGFR, KRAS, and ALK.• We analyzed 205 cytological samples including 183 FNA, 8 pleural fluids, and 14 samples received for consultation. Sample procurement

details are summarize in Table 1. • ROSE was performed in all cases to ensure a correct management of the samples. (Figure 1 )• Analysis of BRAF was performed using Cobas 4800 Braf mutation test in 96 cases (46.8%), direct sequencing in 72 (35.1%), and

pyrosequencing in 28 (13.7%). Nine of these cases were analyzed in duplicated by Cobas and direct sequencing (Table 2).• Clinicopathological characteristics are summarize in Table 3 and Table 4

CONCLUSIONSTo our knowledge this is the first clinicopathological study that includes BRAF in a routine comprehensive diagnostic panel of molecular drivers in serial cytological samples of NSCLC. The frequency of BRAF mutation is 2%. All mutations were found in adenocarcinomas. Contrary to date reported from most retrospective larger surgical series, mutations of BRAF occur in males and smokers. Clinical trails show promising results in BRAF mutated patients. This study supports the value of cytological samples for comprehensive molecular panels. Adequate management of samples is mandatory.

Cytological samples N %

FNA-Bronchoscopy 54 26,3%

FNA-EBUS 38 18,5%

FNA-EUS 39 19%

FNA-CT 19 9,2%

FNA-Ultrasonography 17 8,2%

Direct superficial FNA 16 7,8%

Pleural Fluid 8 3,9%

Consultant cases* 14 6,8%

Total 205 100%

Positive cases Age Gender Histological subtype Stage Smoking

History Status / Follow-

up

1 69 Male Adenocarcinoma IV 60 pack/year Dead*

2 72 Male Adenocarcinoma IV 40 pack/year Alive (7 months)

3 57 Male Adenocarcinoma IV 35 pack/year Alive (14 months)

4 59 Male Adenocarcinoma IV 60 pack/year Alive (16 months)

Gender # %

Male 148 72,2%

Female 57 27,8%

Total 205 100%

Smoking Status

Non-smoker 40 19,5%

Smoker ≤ 10 pack/year 14 6,8%

Smoker > 10 pack year 140 68,3%

Missing information 11 5,4%

Total 205 100%

BRAF

WT 200 97,6%

V600E mutation 4 2%

Invalid 1 0,5%

Total 205 100%

Method for BRAF analysis N %

Cobas 96 46,8%

Direct Sequencing 72 35,1%

Pyrosequencing 28 13,7%

Cobas & Direct sequencing 9 4,4%

Total 205 100%

TTF-1

*11 Stained Smears and 3 cell blocks

* Disseminated intravascular coagulation

Table 1: Sample procurement

Figure 1: ROSE (Rapid On Site Evaluation)

Table 2: BRAF mutation test

Table 4: Gender Table 3: Smoking history

Table 5: BRAF status

Table 6: Clinicopathological Characteristics of four BRAF mutated patients

Figure 2:Cytological findings in one of the cases harboring BRAF V600E mutation

V600E confirmed mutation by direct sequencing

RESULTS• BRAF wild type was found in 200 cases (97.6%), one was invalid due to paucity of DNA (0.5%) (Table 5). Four cases (2 %) harbored BRAF V600

mutation. DNA was obtained from Papanicoalou stained smears in two cases, one pleural fluid, and one cell block from a case for consultation. In these positive cases Cobas results were confirmed by direct sequencing. All harbored the V600E mutation (Figure 2). No concomitant EGFR and KRAS mutations were detected. ALK rearrangements were not observed in the four BRAF mutated cases.

• All cases were stage IV adenocarcinoma, all patients were males and smokers. (Table 6)• They were enrolled in a clinical trial using selective inhibitors of mutant BRAF. All four patients had partial response and three persist up to date in

disease stabilization at 7, 14, and 16 moths.

Positive cases Age

Gender

Histological subtype Stage Smoking

History Status /

Follow-up 1 69 Male Adenocarcinoma IV 60 pack/year Dead*

2 72 Male Adenocarcinoma IV 40 pack/year Alive (7 months)

3 57 Male Adenocarcinoma IV 35 pack/year Alive (14 months)

4 59 Male Adenocarcinoma IV 60 pack/year Alive (16 months)

BRAFWT 200 97,6%

V600E mutation 4 2%

Invalid 1 0,5%Total 205 100%

Table 5: BRAF status

Table 6: Clinicopathological Characteristics of four BRAF mutated patients

CONCLUSIONSTo our knowledge this is the first clinicopathological study that includes BRAF in a routine comprehensive diagnostic panel of molecular drivers in serial cytological samples of NSCLC. The frequency of BRAF mutation is 2%. All mutations were found in adenocarcinomas. BRAF Mmutations occur in males and smokers. Clinical trails show promising results in BRAF mutated patients. This study supports the value of cytological samples for comprehensive molecular panels. Adequate management of samples is mandatory.

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